Synthesis of a library of fucosylated glycoclusters and determination of their binding toward pseudomonas aeruginosa lectin B (PA-IIL) using a DNA-based carbohydrate microarray

Article abstract of DOI:10.1021/bc2006434

Pseudomonas aeruginosa (PA) is a Gram negative opportunistic pathogen and is the major pathogen encounter in the cystic fibrosis (CF) lung airways. It often leads to chronic respiratory infection despite aggressive antibiotic therapy due to the emergence of resistant strains and to the formation of biofilm. The lectin PA-IIL (LecB) is a fucose-specific lectin from PA suspected to be involved in host recognition/adhesion and in biofilm formation. Thus, it can be foreseen as a potential therapeutic target. Herein, 16 fucosylated glycoclusters with antenna-like, linear, or crown-like spatial arrangements were synthesized using a combination of DNA solid-phase synthesis and alkyne azide 1,3-dipolar cycloaddition (CuAAC). Their binding properties toward PA-IIL were then evaluated based on DNA directed immobilization (DDI) carbohydrate microarray. Our results suggested that the antenna-like scaffold was preferred to linear or crown-like glycoclusters. Among the crown-like carbohydrate centered fucosylated glycoclusters, mannose-based core was better than glucose- and galactose-based ones. The influence of the linker arm was also evaluated, and long linkers between fucoses and the core led to a slight better binding than the short ones.

Full text of DOI:10.1021/bc2006434

Article
pubs.acs.org/bc
Synthesis of a Library of Fucosylated Glycoclusters and
Determination of their Binding toward Pseudomonas aeruginosa
Lectin B (PA-IIL) Using a DNA-Based Carbohydrate Microarray
†
‡
†
†
§
∥
Bea
́
trice Gerland, Alice Goudot, Gwladys Pourceau, Albert Meyer, Vincent Dugas, Samy Cecioni,
∥
‡
†
,‡
,†
Sebastien Vidal, Eliane Souteyrand, Jean-Jacques Vasseur, Yann Chevolot,* and Franc o̧ is Morvan*
́
†
Institut des Biomolec
Eugene Bataillon, CC1704, 34095 Montpellier Cedex 5, France
Institut des Nanotechnologies de Lyon (INL), UMR 5270 CNRS, Ecole Centrale de Lyon, 36 Avenue Guy de Collongue, 69134
Ecully Cedex, France
́
ules Max Mousseron (IBMM), UMR 5247 CNRSUniversite
́
Montpellier 1Universite
́
Montpellier 2, Place
̀
‡
§
Laboratoire des Sciences Analytiques, UMR 5180 CNRS, Universite
9622 Villeurbanne Cedex, France
́
Claude Bernard Lyon 1, 43 Boulevard du 11 Novembre 1918,
6
∥
Institut de Chimie et Biochimie Molec ulaires (ICBMS), Laboratoire de Chimie Organique 2Glycochimie,
́
ulaires et Supramolec
́
UMR 5246 CNRS, Universite
S Supporting Information
́
Claude Bernard Lyon 1, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne, France
*
ABSTRACT: Pseudomonas aeruginosa (PA) is a Gram
negative opportunistic pathogen and is the major pathogen
encounter in the cystic fibrosis (CF) lung airways. It often
leads to chronic respiratory infection despite aggressive
antibiotic therapy due to the emergence of resistant strains
and to the formation of biofilm. The lectin PA-IIL (LecB) is a
fucose-specific lectin from PA suspected to be involved in host
recognition/adhesion and in biofilm formation. Thus, it can be
foreseen as a potential therapeutic target. Herein, 16
fucosylated glycoclusters with antenna-like, linear, or crown-
like spatial arrangements were synthesized using a combination
of DNA solid-phase synthesis and alkyne azide 1,3-dipolar
cycloaddition (CuAAC). Their binding properties toward PA-IIL were then evaluated based on DNA directed immobilization
(
DDI) carbohydrate microarray. Our results suggested that the antenna-like scaffold was preferred to linear or crown-like
glycoclusters. Among the crown-like carbohydrate centered fucosylated glycoclusters, mannose-based core was better than
glucose- and galactose-based ones. The influence of the linker arm was also evaluated, and long linkers between fucoses and the
core led to a slight better binding than the short ones.
INTRODUCTION
This lectin has become an attractive target for therapeutics,
and several groups have developed new glycomimetics with
high affinity for PA-IIL. Thus, it is claimed that they may
compete with host glycans and hence could inhibit the
■
Carbohydrate−lectin interactions play an important role in
several biological processes such as cell−cell communication,
inflammation, innate system, and infections by pathogens (i.e.,
adhesion of the bacteria. Indeed, carbohydrates and their
1
−4
viruses or bacteria).
Pseudomonas aeruginosa (PA) is the
17,18
mimetics could be developed as antiadhesive drugs
against
causative agent of lung infections and the major cause of
morbidity and mortality among cystic fibrosis patients but also
among patients hospitalized in intensive care units. PA
expresses two soluble lectins, PA-IL (LecA) and PA-IIL
LecB). PA-IIL has been proposed to be implicated in host
recognition and adhesion and also in biofilm formation. This
pathogens as an alternative to antibiotics. Because antiadhesive
6,9−16
drugs do not affect the viability of bacteria,
it is rather
improbable that bacteria will develop a resistance to such drugs.
As the lectin avidity is relatively low when only one
carbohydrate residue is involved, Nature uses a cluster effect
where several weak interactions yield to a higher affinity than
(
5
,6
4,19−21
tetrameric lectin presents four carbohydrate recognition
domains and binds L-fucose with micromolar affinity. It is
the sum of individual interactions.
The design of
7
more or less a two-tilted cobblestone-shaped protein leading to
a tetrahedral spatial distribution with a distance of 40 Å
between two carbohydrate binding sites.
Received: November 28, 2011
Revised: June 20, 2012
Published: July 16, 2012
8
©
2012 American Chemical Society
1534
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Bioconjugate Chemistry
Article
glycoclusters has been extensively investigated to discover
synthetic glycomimetics able to show high avidity for lectins.
(Cap A, Ac O/pyridine/THF, 10:10:80; Cap B, 10% N-
2
22
methylimidazole in THF) for 15 s. Each oxidation was
performed for 15 s. Detritylation was performed with 2.5%
DCA in CH Cl for 35 s.
Herein, 16 different fucosylated glycomimetics exhibiting
from one to eight fucose residues were synthesized with
antenna-like, linear (or comb-like), or crown-like spatial
2
2
Immobilization on Azide Solid Support 4 of 1-O-
Propargyl Hexoses 15a−c by Cu(I)-Catalyzed Alkyne
Azide 1,3-Dipolar Cycloaddition. An aqueous solution of 1-
O-propargyl hexose (α-mannose 15a, β-galactose 15b, β-
glucose 15c) (100 mM, 175 μL), freshly prepared aqueous
2
3
arrangements. Next, their binding properties toward PA-IIL
were determined using DDI-based carbohydrate micro-
2
4,25
26,27
array.
DNA directed immobilization (DDI)
uses
DNA/DNA hybridization between an immobilized single-
stranded oligonucleotide at the surface of a solid (DNA chip)
and a DNA tagged molecule. In our case, fucosylated
glycoclusters carry a DNA tag for their DDI immobilization
as well as a Cy3 fluorescent dye as an immobilization quality
control reporter. Binding properties of the lectin to the
designed glycoclusters were measured using Alexa-647 labeled
PA-IIL.
solutions of CuSO (100 mM, 14 μL) and sodium ascorbate
4
(500 mM, 14 μL), water (147 μL), and MeOH (350 μL) were
added to 3.5 μmol of azide solid support 4. The resulting
mixture was treated in a sealed tube with a microwave
synthesizer at 60 °C for 45 min (premixing time: 30 s). The
temperature was monitored with an internal infrared probe.
The solution was removed, and CPG beads were washed with
H O (3 × 2 mL), MeOH (3 × 2 mL), and CH CN (3 × 2
2
3
EXPERIMENTAL PROCEDURES
mL), and dried, affording 16a−c.
■
Immobilization on Alkyne Solid Support 1 of 3-
Azidopropyl-α-D-mannopyranoside 24 by Cu(I)-Cata-
lyzed Alkyne Azide 1,3-Dipolar Cycloaddition. A solution
of 3-azidopropyl-α-D-mannopyranoside 24 (100 mM in
MeOH, 300 μL), freshly prepared aqueous solution of
General Procedure for the Elongation by Hydro-
genophosphonate Chemistry. Elongation was performed
on a DNA synthesizer (ABI 394) using a H-phosphonate
chemistry cycle starting from a 1,3-propanediol solid support 7
(
1 μmol). The detritylation step was performed with 2.5%
CuSO (100 mM, 24 μL), and sodium ascorbate (500 mM,
DCA in CH Cl2 for 35 s. Then dimethanolcyclohexane
4
2
2
4 μL), water (552 μL), and MeOH (300 μL) were added to 6
(
DMCH) H-phosphonate monoester 9 (60 mM in anhydrous
μmol of alkyne solid support 1. The tube containing the
resulting suspension was sealed and placed in an oil bath at 60
CH CN/C H N 1:1 v/v) and pivaloyl chloride as activator
3
5
5
(
200 mM in anhydrous CH CN/C H N 1:1 v/v) were passed
3 5 5
°
C for 1 h. After the reaction, the solution was removed, and
six times through the column alternatively for 5 s, (30 molar
excess). The cycle was repeated as required to afford 12a−d.
General Procedure for the Elongation Mixing Hydro-
genophosphonate and Phosphoramidite Chemistries.
The solid-supported hydrogenophosphonate diester scaffold
the CPG beads were washed with H O (3 × 2 mL), MeOH (3
2
×
2 mL), and CH CN (3 × 2 mL), and dried, affording 25.
3
General Procedure for Introduction of Pent-4-ynyl,
Bis-pent-4-ynyl, Bromohexyl, or 3,6-Dioxanon-8-yn-1-yl
Phosphoramidites on Mannose, Glucose, or Galactose
Hydroxyls of 16a−16c or 25. Solid-supported hexose
derivatives 16a−16c or 25 (1 μmol scale) were treated by
phosphoramidite chemistry, on a DNA synthesizer, with
pentyn-4-yl phosphoramidite 17, 6-bromohexyl phosphorami-
dite 22, or 3,6-dioxanon-8-yn-1-yl phosphoramidite 26. Only
coupling and oxidation steps were performed. For the coupling
step, benzylmercaptotetrazole was used as activator (0.3 M in
1
2m was synthesized at the 1 μmol scale on a DNA synthesizer
(
ABI 394). First, H-phosphonate monoester 9 was coupled as
28
described above. Then, the DMCH phosphoramidite 11
(
0.09 M in anhydrous CH CN) was introduced using a
3
phosphoramidite chemistry cycle with benzylmercaptotetrazole
as activator (0.3 M in anhydrous CH CN) with a 120 s
3
coupling time. No capping step was performed to avoid O-
acylation. Oxidation was performed with a solution of tBuOOH
(
1.1 M in anhydrous CH Cl ) for 60 s to avoid the oxidation of
anhydrous CH
M in anhydrous CH
μmol) with a 180 s coupling time. Oxidation was performed
3
CN), and phosphoramidite 17, 22, or 26 (0.2
2
2
H-phosphonate diester. Detritylation was performed with 2.5%
DCA in CH Cl for 35 s. The last two H-phosphonate
CN) was introduced three times (120
3
2
2
monoesters 9 were coupled using the H-phosphonate
chemistry cycle, affording 12m.
General Procedure for Amidative Oxidation. The solid-
with commercial solution of iodide (0.1 M I
water 90:5:5) for 15 s.
2
, THF/pyridine/
General Procedure for Azidation. The solid-supported
oligonucleotides bearing the tetrabromohexyl hexoses (1 μmol)
supported H-phosphonate diesters scaffolds 12a−12d and 12m
(
1 μmol) were treated back and forth using two syringes with 2
were treated with a solution of TMG-N
and NaI (30 mg, 200 equiv) in DMF (1 mL) for 1 h at 65 °C.
The beads were washed with DMF (3 × 2 mL), H O (3 × 2
CN (3 × 2 mL) and then dried by flushing with
3
(31.6 mg, 200 equiv)
mL of a solution of 10% of propargylamine in CCl /C H N
4
5
5
(
1:1 v/v) for 30 min. The CPG beads were washed with
2
C H N (2 × 2 mL) and CH CN (3 × 2 mL) and then dried by
flushing with argon.
mL) and CH
argon.
3
5
5
3
General Procedure for Elongation of DNA Sequences
and Labeling with Cy3. The DNA sequences were
synthesized on the solid-supported scaffolds at the 1 μmol
scale on a DNA synthesizer (ABI 394) by standard
phosphoramidite chemistry. For the coupling step, benzylmer-
captotetrazole was used as activator (0.3 M in anhydrous
General Procedure for Deprotection of Solid-Sup-
ported Oligonucleotides. The CPG beads bearing modified
oligonucleotides were transferred to a 4 mL screw top vial and
treated with 2 mL of concentrated aqueous ammonia for 15 h
at room temperature and warmed to 55 °C for 2 h. For each
compound, the supernatants were withdrawn and evaporated to
dryness. Residues were dissolved in water.
CH CN), and commercially available nucleosides phosphor-
3
amidites (0.09 M in anhydrous CH CN) were introduced with
a 20 s coupling time and Cy3 amidite (0.06 M in anhydrous
General Procedure for CuAAC Reaction. Procedure for
Introduction of Azide L-Fucose Derivative 3. To a solution of
5′-fluorescent-3′-alkyne oligonucleotide 2, 14a−14d, 14m,
3
CH CN) with a 180 s coupling time. The capping step was
3
performed with acetic anhydride using commercial solution
18a−18c, 20, and 27 (100 nmol in 100 μL of H O) were
2
1
535
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Bioconjugate Chemistry
Article
added 8-azido-3,6-dioxaocta-1-yl 2,3,4-tri-O-acetyl-L-fucopyra-
noside 3 (3 equiv per alkyne function, 100 mM in MeOH), 1
mg of Cu(0) nanopowder, triethylammonium acetate buffer 0.1
M, pH 7.7 (25 μL), water, and MeOH to obtain a final volume
of 250 μL (water MeOH, 1:1 v/v). The tube containing the
resulting preparation was sealed and placed in a microwave
synthesizer Initiator from Biotage with a 30 s premixing time at
mL) was added, and the mixture was evaporated under reduced
pressure and the residue dissolved in ethyl acetate (40 mL).
The organic layer was washed with saturated ammonium
chloride (40 mL), the aqueous layer was extracted with ethyl
acetate (2 × 20 mL), and the combined organic layers were
evaporated under vacuum. The dry residue was purified via
silica gel column chromatography (eluting with c-C H :EtOAc
6
12
6
0 °C for the indicated time (see Table S1 in Supporting
7:3 to 2:8) to give 3,6-dioxanon-8-yn-1-ol (1.15 g, 67%) as a
1
Information).
clear oil. H NMR (300 MHz, CDCl ): δ = 4.15 (2 H, d, J = 2.4
3
Procedure for Introduction of 1-O-propargyl-L-Fucose 6.
Hz, CH -CCH), 3.74−3.49 (8 H, m, CH ), 2.38 (1 H, t, J = 2.4
2
2
+
To a solution of 5′-fluorescent-3′-azide oligonucleotide 5 and
Hz, HCC−). LRMS (ESI+): for C H NO [M + NH ] calcd
7
16
3
4
2
3a−23c (100 nmol in 100 μL of H O) were added 1-O-
162.1, found 162.1.
2
propargyl 2,3,4-tri-O-acetyl-L-fucopyranoside 6 (3 or 5 equiv
per azide function, 100 mM in MeOH), 1 mg of Cu(0)
nanopowder, triethylammonium acetate buffer 0.1 M, pH 7.7
O-2-Cyanoethyl-O′-(3,6-dioxanon-8-ynyl)-N,N-diisoprop-
yl-phosphoramidite 26. 2-Cyanoethyl-N,N-diisopropylchloro-
phosphoramidite (500 mg, 2.11 mmol) was added to a solution
of 3,6-dioxanon-8-yn-1-ol (253 mg, 1.76 mmol), 4 Å molecular
sieves and N,N′-diisopropylethylamine (DIEA) (489 μL, 2.81
mmol) in anhydrous dichloromethane (20 mL). The resulting
mixture was stirred at room temperature for 5 h 30, 1 mL of
H O was added, and then the solution was diluted with CH Cl
2
(
(
25 μL), water, and MeOH to obtain a final volume of 250 μL
water MeOH, 1:1 v/v). The tube containing the resulting
preparation was sealed and placed in a oil bath with magnetic
stirring at 60 °C for the indicated time (see Table S1 in
Supporting Information).
2
2
Workup of CuAAC Reactions and HPLC Purifications.
EDTA (400 μL) was added to the mixtures, and after
centrifugation, the supernatants were withdrawn to eliminate
Cu(0) and were desalted by size-exclusion chromatography on
NAP10. After evaporation, the 5′-fluorescent 3′-acetyl-glyco-
mimetic oligonucleotides were dissolved in water and purified
by reversed-phase preparative HPLC. Pure compounds were
treated with concentrated aqueous ammonia (3 mL) for 2 h at
room temperature to remove acetyl groups and evaporated to
dryness (purity >97%). Final compounds were purified again by
reversed-phase preparative HPLC using a linear gradient from
(15 mL). The organic layer was washed with an aqueous
saturated NaHCO solution (30 mL). The resulting aqueous
3
layer was then extracted with CH Cl (2 × 30 mL). The
2
2
organics were collected, washed with brine (30 mL), and dried
(MgSO ), filtered, and evaporated. The dry residue was purified
4
via silica gel column chromatography (eluting with c-
C H :EtOAc: 8:2:0.5 to 0:10:0.5) to give the title compound
6
12
26 (379 mg, 63%) as a clear oil. R : 0.51 (c-C H /AcOEt/Et N
f
6
12
3
1
6:4:0.1). H NMR (200 MHz, CDCl ): δ 4.14 (d, J = 2.4 Hz, 2
3
H, CH -CCH), 3.87−3.45 (m, 12 H, 3 × CH CH O, CH OP,
2
2
2
2
2 × CH(iPr), CH CH CN), 2.59 (t, J = 6.6 Hz, 2 H,
2
2
8% to 32% of acetonitrile in TEAAc buffer pH 7 over 20 min.
CH CH CN), 2.36 (t, J = 2.4 Hz, 1 H, HCC−), 1.11 (dd,
J = 2.0, 6.8 Hz, 12 H, iPr ). C NMR (300 MHz, CDCl ) δ
2 3
2
2
13
Residues were dissolved in water for subsequent analyses (see
Supporting Information).
117.7 (CN), 79.7 (CCH), 74.5 (CCH), 71.3, 71.2, 70.4,
Bis-(pent-4-ynyl) N,N-diisopropyl phosphoramidite 19. To
a solution of anhydrous 4-pentyn-1-ol (5.4 mmol, 500 μL) and
anhydrous Et N (6 mmol, 826 μL) in dry CH Cl (5 mL),
69.2 (CH CH OCH CH ), 62.7, 62.5 (CCH O), 58.7, 58.4
2
2
2
2
2
(POCH CH CN), 43.2, 43.0 (NCMe ), 24.7, 24.6, 24.6,
2
2
2
3
1
3
2
2
24.3 (2 × C(CH )2), 20.4, 20.3 (POCH CH CN).
3
2
2
P
stirring at 0 °C under argon, was added a solution of
diisopropylphosphorodichloridite (2 mmol, 550 mg) in dry
CH Cl (5 mL). The resulting mixture was stirred for 4 h at
NMR (121 MHz, CDCl ):δ 148.5.
3
Fabrication of Microarrays. Fabrication of Microstruc-
tured Slides. Photolithography and wet etching process were
employed for the fabrication microreactors onto flat glass slides.
2
2
room temperature, diluted with CH Cl (100 mL), filtered to
2
2
3
0,31
remove triethylammonium chloride salt, and washed with
Theses methods are detailed elsewhere.
NaHCO3 (1 × 150 mL). The organic layer was dried
Silanization of the Glass Slides. Borosilicate glass slides
(Schott) featured 52 round microreactors (2 mm diameter,
65−100 μm deep with less than 1% variation in depth for one
etching lot) with a volume below 1 μL. They were
(
Na SO ), filtered, and evaporated to dryness. The crude
2 4
product was purified by silica gel column chromatography (0−
5
0% CH CL in cyclohexane containing 10% triethylamine) to
2
2
afford the title compound (500 mg, 83%) as clear oil. TLC R :
0
functionalized according to the protocol developed by Dugas
f
1
32,33
.60 (cyclohexane/AcOEt/Et N: 5:4:1, v/v/v). H NMR
et al.
In brief, after piranha treatment, the slides were
3
(
1
CD CN, 300 MHz): δ 1.18 (d, 12H, J = 6.8 Hz, 4 × CH ),
heated under dry nitrogen at 150 °C for 2 h. Next, dry pentane
and tert-butyl-11-(dimethylamino)silylundecanoate were added
at room temperature. After 2 h of incubation, the pentane was
evaporated and the slides were heated at 150 °C overnight.
Functionalized slides were obtained after washing in THF and
rinsing in water. The ester function was converted into the
corresponding acid using formic acid for 7 h at room
temperature. Acid group bearing slides were activated for
amine coupling with N-hydroxysuccinimide (0.1 M) and
di(isopropyl) carbodiimide (0.1 M) in dry THF, overnight at
room temperature. The slides were rinsed in THF and
dichloromethane.
3
3
.73−1.82 (m, 4H, 2 × CH CH CH ), 2.16−2.19 (t, 2H, J =
2
2
2
2
.6 Hz 2 × CCH), 2.29 (dt, 4H, J = 2.6 Hz, J = 7.1 Hz, 2 ×
d
t
CH CH CCH), 3.54−3.77 (m, 6H, 2 × O−CH , 2 × CHMe ).
2
2
2
2
1
3
C NMR (CDCl , 300 MHz): δ 14.2, 23.6, 29.8, 42.3, 61.2,
3
6
8.6, 83.5. 31P NMR (CD CN): δ 146.25 ppm. HRMS ESI+
3
+
m/z [M + H] for C H O NP calcd 298.1915, found
1
6
29
2
2
98.1936.
3,6-Dioxanon-8-yn-1-ol. 3,6-Dioxanon-8-yn-1-ol was syn-
2
9
thesized by a slightly modified procedure.
hydroxyethyl)ether (1.19 g, 14.1 mmol) and sodium hydride
471 mg, 11.8 mmol) were dissolved in anhydrous DMF (80
Bis(2-
(
mL), and the mixture was stirred at 0 °C for 30 min. Then
propargyl bromide (1.30 mL, 11.8 mmol) was added dropwise,
and the resulting mixture was stirred for 6 h at rt. Methanol (5
Immobilization of Amino-Modified Oligonucleotides. 3′-
Amino-modified oligonucleotides were purchased from Euro-
gentec. Two sequences were used: S1: 5′-GTG AGC CCA
1
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Figure 1. Structures of the linear dimethylcyclohexane-tri-β-D-galactomimetic G0 and the fucomimetics F1−F16.
GAG GCA GGG-5CH ) -NH and S2: 5′-GCT AAT CCA
performed with BSA 4% solution in PBS 1× (pH 7.4) at 37 °C
for 2 h. The washing steps were: 3 × 3 min in PBS-Tween 20
(0.05%) followed by 3 × 3 min in PBS 1×, and finally the
glasses were rinsed with deionized water before being dried by
centrifugation.
Hybridization of the Glycoconjugates on DNA Array. First,
1 μL of a solution of each glycoconjugate bearing a DNA tag at
1 μM in PBS 1× (pH 7.4) were placed at the bottom of the
corresponding well and allowed to hybridize overnight at room
temperature in a water vapor saturated chamber. Then the
samples were washed in saline−sodium citrate 2× (SSC 2X),
2
7
2
ACG CGG GCC AAT CCT T-(CH ) -NH . One μL of the
2
7
2
desired sequence (S1 or S2) at 25 μM in PBS 10× (pH 8.5)
were deposited at the bottom of the corresponding well. The
substitution reaction was performed overnight at room
temperature in a water saturated atmosphere, and then water
was allowed slowly to evaporate. Sequential washing of the
slides was performed with SDS (0.1%) at 70 °C for 30 min and
deionized water.
Blocking. After ssDNA immobilization, all slides were
blocked with bovine serum albumin (BSA). Blocking was
1
537
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SDS (0.1%) at 51 °C for 1 min, followed by SSC 2X at room
temperature for an additional 5 min, and finally rinsed with
deionized water before being dried by centrifugation.
Blocking. After hybridization, all slides were blocked again
with bovine serum albumin (BSA). Blocking was performed
with BSA 4% solution in PBS 1× (pH 7.4) at 37 °C for 1 h.
Same washing steps: 3 × 3 min in PBS-Tween 20 (0.05%),
followed by 3 × 3 min in PBS 1×, and briefly rinsed with
deionized water before dried by centrifugation.
The 16 different fucomimetics F1−F16 exhibiting from one
to eight α-L-fucose residues were synthesized through the
introduction of alkyne or azide functions in several scaffolds by
means of phosphoramidite or H-phosphonate chemistries in
combination with the copper catalyzed alkyne azide 1,3-dipolar
3
6,37
cycloaddition (CuAAC reaction).
This strategy allows for
the multiple conjugations of oligonucleotide-like structures with
azido-functionalized or alkynylated carbohydrate derivatives.
For this study, we used a linear dimethanolcyclohexane
(DMCH) trigalactosylated glycocluster G0 as a negative
control (Figure 1) in order to demonstrate the absence of
Alexa-647 Labeling of PA-IIL Lectin. PA-IIL lectin was
labeled with Alexa Fluor 647 microscale protein labeling kit
24
(
A30009) from Invitrogen. In brief, 100 μL of a 1 mg/mL
nonspecific interactions of the lectin PA-IIL. The antenna-like
fucomimetic F1 with a bis-pentaerythrityl (Pe) scaffold and
solution of PA-IIL (MW: 47 kDa, PA-IIL was kindly provided
25
by Dr. Anne Imberty, CERMAV, Grenoble) diluted in PBS 1×
four residues was previously reported by us (Figure 1). For
the linear glycomimetics F3−F7, with a DMCH scaffold, two to
five fucose residues were introduced to evaluate the effect of
valency on their affinity for PA-IIL. For the carbohydrate-
centered fucosylated clusters (crown-like structures) F8−F11
and F13−F16, a hexose core was used with three different
configurations using mannose, galactose, or glucose residues.
Four fucose residues were introduced on each carbohydrate
core with different linkers to evaluate the effects of both the
spatial orientation and the nature of the linker. Among them, a
mannosyl-centered octa-fucosylated glycocluster F11 was
synthesized to evaluate the effect of an increased valency
from tetravalent to octavalent.
(
(
pH 7.4) was mixed with 10 μL of 1 M sodium bicarbonate
pH 8.3). Separately, 10 μL of water was added to the vial of
Alexa Fluor 647 succinimidyl ester in order to have a reactive
dye stock solution at 7.94 nmol/μL. The appropriate volume of
reactive dye solution was transferred into the reaction tube
containing the pH-adjusted protein. Reaction mixture was
mixed for 15 min at room temperature before purification on a
spin column (gel resin container) in order to separate the
labeled protein from unreacted dye. Lectin concentration and
the dye to lectin ratio were estimated by optical density
(
nanodrop) reading the absorbance at 281 and 650 nm. PA-IIL
concentration was estimated to be 3.86 μM bearing an average
of 0.49 dyes per PA-IIL.
Three linker lengths were evaluated with 13, 17, or 21 atoms
between the alcohol functions of the hexose platform and the
exo-anomeric oxygen atom of the fucose moiety for F8−F11
and F13−F16 (Figure 2). Furthermore, glycoclusters F13−F15
Probing Microarrays. On Chip Biological Recognition.
First, 1 μL of a solution in PBS 1× containing labeled lectin
diluted (final concentration 0.12 μM), CaCl (final concen-
2
tration 5 μM), and BSA 20% (final concentration 2%) was
placed at the bottom of each well. Then slides were incubated
at 37 °C in a water vapor saturated chamber for 3 h. The
washing steps are: PBS-Tween 20 (0.02%), 5 min at 4 °C, then
briefly in deionized water.
IC50 Measurement Assay. For IC50 experiments, fucose was
added to the solution of labeled PA-IIL at different final
concentrations (from 0 to 50000 μM maximum of fucose as
represented below). Each solution (1 μL) was placed at the
bottom of each well and incubated at 37 °C in a water vapor
saturated chamber for 3 h. The slides were then washed in PBS-
Tween 20 (0.02%) for 5 min at 4 °C and finally rinsed with
deionized water before being dried by centrifugation.
Fluorescence Scanning. Slides were scanned with the
Microarray scanner, GenePix 4100A software package (Axon
Instruments; λex 532/635 nm and λem 575/670 nm). The
fluorescence signal of each conjugate was determined as the
average of the mean fluorescence signal of four spots.
Figure 2. Structures of linkers used for the construction of
glycoclusters F8−F11, F13−F16, and distances between L-fucose
and triazole ring estimated in number of atoms. MTzHexPO:
RESULTS AND DISCUSSION
We have previously reported a family of galactomimetics with
Methylene triazole hexyl phosphodiester, EG TzProPO: triethylene
3
■
glycol triazole propyl phosphodiester, EG TzMEG PO: triethylene
3
2
glycol triazole methylene diethylene glycol phosphodiester.
different spatial arrangements, i.e., linear, antenna-like, or
2
4,34
crown-like (calixarene core)
and different electrostatic
35
behaviors (i.e., neutral, positively, or negatively charged).
display a triazole aromatic ring (MTz) at one atom distance
from the fucose moiety (with a methylene bridge) while the
triazole ring is located much further away at eight atoms
Their binding to galactose-binding lectins (i.e., RCA120 and
PA-IL) was highly dependent on the architecture of the
glycomimetics, and the influence of valency proved less
important. Herein, we have synthesized a series of 16 new
fucosylated derivatives in order to identify new glycomimetics
presenting high affinity for PA-IIL. Each of these glycoclusters
was labeled with a Cy3-DNA sequence for their immobilization
on a DNA microarray through DNA directed immobilization
distance (EG Tz) for the other glycoclusters. It has been
3
reported that p-nitrophenyl α-L-fucoside displays a better
affinity than L-fucose, probably to a π−π stacking interaction
7
of the phenyl ring with aromatic residues of the lectin. The
present library of glycoclusters will provide insights on the
influence of an aromatic moiety in close vicinity to the fucose
epitope on the affinity for the PA-IIL lectin. Finally, two
27
(
DDI) for a rapid screening of their binding to PA-IIL.
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a
Scheme 1. Synthesis of the Monovalent Fucosylated Conjugate F2
a
SPOS: solid phase oligonucleotide synthesis: (1) 0.09 M phosphoramidite derivative in dry CH CN + 0.3 M benzylthiotetrazole in dry CH CN;
3
3
(
2) Ac O, N-methyl-imidazole, 2,6-lutidine; (3) 0.1 M I THF/H O/pyridine; (4) 2.5% dichloroacetic acid in CH Cl .
2
2
2
2
2
Scheme 2. Synthesis of the Monovalent Fucosylated Oligonucleotide Conjugate F12
4
0
monovalent constructions F2 and F12 with only one fucose
residue but with different linkers were prepared as control for
the evaluation of the multivalency effect (Schemes 1 and 2).
Synthesis of Monovalent Fucosylated Oligonucleo-
tide Conjugates F2 and F12. A monovalent fucosylated
oligonucleotide conjugate with a triethyleneglycoltriazole
microwave assistance. After centrifugation, the supernatant
was desalted by size-exclusion chromatography (SEC). The
resulting acetylated conjugate was treated with ammonia,
providing the monovalent fucosylated conjugate F2 after
purification by HPLC.
The monovalent fucosylated oligonucleotide conjugate F12
(
EG Tz) linker was synthesized starting from the propargylated
with a methylene triazole (MTz) linker was synthesized
similarly starting from the azidohexyl solid support 4 on
3
3
8
41
solid support 1 on which the oligonucleotide sequence was
assembled and then labeled with Cy3 fluorescent dye using
standard phosphoramidite chemistry on a DNA synthesizer
which the oligonucleotide was elongated then labeled with Cy3
and subsequently deprotected with ammonia (Scheme 2). The
resulting 3′-azide oligonucleotide 5 was conjugated with
(
Scheme 1). After treatment with concentrated ammonia for
deprotection and release from the solid support, the resulting
′-Cy3 oligonucleotide with a 3′-alkyne function 2 was
conjugated with the 8-azido-3,6-dioxaocta-1-yl 2,3,4-tri-O-
42
propargyl 2,3,4-tri-O-acetyl-α-L-fucopyranoside 6, affording
the monovalent conjugate F12 after ammonia treatment and
HPLC purification.
5
12
acetyl-α-L-fucopyranoside 3 by CuAAC using Cu(0) nano-
powder in a mixture of methanol and TEAAc buffer under
Synthesis of Linear DMCH Fucosylated Glycoclusters
F3−F7. The linear fucosylated oligonucleotide conjugates were
3
9
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Scheme 3. Synthesis of Linear DMCH Fucosylated Glycoclusters F3−F7
44,45
synthesized using H-phosphonate chemistry combined with
compound 10m.
Then, the synthesis was continued
amidative oxidation to introduce the alkyne functions as
through H-phosphonate chemistry for the two subsequent
coupling cycles, providing 12m followed by the amidative
40
previously reported (Scheme 3). The oligonucleotide was
then elongated and labeled with Cy3 by phosphoramidite
chemistry (Scheme 3). After treatment with ammonia to release
the conjugate from the solid support and deprotect the
polyalkyne oligonucleotide conjugate, the fucose moieties were
introduced in solution by CuAAC reaction as described for
monovalent glycomimetics. Starting from the 1,3-propanediol
solid-support 7, deprotection of the dimethoxytrityl group
afforded alcohol 8. Two, three, four, or five DMCH H-
phosphonate monoesters 9 were then introduced, affording
four different scaffolds 12a−12d with two to five H-
oxidation with CCl /propargylamine (leading to 13m), DNA
4
elongation, and Cy3 labeling (leading to 14m). After ammonia
deprotection, the three fucose residues were introduced in
solution by CuAAC reaction, affording the desired trifucosy-
lated glycocluster F7.
Synthesis of Crown-Like Fucosylated Oligonucleotide
Conjugates F8−F11, and F13−F16. The tetra- and octavalent
crown-like fucosylated glycoclusters were synthesized according
39
to the strategy recently reported, starting from an azido-
41
functionalized solid support 4 on which propargyl α-D-
4
3
46
phosphonate diesters. Amidative oxidation of the H-
phosphonate linkages by carbon tetrachloride in the presence
of propargylamine led to multialkynylated scaffolds 13a−13d,
which were then elongated by phosphoramidite chemistry and
labeled with Cy3. The 5′-Cy3−3′-polyalkyne oligonucleotides
mannopyranoside 15a, propargyl β-D-galactopyranoside
47
47
15b, or propargyl β-D-glucopyranoside 15c was immobi-
lized by CuAAC with copper sulfate and sodium ascorbate
under microwave assistance (Scheme 4), affording the
corresponding solid-supported hexose-based platforms 16a−
16c, respectively. Then, four pent-4-ynyl chains were coupled
on the four hydroxyls of the solid supported hexoses 16a−16c
using the pent-4-ynyl phosphoramidite 17 on a DNA
synthesizer followed by the oligonucleotide elongation and
Cy3 labeling. After an ammonia treatment, the resulting 5′-
Cy3-oligonucleotides with a tetra-alkynylated hexose moiety
18a−18c were conjugated by CuAAC with azido-functionalized
α-L-fucose 3 as described above to give the fucosylated
1
4a−14d were obtained in solution after deprotection by
ammonia treatment. Each modified oligonucleotide was
conjugated with the azido functionalized fucose derivative 3
39
by CuAAC reaction using Cu(0) nanopowder as described
earlier. Acetyl groups were hydrolyzed by ammonia treatment,
and the conjugates were purified by C18 reverse phase HPLC.
Among the linear fucosylated glycoclusters, a trifucosylated
conjugate with a second DMCH linker between two fucose
residues was synthesized to evaluate the impact of the distance
between fucose residues on its affinity toward PA-IIL. Hence,
after the incorporation of the first H-phosphonate DMCH 9 to
afford 10, an additional phosphoramidite DMCH building
block 11 was introduced by phosphoramidite chemistry using
tert-butylhydroperoxide as an oxidizing agent to avoid the
oxidation of the H-phosphonate diester moiety present in
glycoclusters F8−F10 with the EG Tz linker.
3
The octavalent glycocluster F11 was synthesized on a
mannose platform 16a on which eight alkyne functions were
introduced using the bis-pent-4-ynyl phosphoramidite 19
conjugated with each alcohol of the mannose residue by
phosphoramidite chemistry (Scheme 4). Compound 19 was
prepared by reaction between pent-4-yn-1-ol and diisopropy-
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Scheme 4. Crown-Like Tetra-fucosylated Glycoclusters Based on Mannose F8, Galactose F9, and Glucose F10 Cores and Octa-
fucoylated Glycocluster Based on Mannose Core F11 with EG Tz Linkers
3
lamino phosphorodichloride in the presence of triethylamine in
dichloromethane (Scheme 5). The oligonucleotide was then
elongated and labeled with Cy3. The ammonia treatment led to
the intermediate 20, which was conjugated with the azido-
functionalized fucose derivative 3. However, our first attempts
to introduce the eight fucose residues 3 in TEAAc buffer and
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Scheme 5. Synthesis of the Bis-pent-4-ynyl Phosphoramidite 19 and 3,6-Dioxanon-8-yn-1-yl 2-Cyanoethyl N,N-Diisopropyl
Phosphoramidite 26
Scheme 6. Crown-Like Tetra-fucosylated Glycoclusters on Mannose F13, Galactose F14, and Glucose F15 Cores with MTz
Linkers
methanol gave an incomplete reaction. The solubility of the
intermediates most probably decreased due to the introduction
of nonpolar residues during the progress of the successive
CuAAC conjugations. To overcome this limitation, the acetyl
groups of 3 were hydrolyzed with aqueous ammonia, affording
water/methanol. The eight conjugations with 21 could be
performed smoothly in TEAAc buffer and methanol (3:1, v/v)
using Cu(0). The resulting 3′-mannose-centered octa-L-fucose
5′-Cy3-oligonucleotide conjugate F11 was purified by HPLC.
The fucosylated mimetics with MTz linkers were prepared
starting from the hexose-based solid supported platforms 16a−
21, which was directly used after evaporation and dissolution in
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Scheme 7. Crown-Like Tetra-fucosylated Glycocluster Based on a Mannose Core F16 with EG TzMEG PO Linkers
3
2
3
8,48
1
6c on which the 6-bromohexyl phosphoramidite 22
was
each well for binding to the glycoclusters. After washing, the
fucomimetic/PA-IIL interaction was visualized by fluorescence
scanning.
Immobilization was controlled by fluorescence scanning at
537 nm (excitation) due to the presence of Cy3, allowing the
control of the relative surface densities of the immobilized
conjugated on each alcohol function, and the oligonucleotide
was then elongated and labeled with Cy3 (Scheme 6). The four
bromine atoms were then substituted with azides by means of
tetramethylguanidine azide (TMGN ), and the Cy3-oligonu-
cleotides 23a−23c with the tetra-6-azidohexyl platforms were
obtained in solution after ammonia treatment. The fucose
moieties were introduced using the propargyl derivative 6 by
CuAAC reaction as described above using Cu(0) nanopowder
and microwaves. The resulting acetylated fucosylated glyco-
clusters were purified by HPLC and deprotected by ammonia,
affording glycoclusters F13−F15 incorporating MTz linkers.
A fucosylated glycocluster based on a mannose core with a
longer linker (EG TzMEG PO) was also synthesized to
3
glycomimetics G0 and F1−F16 in the absence of Alexa-647−
4
2
24,50
PA-IIL.
The variation of the average Cy3 fluorescence
signals (from four repetitions) between fucomimetics was
within 6−7% except for fucomimetics F1, F2, F13, and F16,
where the observed signal deviated by 9%, 12%, 33%, and 19%,
respectively. The lectin/glycomimetic interaction was visualized
after incubation with Alexa-647 labeled PA-IIL and fluorescence
scanning at 635 nm (excitation). The corresponding Alexa-647
signal observed upon interaction with the labeled lectin was
normalized based on the Cy3 fluorescence signal.
3
2
evaluate the impact of the linker length on the affinity for the
PA-IIL lectin (Figure 2). The phosphoramidite 26 with a longer
2
9
alkyne linker was prepared from 3,6-dioxanon-8-yn-1-ol by
reaction with the N,N-diisopropylcyanoethyl chlorophosphor-
amidite in presence of N,N-diisopropylethylamine in dry
dichloromethane (Scheme 5). The solid-supported mannose
platform 25 was prepared by CuAAC reaction between the
propargylated solid support 1 and the 3-azido-prop-1-yl α-D-
This qualitative information was complemented with
quantitative IC50 values measured by incubation of the PA-
IIL lectin with an increasing concentration of L-fucose used as a
reference ligand of this lectin, therefore, the higher the binding
of PA-IIL for the fucomimetic, the higher the concentration of
L-fucose required to obtain a 50% reduction of the Alexa-647
49
mannopyranoside 24 using CuSO and sodium ascorbate in
fluorescence signal. Hence, the higher the IC value, the higher
4
50
50
methanol and water under microwave assistance (Scheme 7).
The long linker phosphoramidite 26 was then introduced,
followed by the oligonucleotide elongation and labeling. After
ammonia treatment, the Cy3-oligonucleotide mannose with
four long alkyne linkers 27 was conjugated with fucosylated
azide 3 and purified by HPLC. The fucosylated glycocluster
F16 was obtained after deacetylation by ammonia.
the binding of the fucomimetic for the lectin.
The fluorescence intensity of Alexa-647 signal for the 17
glycomimetics (G0 and F1−F16) was measured by direct
fluorescence scanning (Figure 3). The very low fluorescence
intensity observed for the galactosylated glycomimetic G0
demonstrated the absence of nonspecific binding of PA-IIL to
the carbohydrate microarray surface. As previously reported,
direct fluorescence scanning is qualitative and a more precise
Binding Assays Using a Carbohydrate Microarray. The
DNA microarray was prepared by covalent immobilization of a
34,50
binding efficacy is obtained by determination of IC values.
5
0
3
′-amino-oligonucleotide by reaction on NHS ester modified
The monovalent fucosides F2 and F12 displayed a similar
fluorescence signal around 15000 au lower than all other
multivalent fucomimetics. This result shows that the presence
of the triazole ring in vicinity to the anomeric center of the L-
fucose has no or very little positive influence on the binding to
PA-IIL.
glass slides divided in microwells. The carbohydrate−
oligonucleotide conjugates G0 and F1−F16 with their
complementary Cy3-oligonucleotide tag were then immobilized
in each microwell through DNA/DNA duplex formation. After
washing, Alexa-647 labeled PA-IIL lectin was then incubated in
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Figure 3. Binding of Alexa-647−PA-IIL to the glycomimetics
evaluated by a carbohydrate microarray.
PA-IIL is a tetrameric lectin exhibiting four carbohydrate
8
binding domains. F1, F5, F8−F10, and F13−F16 exhibit four
fucose residues in antenna-like (F1), linear (F5), or crown-like
arrangements (F8−F10 and F13−F16) (Table 1). Among the
carbohydrate-centered fucomimetics (crown-like arrangement),
three different carbohydrate cores were tested: mannose (F8,
F13, and F16), galactose (F9 and F14), and glucose (F10 and
F15). For these crown-like fucomimetics, three different linkers
(
MtzHexPO, EG TzProPO, and EG TzMEG PO, Figure 2)
3 3 2
were also tested to study the influence of the distance between
the fucose and triazole group (1 or 8 atoms) as well as the
distance between the fucose residues and the hydroxyl of the
carbohydrate core (13, 17, or 21 atoms, Figure 2).
The fluorescence signals observed were above 30000 au for
F1, near 25000 au for F5, F13, and F16, and near 20000 au for
F9, F10, F14, and F15 (Figure 3). This data suggested that the
antenna-like topology (Pe) was preferred by PA-IIL than the
linear or the crown-like arrangements. These observations were
further confirmed by IC50 measurements where the fucomi-
metics competed with an increasing concentration of L-fucose.
Glycomimetics F1, F5, and F8 exhibiting the same linker
displayed IC50 values of 15, 5, and 11 μM, respectively.
Independently from the nature of the linker, the fluorescence
signals on crown-like fucomimetics were higher for mannose-
centered fucomimetics compared to galactose- or glucose-
centered glycoclusters. These results were further corroborated
by IC50 measurements on the carbohydrate microarray.
Glycomimetics F8, F9, and F10 displayed IC50 values of 11,
1
, and 2 μM, respectively, confirming that mannose-centered
molecules displayed a better binding than glucose- and
galactose-centered fucomimetics while the glucose and
galactose cores led to similar properties. In addition, the linear
pentavalent fucomimetics F6 and the mannose crown-like
fucomimetics (F8 and F13) displayed a similar binding.
Glycomimetics F8, F13, and F16 are mannose-centered
fucomimetics bearing MtzHexPO, EG TzProPO, and
3
EG TzMEG PO linkers, respectively. The fluorescence signals
3
2
measured were similar for the two first linkers used but with a
slight increase for the latter and longer linker. This result
corroborates the data obtained for the monofucomimetics F2
and F12, showing that the presence of the triazole close to
fucose has little to no influence on the binding to PA-IIL. In
addition, the increased length of the linker from 13 to 17 atoms
led to the same binding, while a linker of 21 atoms was found
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6
slightly better. Similar trends were observed for F9/F14 and
F10/F15, the crown-like fucomimetics bearing the
chelate binding mode. In another recent study, Reymond et al.
studied the interaction of PA-IIL with fucosylated peptide-
based dendrimers. The best ligand identified in this study
displayed its fucose residues separated by 7 amino acids.
Similarly, molecular modeling demonstrated that the glycoden-
drimer was too small to interact in a chelate binding mode.
In the present study, the relative potency per fucose residues
remained similar for mannose centered fucomimetics F8 and
F11, slightly increased as a function of fucose residues for linear
fucomimetics F3−F6, and displayed a neat increase for the
mannose centered fucomimetics F13, F8, and F16 according to
the increase length of the linker. However, the distance
between the fucose residues is expected to be in the same range
as those of the glycoclusters from Roy or Reymond
laboratories. Therefore, similarly to these two precedent
studies, the distance between each fucose epitope in our
glycoclusters was not sufficient to bind two distinct binding
sites on the same lectin tetramer, preventing a chelate effect to
occur.
EG TzProPO/MTzHexPO linkers with a galactose- and
3
glucose-based core, respectively. Determination of IC50 values
gave a confirmation of these results. The mannose crown-like
fucomimetics F13, F8, and F16, exhibiting increasing length of
linker with 13, 17, and 21 atoms, respectively, displayed an
increasing relative potency of 8.8, 13.8, and 17.5, respectively,
showing the beneficial effect of a longer linker on the binding
(
Figure 4).
CONCLUSION
■
The binding affinity of 16 fucomimetics for PA-IIL using
minute amounts (few picomoles) of glycoclusters could be
rapidly and efficiently screened using a carbohydrate micro-
array. According to our results, both monofucose derivatives
Figure 4. Relative potency per fucose of some fucomimetics.
The influence of the number of carbohydrates was then
studied by increasing the number of fucose residues from 2 to 5
for linear fucomimetics (F3−F6). The fluorescence signal
increased from 20270 au to 24460 au, matching the increasing
number of fucose epitopes. Likewise, the relative potency per
fucose residues was slightly increased for fucomimetics F3−F6
(
F2 and F12) displayed a lower binding affinity for PA-IIL than
all the other oligofucomimetics, showing the local concen-
tration effect of glycoclusters on binding. The best binding
properties were observed for the antenna-like fucomimetic F1
bearing four residues and for the mannose-centered fucomi-
metics F16 and F11 bearing four and eight fucosyl residues,
respectively. Among the carbohydrate-centered fucomimetics
exhibiting four residues (F8−F10 and F13−F16), the mannose
core showed the best binding (F8, F13 and F16) and a better
effect was observed with the longer linker (F16). The linear
fucomimetics showed an increase of binding correlated with the
number of residues.
Our results suggested that our fucomimetics were not able to
bind more than one site per lectin resulting, no chelate effect
was observed.. The increase bindings most probably were the
result of increased fucose local concentration for linear
fucomimetics F3−F6, while the increase of binding for the
mannose-centered fucomimetics F8, F13, and F16 may be due
to additional interactions or to steric stabilization, preventing
the approach of free fucose
From all studied glycomimetics, the present study allowed us
to select two fucomimetics, F1 and F16, bearing four fucose
moieties exhibiting the best binding affinity for PA-IIL (relative
potency per fucose around 18). Work is in progress to
synthesize fucomimetics with longer linker with the expectation
to bind two PA-IIL sites within the same lectin in order to
obtain chelate effect. Furthermore, F1 and F16 without the
DNA sequence will be synthesized at a higher scale for
biophysical and biological studies.
(
from 5 to 8, Table 1, Figure 4). The spacing between fucose
residues was also studied for three fucoses bearing glycomi-
metics (F4 and F7), but no significant difference in the
fluorescence signal could be observed (Figure 3). On the basis
of the relative potency values, the pentavalent fucomimetic F6
displayed the best binding of the linear ones but lower than the
antenna-like tetravalent fucomimetic F1 and the mannose
crown-like fucomimetics (F8, F13, and F16) whatever their
linker.
Similarly to comb-like fucomimetics, the binding of PA-IIL
was improved by increasing to eight the number of fucose
residues at the periphery of a mannose-centered glycomimetics
(
F11 vs F8). The IC values measured for F8 and F11 were 11
50
and 16 μM, respectively, showing a better binding for the F11
but a lower potency per fucose residue (13.8 and 10 for F8 and
F11, respectively, Figure 4).
The binding properties toward PA-IIL of divalent and
a
trivalent glycoclusters bearing the Lewis a trisaccharide (Le ) as
epitopes were studied by enzyme linked lectin assay (ELLA)
and by isothermal titration microcalorimetry (ITC) by Roy et
1
1
al. The fucosylated glycoclusters were assembled on linear or
aromatic platforms by Cu(I) alkyne−azide 1,3-dipolar cyclo-
addition (CuAAC). The triazole group did not favor or
hampered the interaction between the lectin and the clusters.
The distance between the fucose residues in their extended
conformation was estimated by molecular modeling of 22−41
Å. Their results showed that the molecules interacted with only
one binding site within the same lectin. No influence of the
length of the spacer was observed, maybe because the length
and structure of the linker did not allow the interaction of
glycoclusters with two binding sites on the same tetramer in a
ASSOCIATED CONTENT
■
*
S
Supporting Information
General procedures, HPLC data of fucomimetics, NMR data of
compounds 19 and 26. This material is available free of charge
via the Internet at http://pubs.acs.org.
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Dendrimers with High Affinity for the Fucose-Binding Lectin LecB
AUTHOR INFORMATION
■
from Pseudomonas aeruginosa. ChemMedChem 4, 562−569.
Corresponding Author
(14) Andreini, M., Anderluh, M., Audfray, A., Bernardi, A., and
*
For F.M.: phone, +33/467 144 961; fax, +33/467 042 029; E-
mail morvan@univ-montp2.fr. For Y.C.: phone, +33/472 186
40; fax, +33/4 78 43 35 93; E-mail, yann.chevolot@ec-lyon.fr.
Imberty, A. (2010) Monovalent and bivalent N-fucosyl amides as high
affinity ligands for Pseudomonas aeruginosa PA-IIL lectin. Carbohydr.
Res. 345, 1400−1407.
2
(15) Johansson, E. M. V., Kadam, R. U., Rispoli, G., Crusz, S. A.,
Notes
Bartels, K. M., Diggle, S. P., Camara, M., Williams, P., Jaeger, K. E.,
Darbre, T., and Reymond, J. L. (2011) Inhibition of Pseudomonas
aeruginosa biofilms with a glycopeptide dendrimer containing D-amino
acids. MedChemComm 2, 418−420.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
0
(16) Consoli, G. M. L., Granata, G., Cafiso, V., Stefani, S., and Geraci,
This work was financially supported by ANR-08-BLAN-0114-
C. (2011) Multivalent calixarene-based C-fucosyl derivative: a new
1, Lyon Biopole, and Vaincre la Mucoviscidose. G.P. thanks
ion Languedoc−Roussillon for the
award of a research studentship. F.M. is from Inserm.
Programme Cible 2010 “region Rhone-Alpes” is acknowledged.
Pseudomonas aeruginosa biofilm inhibitor. Tetrahedron Lett. 52, 5831−
the CNRS and the Reg
́
5
834.
(
17) Sharon, N. (2006) Carbohydrates as future anti-adhesion drugs
for infectious diseases. Biochim. Biophys. Acta 1760, 527−537.
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