Synthesis of novel mannoside glycolipid conjugates for inhibition of HIV-1 trans -infection

Article abstract of DOI:10.1021/bc200644d

Mannose-binding lectins, such as dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN), are expressed at the surface of human dendritic cells (DCs) that capture and transmit human immunodeficiency virus type-1 (HIV-1) to CD4⁺ cells. With the goal of reducing viral trans-infection by targeting DC-SIGN, we have designed a new class of mannoside glycolipid conjugates. We report the synthesis of amphiphiles composed of a mannose head, a hydrophilic linker essential for solubility in aqueous media, and a lipid chain of variable length. These conjugates presented unusual properties based on a cooperation between the mannoside head and the lipid chain, which enhanced the affinity and decreased the need for multivalency. With an optimal lipid length, they exhibited strong binding affinity for DC-SIGN (K_d in the micromolar range) as assessed by surface plasmon resonance. The most active molecules were branched trimannoside conjugates, able to inhibit the interaction of the HIV-1 envelope with DCs, and to drastically reduce trans-infection of HIV-1 mediated by DCs (IC_50s in the low micromolar range). This new class of compounds may be of potential use for prevention of HIV-1 dissemination, and also of infection by other DC-SIGN-binding human pathogens.

Full text of DOI:10.1021/bc200644d

Article
pubs.acs.org/bc
Synthesis of Novel Mannoside Glycolipid Conjugates for Inhibition of
HIV‑1 Trans-Infection
Laure Dehuyser,†,§ Evelyne Schaeffer,*,‡,§ Olivier Chaloin,‡ Christopher G. Mueller,‡ Rachid Baati,*,†
and Alain Wagner†
†Laboratory of Functional Chemo Systems, CNRS-UdS UMR 7199, Faculte
Rhin, 67400 Illkirch, France
́
de Pharmacie, Universite
́
de Strasbourg, 74 route du
‡Laboratory of Immunology and Therapeutic Chemistry, CNRS UPR 9021, Institut de Biologie Molec
́
ulaire et Cellulaire, 15 rue Rene
́
Descartes, 67000 Strasbourg, France
S
* Supporting Information
ABSTRACT: Mannose-binding lectins, such as dendritic cell-
specific ICAM-3-grabbing non-integrin (DC-SIGN), are expressed
at the surface of human dendritic cells (DCs) that capture and
transmit human immunodeficiency virus type-1 (HIV-1) to CD4+
cells. With the goal of reducing viral trans-infection by targeting
DC-SIGN, we have designed a new class of mannoside glycolipid
conjugates. We report the synthesis of amphiphiles composed of a
mannose head, a hydrophilic linker essential for solubility in
aqueous media, and a lipid chain of variable length. These
conjugates presented unusual properties based on a cooperation
between the mannoside head and the lipid chain, which enhanced
the affinity and decreased the need for multivalency. With an optimal lipid length, they exhibited strong binding affinity for DC-
SIGN (Kd in the micromolar range) as assessed by surface plasmon resonance. The most active molecules were branched
trimannoside conjugates, able to inhibit the interaction of the HIV-1 envelope with DCs, and to drastically reduce trans-infection
of HIV-1 mediated by DCs (IC50s in the low micromolar range). This new class of compounds may be of potential use for
prevention of HIV-1 dissemination, and also of infection by other DC-SIGN-binding human pathogens.
that DCs transmit HIV-1 through a DC-SIGN-independent
exocytic pathway that involves HIV-associated exosomes.15
DC-SIGN is a type II membrane protein with a short amino-
terminal cytoplasmic tail, a neck region, and a single C-terminus
carbohydrate recognition domain (CRD). The extracellular
CRD is a tetramer stabilized by an α-helical stalk, which
specifically recognizes glycosylated proteins, and binds ligands
bearing mannose and related sugars. The interaction of this
domain with high-mannose structures of glycoproteins is
multivalent and calcium-dependent.16,17 It interacts with
envelope glycoproteins from distinct viruses such as HIV-1,
Ebola, Hepatitis C, and Dengue,18−22 but also with
mycobacteria,23 fungi,24 and parasites.25 The HIV-1 envelope
consists of the gp120 glycoprotein, exposed on the surface of
the virion, and the transmembrane protein gp41. The highly
glycosylated gp120 interacts with the mannose-binding domain
of DC-SIGN at the surface of DCs.26−28
INTRODUCTION
■
Since the beginning of the AIDS pandemia in the early 1980s,
there are 32 antiretroviral drugs approved for therapy. They
target different stages of the human immunodeficiency virus
type-1 (HIV-1) life cycle, such as entry, reverse transcription,
integration, and maturation.1 Their drawbacks are virus-induced
drug resistance and drug-induced side effects. A complementary
approach is now dedicated to the search of inhibitors able to
prevent mucosal infection and transmission, since the epidemic
disease has been largely maintained by mucosal transmission.2,3
After crossing the mucosal barrier, HIV-1 infects dendritic cells
(DCs), macrophages, and T lymphocytes, residing underneath
epithelial layers. DCs are one of the first cell types to be
infected by HIV-1, mostly the R5 strain.4−7 In addition, DCs
contribute to early-stage viral dissemination and transmission
by their ability to capture, transport virions, and mediate
infection of new target cells.8−10 This so-called trans-infection
can occur by distinct pathways. The first mechanism implies the
DC-specific intercellular adhesion molecule 3 [ICAM-3]-
grabbing nonintegrin (DC-SIGN), a C-type lectin highly
expressed at the surface of DCs.11−13 DCs capture HIV-1
through DC-SIGN and transfer them to target CD4+ cells
through cell−cell junctions.10 The vast majority of virions
transmitted in trans originate from the cell plasma membrane
rather than from intracellular vesicles.14 Other studies found
Previous studies aimed at preventing HIV transmission by
targeting DC-SIGN have reported the synthesis of multivalent
carbohydrate-containing molecules. Indeed, monovalent carbo-
hydrates present weak millimolar binding constants for
Received: November 29, 2011
Revised: June 27, 2012
© XXXX American Chemical Society
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protein−carbohydrate recognition. It has been well recognized
that the multivalent effect improves binding affinities by orders
of magnitude. DC-SIGN binds to Man(9)GlcNAc(2) oligo-
saccharide 130-fold more tightly than mannose.17 Mannose
dendritic polymers were found to interfere with the interaction
between DC-SIGN and gp120 proteins.29,30 Two recent
reports have described that multivalent mannoside polymers
inhibit HIV transmission mediated by cell lines engineered to
express DC-SIGN. First, gold mannoglyco-nanoparticles were
shown to inhibit HIV trans-infection mediated by Raji-DC-
SIGN cells.31 Second, mannosyl glycodendritic structures,
based on second and third generations of Boltorn hyper-
branched dendritic polymers functionalized with mannose,
inhibited HIV trans-infection mediated by THP-1-DC-SIGN
cells.32
Distinct studies have described that the interaction between
C-type lectins and natural lipomannans, a complex of four-
domain amphipathic molecules, is influenced by the presence of
a lipid moiety. Indeed, the major cell-wall lipoglycans of
Mycobacterium tuberculosis are high-molecular-weight mannosy-
lated lipoarabinomannans (ManLAM) that strongly interact
with DC-SIGN. Their lipid appendages, which are not directly
linked to the mannose moieties, are thought to be involved in a
supramolecular organization of the molecules by formation of
micelles that increase ManLAM avidity for DC-SIGN.33,34
These data highlight the crucial role of lipids for creating a
multivalent mannose presentation via structure formation into
dynamic micelles.
Surface Plasmon Resonance (SPR). SPR experiments
were performed using a Biacore 3000. Immobilization of
human DC-SIGN (Lys62-Ala404, from R&D Systems) on a
CM5 sensor chip (GE-Healthcare, Uppsala, Sweden) was
performed by injecting 70 μL of DC-SIGN (50 μg/mL in
formate buffer, pH 4.3) onto the surface activated with N-ethyl-
N′-dimethylaminopropyl carbodiimide (EDC)/N-hydroxysuc-
cinimide (NHS), which gave a signal of approximately 8000
RU, followed by 20 μL of ethanolamine hydrochloride, pH 8.5,
to saturate the free activated sites of the matrix. Biosensor
assays were performed with HEPES-buffered-saline (10 mM
HEPES, 150 mM sodium chloride, pH 7.4) containing 0.005%
surfactant P20 and 5 mM CaCl2, as running buffer. All binding
experiments were carried out at 25 °C with a constant flow rate
of 20 μL/min. The compounds dissolved in the running buffer
(0.39 to 50 μM) were injected for 3 min, followed by a
dissociation phase of 3 min. The sensor chip surface was
regenerated after each experiment by injecting 10 μL of EDTA
0.5 M pH 8.0. A simple channel activated by EDC/NHS and
deactivated with ethanolamine was used as control. The kinetic
parameters were calculated using the BIAeval 4.1 software.
Analysis was performed using the simple Langmuir binding
model with separate ka/kd (kon/koff). The specific binding
profiles were obtained after subtracting the response signal
from the control channel and from blank-buffer injection. The
fitting to each model was judged by the χ2 value and
randomness of residue distribution compared to the theoretical
model (Langmuir binding 1:1).
Cell Culture. MAGI-CCR5 cells are HeLa-CD4-LTR-LacZ
cells expressing both CXCR4 and CCR5 coreceptors (received
from NIH AIDS Research and Reference Program, from Dr.
Julie Overbaugh37). HEK293T cells were used for HIV-1
production. MCF-7 cells were used for viability tests. All cell
lines were cultured at 37 °C and 5% CO2 in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with
gentamycin and 10% (v/v) heat-inactivated fetal bovine
serum (FBS).
Therefore, with the goal of inhibiting HIV-1 trans-infection
by blocking interactions with DC-SIGN, we decided to
investigate the activity of low-molecular-weight mannose-
based glycolipids. We designed a novel class of conjugates,
formed by three building blocks that combine a polar mannose
epitope moiety, a hydrophilic linker essential for water
solubility, and a hydrophobic saturated lipid chain of variable
length. Here, we describe their synthesis, their chemical and
biological characteristics, namely, their affinity for DC-SIGN,
their capacity to inhibit the binding of gp120 to DCs, and their
ability to reduce HIV-1 trans-infection mediated by infected
primary human immature DCs. Our findings revealed a
branched trimannoside glycolipid conjugate able to drastically
limit HIV-1 trans-infection.
Generation of Human Monocyte-Derived Dendritic
Cells. Elutriated human monocytes were obtained from the
French Blood Bank (Etablissement Franca̧ is du Sang,
Strasbourg, France). To generate monocyte-derived dendritic
cells, monocytes were cultured at 37 °C and 5% CO2 in RPMI
1640 medium supplemented with 10% FBS, gentamycin, 50
ng/mL of recombinant human granulocyte macrophage colony
stimulating factor (rhGM-CSF; ImmunoTools), and 10 ng/mL
of recombinant human interleukin-4 (rhIL-4; ImmunoTools),
with readdition of cytokines at day 3. Cells were harvested on
day 5, and specific cell marker expression (CD1a, DC-SIGN)
was characterized by flow cytometry on a FACS Calibur flow
cytometer (Beckton-Dickinson) and analyzed with the
CellQuest Pro software (BD Biosciences).
EXPERIMENTAL PROCEDURES
■
Compounds synthesis and characterization are described in
Supporting Information.
Critical Micelle Concentrations (CMC) and Dynamic
Light Scattering (DLS). The CMC were measured using
pyrene as an extrinsic fluorescent probe, as described.35 Briefly,
a range of glycolipid samples were dissolved in water (1 mL),
with concentrations ranging from 1.0 to 0.001 mg/mL. A stock
solution of pyrene (1 mM) in dimethyl sulfoxide was added to
each sample. Fluorescent emission of pyrene was measured at
25 °C using a Fluorolog spectrofluorometer (Jobin Yvon,
Horiba). The intensities of fluorescence emission were
determined at 374 nm (I374) and at 383 nm (I383), with an
excitation at a 339 nm wavelength. The ratios I374/I383 were
calculated and plotted against the glycolipid concentration. The
CMC value was determined at the intersection of the two
straight lines. The hydrodynamic diameters of micelles were
measured in water by DLS measurements using a Zetasizer
Nano ZS system (Malvern Instruments) as described.36
Flow Cytometry. Expression of cell markers (CD1a, DC-
SIGN) was determined in FACS buffer (1% fetal bovine serum
in PBS) at 4 °C for 20 min, using specific antibodies and their
corresponding isotype controls (from BD-Pharmingen): CD1a-
allophycocyanin (HI149), CD209/DC-SIGN-PerCP-Cy5.5
(DCN46).
Competition Assays between TriManC24 and HIV-1
gp120. Human DCs (1 × 105 in 100 μL) were aliquoted in a
96-well microtiter plate in RPMI 1640 medium (without FBS).
To assess the binding of HIV-1 glycoprotein 120, FITC-
conjugated recombinant gp120 HIV-1 IIIB (ImmunoDiagnos-
tics Inc., Woburn, MA, USA) was added to the cells (final
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Figure 1. Structure of mannoside glycolipid conjugates 1−8.
concentration of 5 μg/mL) and incubated for 45 min at 37 °C.
For competition assays, cells were pretreated with either
mannan (100 μg/mL final, corresponding to 620 μM of
mannose units) or TriMan (100 μM) for 30 min at 37 °C,
followed by incubation with gp120-FITC for 45 min at 37 °C.
After washing with HBSS buffer (Lonza), expression of
fluorescence intensity was analyzed on a FACS Calibur flow
cytometer with the CellQuest Pro software.
HIV-1 Production. Molecular clones of HIV-1 NL4−3 (X4
strain) and an NL4−3 R5-tropic version38 were transfected into
HEK293T cells and viruses harvested from the supernatants 48
h later. All transfections were performed using calcium
phosphate for precipitation of DNA. Viral stocks were
normalized based on their p24 Gag content, measured in an
enzyme-linked immunosorbent assay (ELISA) using the
Innotest HIV antigen mAb kit (Innogenetics).
concentrations of compounds, mannan (Sigma), or culture
medium alone in RPMI 1640 medium containing 10% FBS for
45 min at 37 °C, prior to infection with HIV-1 (100 ng of
p24Gag) for 3 h. Excess free virus and molecules were removed
by three washes. DCs were then cocultured with MAGI-CCR5
cells (1 × 105) at 37 °C plated in a 48-well plate. After two
days, viral infection of MAGI-CCR5 cells was quantitated
following β-galactosidase assays and enumerating blue cells by
light microscopy. The IC50s were determined by serial dilution
of each compound. The trans-infection assays were repeated a
minimum of three times, and the IC50s determined using
Kaleidagraph.
RESULTS AND DISCUSSION
■
Synthesis of Monovalent Glycolipids Conjugates 1−4.
Synthesis of the monosaccharide conjugates 1−4 (Figure 1)
was achieved as depicted in Figure 2A. The linker between the
mannose and the lipid chain was introduced for assuring
HIV-1 Trans-Infection. Mannoside glycolipids (1.0 or 0.5
mM stock solution in water) were serially diluted in water.
Human DCs (1 × 105 in 200 μL) were pretreated with different
C
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Figure 2. Synthesis of linear and ramified mannoside glycolipids. (A) Synthesis of mannosides 1−4. Conditions: (i) TMSOTf, DCM, 0 °C to RT,
overnight, 47%; (ii) PPh3, THF, H2O, RT, overnight, 81%; (iii) CH3COCl, THF, 0 °C to RT, overnight, 25%; (iv) NaOCH3, MeOH, RT,
quantitative yield; (v) DCC, HOBT, THF, reflux, overnight, 9′, (14%); (vi) PPh3,THF, H2O, RT, overnight, 49%; (vii) DCC, HOBT, THF, reflux,
overnight; lauric acid 17 (98%), stearic acid 18 (78%), pentacosanoic acid 19 (88%); (viii) NaOCH3, MeOH, RT, overnight, quantitative yield. (B)
Synthesis of trivalent mannoside 7. Conditions: (i) Pentacosanoic acid 19, HOBt, DCC, THF, reflux, overnight, 77%; (ii) NaOH 4 N, THF/EtOH,
RT, overnight, 94%; (iii) HOBt, HBTU, DIPEA, THF, reflux, overnight, 63%; (iv) NaOCH3, MeOH, RT, overnight, quantitative yield. (C)
Synthesis of trivalent dimannoside 8. Conditions: (i) 9, TMSOTf, NIS, MS 4A, DCM, RT, overnight, 76%; (ii) K2CO3 (cat), MeOH, RT, overnight,
96%; (iii) 29, TMSOTf, NIS, MS 4A, DCM/Et2O, RT, overnight, 58%; (iv) PPh3, THF, H2O, RT, overnight, 61%; (v) 26, HOBt, HBTU, DIPEA,
THF, reflux, overnight, 27%; (vi) K2CO3 (cat), MeOH, RT, overnight, quantitative yield; (vii) H2, Pd(OH)2/C, MeOH, RT, overnight, 97%.
solubility of the molecules in physiological media. The key
intermediates PEG-azido linker 9′ and the glycosyl donor 11
were prepared from tetraethylene glycol and D-mannose 10,
respectively.39,40 Briefly, glycosylation of 11 with alcohol 941
followed by the reduction of the azido function of 1242 afforded
the pivotal amine compound 13, which upon acetylation
conditions and Zemplen final deprotection gave ManC1 1.
Alternatively, amide coupling of 13 with PEG-carboxylic acid 9′
yielded sugar 15. After reduction of the azide function using
Staudinger reduction to afford 16, the product was engaged in
D
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amide bond formation with lauric 17, stearic 18, or
pentacosanoic 19 acids to give, respectively, ManC11 (2),
ManC17 (3), and ManC24 (4) in good chemical isolated yields
(Supporting Information).
dissociation equilibrium constant (KD = koff/kon) (Figure 3).
As expected, mannan efficiently bound to the surface, while
Synthesis of the Multivalent Conjugate TriManC24 7.
Based on previous studies showing that multiple mannose
units, such as dendron-formed structures, are necessary for an
optimal interaction with DC-SIGN, we synthesized multivalent
compounds. To obtain 7 (Figure 2B), the synthesis of the key
branched amine connector 24 was achieved.43 Treatment of 24
with pentacosanoic acid 19 and DCC yielded triester 25, which
was saponified to tricarboxylic acid 26. Compound 26 was then
reacted with sugar 13 under DCC/HOBt conditions to furnish
trimannosylated product 27, which was deprotected to afford
TriManC24 7.
Synthesis of TriDiManC24 8. To further enhance the
multivalent mannose presentation, we produced TriDiManC24
8
(Figure 2C). The glycosyl donor 29 was first synthesized.44−46
After glycosylation with alcohol 9 affording 30, the acetate
function at C2 of 30 was selectively removed to yield the C2
free hydroxyl sugar acceptor 31. Subsequent glycosylation with
glycosyl donor 29 gave the expected disaccharide 32. Selective
reduction of the azido group using PPh3/H2O in standard
conditions led to 33. Further coupling to the tricarboxylic acid
connector 26 afforded the expected fully protected TriDi-
ManC24 molecule. Removal of the acetates and global
deprotection of the benzyl groups afforded TriDiManC24 8.
Synthesis of Monovalent unsaturated Glycolipids
Conjugates 5−6. To examine the importance of a saturated
versus an unsaturated chain, we synthesized ManC24U 5,
containing two triple insaturations, by amide bond formation
between intermediate 16 and 10,12-pentacosadiynoic acid. As a
control for the role of the sugar head, CellC24U 6 containing a
cellobiose (or glucose−glucose) unit (Figure 1) was synthe-
sized, as described in the Supporting Information.
Properties of Mannoside Glycolipid Conjugates. All
conjugates 1−8 could be solubilized in water by simple stirring,
thanks to the hydrophilic linker. The saturated molecules
exhibited no noticeable cytotoxicity on cells, as shown by
viability assays on a human cell line and by dead cell staining of
human DCs. The unsaturated compounds 5 and 6 were non-
cytotoxic up to 250 μM (Supporting Information).
Micelles are dynamic systems that can spontaneously self-
assemble above the critical micelle concentration (CMC), thus
avoiding unnecessary construction steps to afford multivalent
systems. Interestingly, when compounds were dissolved in
water at 0.5 mg/mL, molecules ManC17 3, ManC24 4, and
TriManC24 7 were able to auto-organize in micelles, at a CMC
of, respectively, 112, 97, and 109 μM. The hydrodynamic
diameter of these micelles was determined by dynamic light
scattering and was, respectively, 17, 17, and 39 nm. Unsaturated
ManC24U 5 had a lower CMC of 13 μM, and micelles had a
hydrodynamic diameter of 11 nm. Molecules ManC1 1, ManC11
2, and TriDiManC24 8 were unable to form micelles.
Figure 3. Direct binding by surface plasmon resonance (SPR) of
mannoside glycolipid conjugates to DC-SIGN immobilized on a CM5
sensor chip. (A) Sensorgram of the binding of 7 to DC-SIGN (CRD)
immobilized via amino groups. The binding response (in RU) was
recorded as a function of time. (B) Kinetic parameters of interactions.
kon, kinetic association rate; koff, kinetic dissociation rate; KD,
equilibrium dissociation constant; Rmax (RU), maximal response
expressed as resonance units; χ2, chi square value. 1:1 Langmuir
binding model was used for all compounds, except for compound 3,
for which steady-state affinity was used for calculation. With the
control compound NHPD lacking mannose, no binding was detected
at 100 μM. “N.b.”: No binding until 50 μM.
OEG was unable to bind. We found that ManC1 1 and ManC11
2 were unable to bind, ManC17 3 bound with a low affinity,
while ManC24 4 exhibited an optimal affinity in the
submicromolar range. These data showed that the affinity of
the linear molecules for DC-SIGN correlated with the length of
the lipid chain, suggesting that the lipid moiety of the
moleculeat an optimal size of 24 carbon atomsenhanced
the binding capacity of the mannose polar head in a cooperative
manner.
One mannose unit in ManC24 4 was sufficient for an effective
interaction, with a KD in the submicromolar range. TriManC24
7
and TriDiManC24 8 also efficiently bound to DC-SIGN, with
the KD in the low micromolar range, although they presented
no improvement over ManC24 4. Similarly, ManC24U
5
Affinities for DC-SIGN. Using surface plasmon resonance
(SPR), we assessed the ability of our compounds to bind to the
carbohydrate recognition domain (CRD) of DC-SIGN. A CM5
sensor chip was functionalized with the DC-SIGN CRD, and
increasing concentrations of the various compounds, mannan as
a positive control and octa(ethylene glycol) (OEG) as a
negative control, were injected over the chip surface. By
evaluation of the sensorgrams, we deduced the association (kon)
and dissociation (koff) rate constants and the resulting
containing an unsaturated lipid chain exhibited a KD in the
low micromolar range, showing that these unsaturations did not
improve the binding relative to a saturated chain. When the
mannose head was replaced by a glucose unit (cellobiose), the
binding was lost, which clearly revealed the importance of the
mannose head. Similarly, when the sugar head was lacking, in
NHPD composed of a linker conjugated to a 24-carbon lipid
chain (see Figure 1), no binding was detected, which allowed us
to rule out nonspecific binding. These results stressed the
E
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importance of the mannose head, as well as of the 24-carbon
saturated or unsaturated lipid chain.
Altogether, these data demonstrated that glycolipid con-
jugates, formed by a C24-lipid chain covalently linked to one,
three, or six mannose units, bind to the CRD of DC-SIGN with
a good affinity.
It is worth noting that ManC24 4, ManC24U 5, TriManC24 7,
and TriDiManC24 8 interacted with DC-SIGN at concentrations
below their CMC; the binding was effective below 10 μM, as
visualized on the sensorgram Figure 3A. Thus, although
molecules 4, 5, and 7 are able to form micelles, while 8 is
unable to form micelles, they could all bind to DC-SIGN as
single molecules, and micelle formation was not required. This
contrasts with mycobacterial mannosylated lipoarabino-
mannans (ManLAM), in which terminal mannose residues
and the lipid moiety are critical for binding to the pulmonary
surfactant protein A (SP-A). In this case, lipids are required for
an aggregate/micellar organization of the lipoglycan, since
interaction with SP-A is lost in the presence of a low
concentration of mild detergent (0.0005% of Polysorbate
20).47 In similar detergent conditions, binding of our
mannosylatedC24 lipids to DC-SIGN was conserved, confirming
that a process of supra-macromolecular structure formation is
not required for binding to DC-SIGN.
Binding Competition with HIV-1 gp120. We examined
the ability of our compounds to inhibit the interaction of HIV-1
gp120 with DC-SIGN expressed at the surface of DCs.
Immature DCs were prepared by differentiation of human
monocytes. The expression levels of DC-SIGN were controlled
by flow cytometry. As expected, results showed a high level of
expression on DCs but no expression on monocytes, as
indicated by the mean fluorescence index (MFI) (Figure 4A).
When cells were incubated with fluorescently labeled gp120, we
observed a strong fluorescence for DCs but not for monocytes,
demonstrating more gp120 binding to DCs (Figure 4B). Some
binding to monocytes was observed, likely owing to interaction
with other cell surface proteins.48
Figure 4. TriManC24 7 competes with HIV gp120 binding to dendritic
cells (DCs). (A) Cell surface expression of DC-SIGN on DCs and
monocytes, analyzed by flow cytometry. Filled and gray histograms
represent, respectively, antigen labeling and isotype controls. Values of
mean fluorescence intensity (MFI) for antigen labeling are indicated.
(B) Interaction of gp120-FITC with monocytes and DCs, after
incubation for 45 min at 37 °C (filled histograms). Gray histograms
represent untreated cells, in the presence of IgG1-FITC control. (C)
DCs were exposed to TriManC24 7 (100 μM) or mannan (100 mg/mL
or 620 mM of mannose units) for 30 min or left untreated, followed
by incubation with gp120-FITC for 45 min at 37 °C. Flow cytometry
open and filled histograms correspond to cells binding gp120-FITC,
respectively, in the absence and presence of pretreatment. Results are
from one representative experiment repeated three times.
We then performed competition assays by preincubating
DCs with either compound TriManC24 7 or mannan followed
by addition of fluorescently labeled gp120 (Figure 4C). Flow
cytometry showed that 7 was able to compete with gp120-
FITC for the binding to DCs. Although inhibition was not
complete, 100 μM of TriManC24 7 interfered better than
mannan (620 μM of mannose units) with the binding of gp120
to DCs.
Inhibition of HIV-1 trans-Infection Mediated by DCs.
To investigate the ability of our compounds to inhibit HIV-1
trans-infection, we chose to infect DCs to best mimic in vivo
conditions of viral capture by mucosal DCs. The DCs were
mixed with the different compounds for 45 min at 37 °C,
before exposure to HIV-1. After a 3 h incubation, the cells were
extensively washed to remove unbound virions and mannosides
and then cocultured with MAGI-CCR5 cells. This procedure
allows precise quantification of HIV-1 replication in reporter
MAGI-CCR5 cells by detection of β-galactosidase activity, since
these cells express an integrated copy of the HIV long terminal
repeat (LTR) fused to the β-galactosidase reporter gene.
MAGI-CCR5 express the CD4 receptor and CCR5 and
CXCR4 coreceptors, required for entry of, respectively, HIV-
1 R5 and X4.
2 did not present better inhibitory activity than mannan,
ManC17 3, ManC24 4, and ManC24U 5 clearly reduced trans-
infection in a dose-dependent manner, more efficiently than
mannan (0.6 and 1.5 mM of mannose units). This inhibition
was in the high micromolar range with an IC50 of 120 μM.
Interestingly, CellC24U 6 was unable to affect trans-infection,
which showed the importance of the mannose head in the
inhibition process. Dose−response experiments were then
performed to compare the inhibitory capacity of the linear
ManC24 4 and branched TriManC24 7 and TriDiManC24 8.
When compared with ManC24 4, TriManC24 7 displayed a more
potent inhibitory activity in the submicromolar range (IC50 of
0.5 μM). At the highest concentrations, trans-infection was
drastically reduced, down to a residual level of 20% (Figure
5B). TriDiManC24 8 exhibited a powerful inhibitory activity in
the nanomolar range (IC50 of 38 nM), although, surprisingly,
infection leveled off at 40% (Figure 5C). The residual level of
trans-infection may be explained by the previously reported
distinct mechanisms of viral transmission, besides the transfer
mediated by DC-SIGN.
We further examined the effect of TriManC24 7 on the HIV-1
X4 strain (Figure 5D). Similar to the effect on the R5 strain, it
also inhibited HIV-1 X4 trans-infection down to a residual level
of 15%, although with a higher IC50 (7 μM). These findings
demonstrate that TriManC24 7 is an active mannoside glycolipid
We first exposed DCs to the HIV-1 R5 strain, mostly
involved in mucosal infections,7,49,50 in the presence of the
linear glycolipids 1−6 (Figure 5A). While ManC1 1 and ManC11
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Figure 5. Inhibition of HIV-1 trans-infection by glycolipid conjugates. DCs were pretreated with the indicated compounds for 30 min at 37 °C, and
then infected with HIV-1. After extensive washes, DCs were cocultured with MAGI-CCR5 cells for 2 days. Viral trans-infection was quantified.
Values were normalized relative to 100% infection obtained with medium alone. Values are means SE from data obtained at least from three
independent experiments. (A) Effect of the length of the carbon chain and of the sugar (either mannose or glucose). Mannan was included as a
positive control. (B) Dose−response results for HIV-1 R5 exposed to mono- and trimannosides. (C) HIV-1 R5 exposed to tri- and tri-bis-
mannosides. (D) HIV-1 X4 exposed to TriManC24 7.
conjugate to reduce trans-infection of HIV-1 R5 and X4
mediated by DCs.
other while bound to the same or adjacent DC-SIGN molecules
simulating a bivalent ligand that bridges binding sites. Such a
chelating binding has been shown before to strongly increase
binding affinity.51,52 The precise role of the lipid chain in the
molecular interactions between the compounds and DC-SIGN
remains the subject of further studies. Our findings reveal that,
thanks to this original cooperation, such conjugates are able to
target DC-SIGN and inhibit HIV-1 trans-infection without the
need for synthesis of previously reported complex mannosides
structures.31,32 Nevertheless, it is clear that the multivalent
presentation of 6 and 3 mannoses helped improve the trans-
infection ability.
The most likely hypothesis accounting for inhibition of HIV-
1 trans-infection relies on the shield model: interaction of the
compounds with DC-SIGN at the DC cell membrane prevents
the attachment of HIV-1 to this receptor. In addition, partial
down-regulation of DC-SIGN from the cell surface may also
enhance the protective effect. One could also hypothesize a
direct effect of the compounds on the virus particle. The
molecule may disrupt the viral membrane by insertion of its
lipid moiety, or interact via its mannose units with gp120 at the
surface of the virus. Clearly, further studies are needed to shed
more light on the precise inhibitory mechanism. Finally, the
interest of this novel class of inhibitory molecules also lies in
their potential ability to target other viruses and pathogens that
use DC-SIGN or other C-type lectins for entry and
infection.18−25
CONCLUSIONS
■
Our studies revealed the unexpected properties of a new class
of glycolipid conjugates. These compounds are formed by three
building blocksa mannose head, a linker, and a C24 lipid
chainand are able to efficiently target DC-SIGN. Surpris-
ingly, our data showed that a scaffold offering a multivalent
mannose presentation, to mimic the high mannose structure
occurring in glycoproteins, was not required for obtaining high
binding affinity for DC-SIGN. Our SPR results demonstrated
that the affinity of the mannose moiety for the extracellular
domain of DC-SIGN could be greatly increased by coupling the
mannose unit to a lipid chain of at least 17 carbons.
The activities of these conjugatestheir affinity for DC-
SIGN, their ability to compete for gp120 binding, and their
inhibition of HIV-1 trans-infectionare based on the
cooperation between the lipid chain and the linked mannose
moiety. The lipid chain was originally synthesized for molecular
structure formation into self-organized micelles, and thus afford
a multivalent mannose presentation. Interestingly, we discov-
ered that such a structure formation was not required, neither
for binding to DC-SIGN nor for inhibiting HIV trans-infection,
as revealed by ManC24 4. Indeed, TriManC24 7 and TriDiManC24
8 inhibited viral trans-infection at an IC50 of, respectively, 500
and 38 nM, well below their CMC, showing that they possess
the property of being active not only as micelles, but also as
individual molecules. It is likely that, in addition to the
interaction of the mannose unit with the CRD, the lipid chain
interacts with distinct amino acids of DC-SIGN, thus
optimizing the binding. It could also be that the lipid chains
of two molecules make hydrophobic interactions with each
ASSOCIATED CONTENT
■
S
* Supporting Information
Full synthetic and characterization details of the compounds
including NMR spectra, and cell viability assays are provided.
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(11) Geijtenbeek, T. B., and van Kooyk, Y. (2003) DC-SIGN: a
novel HIV receptor on DCs that mediates HIV-1 transmission. Curr.
Top. Microbiol. Immunol. 276, 31−54.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(12) Arrighi, J. F., Pion, M., Wiznerowicz, M., Geijtenbeek, T. B.,
Garcia, E., Abraham, S., Leuba, F., Dutoit, V., Ducrey-Rundquist, O.,
van Kooyk, Y., Trono, D., and Piguet, V. (2004) Lentivirus-mediated
RNA interference of DC-SIGN expression inhibits human immuno-
deficiency virus transmission from dendritic cells to T cells. J. Virol. 78,
10848−10855.
(13) Chung, N. P., Breun, S. K., Bashirova, A., Baumann, J. G.,
Martin, T. D., Karamchandani, J. M., Rausch, J. W., Le Grice, S. F., Wu,
L., Carrington, M., and Kewalramani, V. N. (2010) HIV-1 trans-
mission by dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-
SIGN) is regulated by determinants in the carbohydrate recognition
domain that are absent in liver/lymph node-SIGN (L-SIGN). J. Biol.
Chem. 285, 2100−2112.
(14) Cavrois, M., Neidleman, J., Kreisberg, J. F., and Greene, W. C.
(2007) In vitro derived dendritic cells trans-infect CD4 T cells
primarily with surface-bound HIV-1 virions. PLoS Pathog. 3, e4.
(15) Wiley, R. D., and Gummuluru, S. (2006) Immature dendritic
cell-derived exosomes can mediate HIV-1 trans infection. Proc. Natl.
Acad. Sci. U.S.A. 103, 738−743.
(16) Feinberg, H., Mitchell, D. A., Drickamer, K., and Weis, W. I.
(2001) Structural basis for selective recognition of oligosaccharides by
DC-SIGN and DC-SIGNR. Science 294, 2163−2166.
(17) Mitchell, D. A., Fadden, A. J., and Drickamer, K. (2001) A novel
mechanism of carbohydrate recognition by the C-type lectins DC-
SIGN and DC-SIGNR. Subunit organization and binding to
multivalent ligands. J. Biol. Chem. 276, 28939−28945.
(18) Alvarez, C. P., Lasala, F., Carrillo, J., Muniz, O., Corbi, A. L., and
Delgado, R. (2002) C-type lectins DC-SIGN and L-SIGN mediate
cellular entry by Ebola virus in cis and in trans. J. Virol. 76, 6841−6844.
(19) Geijtenbeek, T. B., and van Kooyk, Y. (2003) Pathogens target
DC-SIGN to influence their fate DC-SIGN functions as a pathogen
receptor with broad specificity. APMIS 111, 698−714.
(20) Tassaneetrithep, B., Burgess, T. H., Granelli-Piperno, A.,
Trumpfheller, C., Finke, J., Sun, W., Eller, M. A., Pattanapanyasat,
K., Sarasombath, S., Birx, D. L., Steinman, R. M., Schlesinger, S., and
Marovich, M. A. (2003) DC-SIGN (CD209) mediates dengue virus
infection of human dendritic cells. J. Exp. Med. 197, 823−829.
(21) Wang, Q. C., Feng, Z. H., Nie, Q. H., and Zhou, Y. X. (2004)
DC-SIGN: binding receptors for hepatitis C virus. Chin. Med. J. (Engl.)
117, 1395−1400.
(22) Ludwig, I. S., Lekkerkerker, A. N., Depla, E., Bosman, F.,
Musters, R. J., Depraetere, S., van Kooyk, Y., and Geijtenbeek, T. B.
(2004) Hepatitis C virus targets DC-SIGN and L-SIGN to escape
lysosomal degradation. J. Virol. 78, 8322−8332.
(23) Geijtenbeek, T. B., Van Vliet, S. J., Koppel, E. A., Sanchez-
Hernandez, M., Vandenbroucke-Grauls, C. M., Appelmelk, B., and Van
Kooyk, Y. (2003) Mycobacteria target DC-SIGN to suppress dendritic
cell function. J. Exp. Med. 197, 7−17.
(24) Cambi, A., Gijzen, K., de Vries, J. M., Torensma, R., Joosten, B.,
Adema, G. J., Netea, M. G., Kullberg, B. J., Romani, L., and Figdor, C.
G. (2003) The C-type lectin DC-SIGN (CD209) is an antigen-uptake
receptor for Candida albicans on dendritic cells. Eur. J. Immunol. 33,
532−538.
(25) Colmenares, M., Puig-Kroger, A., Pello, O. M., Corbi, A. L., and
Rivas, L. (2002) Dendritic cell (DC)-specific intercellular adhesion
molecule 3 (ICAM-3)-grabbing nonintegrin (DC-SIGN, CD209), a C-
type surface lectin in human DCs, is a receptor for Leishmania
amastigotes. J. Biol. Chem. 277, 36766−36769.
(26) Hong, P. W., Flummerfelt, K. B., de Parseval, A., Gurney, K.,
Elder, J. H., and Lee, B. (2002) Human immunodeficiency virus
envelope (gp120) binding to DC-SIGN and primary dendritic cells is
carbohydrate dependent but does not involve 2G12 or cyanovirin
binding sites: implications for structural analyses of gp120-DC-SIGN
binding. J. Virol. 76, 12855−12865.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: e.schaeffer@ibmc-cnrs.unistra.fr (E.S.); baati@
bioorga.u-strasbg.fr (R.B.).
Author Contributions
§These authors contributed equally to this work.
Funding
This work was supported by the “Centre National de la
Recherche Scientifique” (CNRS). The University of Strasbourg
is acknowledged for a research grant to L.D.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to P. Pev
the A3/L3 platform of the Institut Fed
́
et and S. Reibel-Foisset for the use of
eratif de Recherches
́
́
(IFR37), Strasbourg. We thank the AIDS Research and
Reference Reagent Program, Division of AIDS, NIAID, NIH,
for MAGI-CCR5 cells.
ABBREVIATIONS
■
DC-SIGN, dendritic cell-specific ICAM-3-grabbing noninte-
grin; DCs, dendritic cells; HIV-1, human immunodeficiency
virus type-1; CRD, carbohydrate recognition domain; CMC,
critical micelle concentration; SPR, surface plasmon resonance
REFERENCES
■
(1) Flexner, C. (2007) HIV drug development: the next 25 years.
Nat. Rev. Drug Discovery 6, 959−966.
(2) Hladik, F., and McElrath, M. J. (2008) Setting the stage: host
invasion by HIV. Nat. Rev. Immunol. 8, 447−457.
(3) Haase, A. T. (2010) Targeting early infection to prevent HIV-1
mucosal transmission. Nature 464, 217−223.
(4) Hu, J., Gardner, M. B., and Miller, C. J. (2000) Simian
immunodeficiency virus rapidly penetrates the cervicovaginal mucosa
after intravaginal inoculation and infects intraepithelial dendritic cells.
J. Virol. 74, 6087−6095.
(5) Gurney, K. B., Elliott, J., Nassanian, H., Song, C., Soilleux, E.,
McGowan, I., Anton, P. A., and Lee, B. (2005) Binding and transfer of
human immunodeficiency virus by DC-SIGN+ cells in human rectal
mucosa. J. Virol. 79, 5762−5773.
(6) Wilkinson, J., and Cunningham, A. L. (2006) Mucosal
transmission of HIV-1: first stop dendritic cells. Curr. Drug Targets
7, 1563−1569.
(7) Yamamoto, T., Tsunetsugu-Yokota, Y., Mitsuki, Y. Y., Mizukoshi,
F., Tsuchiya, T., Terahara, K., Inagaki, Y., Yamamoto, N., Kobayashi,
K., and Inoue, J. (2009) Selective transmission of R5 HIV-1 over X4
HIV-1 at the dendritic cell-T cell infectious synapse is determined by
the T cell activation state. PLoS Pathog. 5, e1000279.
(8) Geijtenbeek, T. B., Kwon, D. S., Torensma, R., van Vliet, S. J., van
Duijnhoven, G. C., Middel, J., Cornelissen, I. L., Nottet, H. S.,
KewalRamani, V. N., Littman, D. R., Figdor, C. G., and van Kooyk, Y.
(2000) DC-SIGN, a dendritic cell-specific HIV-1-binding protein that
enhances trans-infection of T cells. Cell 100, 587−597.
(9) Piguet, V., and Steinman, R. M. (2007) The interaction of HIV
with dendritic cells: outcomes and pathways. Trends Immunol. 28,
503−510.
(10) Wu, L., and KewalRamani, V. N. (2006) Dendritic-cell
interactions with HIV: infection and viral dissemination. Nat. Rev.
Immunol. 6, 859−868.
H
dx.doi.org/10.1021/bc200644d | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
(27) Su, S. V., Gurney, K. B., and Lee, B. (2003) Sugar and spice:
viral envelope-DC-SIGN interactions in HIV pathogenesis. Curr. HIV
Res. 1, 87−99.
(28) Feinberg, H., Castelli, R., Drickamer, K., Seeberger, P. H., and
Weis, W. I. (2007) Multiple modes of binding enhance the affinity of
DC-SIGN for high mannose N-linked glycans found on viral
glycoproteins. J. Biol. Chem. 282, 4202−4209.
(29) Tabarani, G., Reina, J. J., Ebel, C., Vives, C., Lortat-Jacob, H.,
Rojo, J., and Fieschi, F. (2006) Mannose hyperbranched dendritic
polymers interact with clustered organization of DC-SIGN and inhibit
gp120 binding. FEBS Lett. 580, 2402−2408.
(30) Wang, S. K., Liang, P. H., Astronomo, R. D., Hsu, T. L., Hsieh,
S. L., Burton, D. R., and Wong, C. H. (2008) Targeting the
carbohydrates on HIV-1: Interaction of oligomannose dendrons with
human monoclonal antibody 2G12 and DC-SIGN. Proc. Natl. Acad.
Sci. U.S.A. 105, 3690−3695.
(44) Floyd, N., Vijayakrishnan, B., Koeppe, J. R., and Davis, B. G.
(2009) Thiyl glycosylation of olefinic proteins: S-linked glycoconju-
gate synthesis. Angew. Chem. 48, 7798−7802.
(45) Xue, J., Shao, N., and Guo, Z. (2003) First total synthesis of a
GPI-anchored peptide. J. Org. Chem. 68, 4020−4029.
(46) Duffels, A., Green, L. G., Ley, S. V., and Miller, A. D. (2000)
Synthesis of high-mannose type neoglycolipids: active targeting of
liposomes to macrophages in gene therapy. Chem.Eur. J. 6, 1416−
1430.
(47) Sidobre, S., Puzo, G., and Riviere, M. (2002) Lipid-restricted
recognition of mycobacterial lipoglycans by human pulmonary
surfactant protein A: a surface-plasmon-resonance study. Biochem. J.
365, 89−97.
(48) Vives, R. R., Imberty, A., Sattentau, Q. J., and Lortat-Jacob, H.
(2005) Heparan sulfate targets the HIV-1 envelope glycoprotein
gp120 coreceptor binding site. J. Biol. Chem. 280, 21353−21357.
(49) David, S. A., Smith, M. S., Lopez, G. J., Adany, I., Mukherjee, S.,
Buch, S., Goodenow, M. M., and Narayan, O. (2001) Selective
transmission of R5-tropic HIV type 1 from dendritic cells to resting
CD4+ T cells. AIDS Res. Hum. Retroviruses 17, 59−68.
(50) Grivel, J. C., Shattock, R. J., and Margolis, L. B. (2011) Selective
transmission of R5 HIV-1 variants: where is the gatekeeper? J. Transl.
Med. 9 (Suppl 1), S6.
(51) Kitov, P. I., Sadowska, J. M., Mulvey, G., Armstrong, G. D., Ling,
H., Pannu, N. S., Read, R. J., and Bundle, D. R. (2000) Shiga-like
toxins are neutralized by tailored multivalent carbohydrate ligands.
Nature 403, 669−672.
(31) Martinez-Avila, O., Hijazi, K., Marradi, M., Clavel, C., Campion,
C., Kelly, C., and Penades, S. (2009) Gold manno-glyconanoparticles:
multivalent systems to block HIV-1 gp120 binding to the lectin DC-
SIGN. Chem.Eur. J. 15, 9874−9888.
(32) Sattin, S., Daghetti, A., Thepaut, M., Berzi, A., Sanchez-Navarro,
M., Tabarani, G., Rojo, J., Fieschi, F., Clerici, M., and Bernardi, A.
(2010) Inhibition of DC-SIGN-mediated HIV infection by a linear
trimannoside mimic in a tetravalent presentation. ACS Chem. Biol. 5,
301−312.
(33) Maeda, N., Nigou, J., Herrmann, J. L., Jackson, M., Amara, A.,
Lagrange, P. H., Puzo, G., Gicquel, B., and Neyrolles, O. (2003) The
cell surface receptor DC-SIGN discriminates between Mycobacterium
species through selective recognition of the mannose caps on
lipoarabinomannan. J. Biol. Chem. 278, 5513−5516.
(52) Schwefel, D., Maierhofer, C., Beck, J. G., Seeberger, S.,
Diederichs, K., Moller, H. M., Welte, W., and Wittmann, V. (2010)
Structural basis of multivalent binding to wheat germ agglutinin. J. Am.
Chem. Soc. 132, 8704−8719.
(34) Riviere, M., Moisand, A., Lopez, A., and Puzo, G. (2004) Highly
ordered supra-molecular organization of the mycobacterial lipo-
arabinomannans in solution. Evidence of a relationship between
supra-molecular organization and biological activity. J. Mol. Biol. 344,
907−918.
(35) Perino, A., Klymchenko, A., Morere, A., Contal, E., Rameau, A.,
́
Guenet, J.-M., Mely, Y., and Wagner, A. (2011) Structure and
behaviour of polydiacetylene-based micelles. Macromol. Chem. Phys.
212, 111−117.
(36) Morin, E., Nothisen, M., Wagner, A., and Remy, J.-S. (2011)
Cationic polydiacetylene micelles for gene delivery. Bioconjugate Chem.
22, 1916−1923.
(37) Chackerian, B., Long, E. M., Luciw, P. A., and Overbaugh, J.
(1997) Human immunodeficiency virus type 1 coreceptors participate
in postentry stages in the virus replication cycle and function in simian
immunodeficiency virus infection. J. Virol. 71, 3932−3939.
(38) Schaeffer, E., Soros, V. B., and Greene, W. C. (2004)
Compensatory link between fusion and endocytosis of human
immunodeficiency virus type 1 in human CD4 T lymphocytes. J.
Virol. 78, 1375−1383.
(39) Khiar, N., Leal, M. P., Baati, R., Ruhlmann, C., Mioskowski, C.,
Schultz, P., and Fernandez, I. (2009) Tailoring carbon nanotube
surfaces with glyconanorings: new bionanomaterials with specific
lectin affinity. Chem. Commun. 27, 4121−4123.
(40) Su, Y., Xie, J., Wang, Y., Hu, X., and Lin, X. (2010) Synthesis
and antitumor activity of new shikonin glycosides. Eur. J. Med. Chem.
45, 2713−2718.
(41) Tietze, L. F., Schmuck, K., Schuster, H. J., Muller, M., and
Schuberth, I. (2011) Synthesis and biological evaluation of prodrugs
based on the natural antibiotic duocarmycin for use in ADEPT and
PMT. Chem.Eur. J. 17, 1922−1929.
(42) Hatch, D. M., Weiss, A. A., Kale, R. R., and Iyer, S. S. (2008)
Biotinylated bi- and tetra-antennary glycoconjugates for Escherichia
coli detection. ChemBioChem 9, 2433−2442.
(43) Kikkeri, R., Garcia-Rubio, I., and Seeberger, P. H. (2009)
Ru(II)-carbohydrate dendrimers as photoinduced electron transfer
lectin biosensors. Chem. Commun. 2, 235−237.
I
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