Multivalent mannose-displaying nanoparticles constructed from poly{styrene-co-[(maleic anhydride)-alt-styrene]}

Article abstract of DOI:10.1039/b817823b

Micellar mannose nanoparticles were constructed from poly{styrene-co- [(maleic anhydride)-alt-styrene]} with fluorophores or quenchers doped into the hydrophobic inner cores, allowing the direct monitoring of multivalent lectin-glycan interactions. The ma

Full text of DOI:10.1039/b817823b

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Multivalent mannose-displaying nanoparticles constructed from
poly{styrene-co-[(maleic anhydride)-alt-styrene]}†
Rongmin Su,a Lei Li,b Xiaoping Chen,a Jiahuai Hanc and Shoufa Han*a
Received 10th October 2008, Accepted 4th December 2008
First published as an Advance Article on the web 17th March 2009
DOI: 10.1039/b817823b
Micellar mannose nanoparticles were constructed from poly{styrene-co-[(maleic anhydride)-
alt-styrene]} with fluorophores or quenchers doped into the hydrophobic inner cores, allowing the
direct monitoring of multivalent lectin–glycan interactions. The mannose-displaying nanoparticles bind
Con A and sperm surface lectins with high affinity, suggesting their broad utility for modulating
protein–carbohydrate interaction mediated cell surface biology.
drugs.12 Similarly, hydrophobic fluorophores can be doped into
the hydrophobic cores, which facilitates the monitoring of lectin–
glycan interactions.
Introduction
Cell surface protein–glycan interactions mediate a variety of
biological processes ranging from viral infections, cancer cell
metastasis, to cell signaling events, etc.1 Lectins, which are glycan-
binding proteins, typically bind their monovalent ligands with low
affinity. Although the intrinsic affinities of lectin–glycan interac-
tions are low, nature utilizes multivalency to achieve stable binding
for the initiation of subsequent biological activities.1 For instance
influenza virus, with kd values at mM levels towards monomeric
sialoside ligands,2 binds cell surface sialosides in a polyvalent
manner with avidity that leads to effective viral infections. Probes
capable of inhibiting cell surface lectin–glycan interactions have
potential therapeutic applications in combating viral infections,
and cancer metastasis, etc.3–5 Currently, the design of ligand-based
probes of lectins largely relies on the display of multivalent glycan
ligands on selected scaffolds including polymers, dendrimers,
nanoparticles, virus, etc.6–11 Some of these systems suffer from
multistep syntheses, low overall yields, moderately enhanced
lectin binding affinity, or a lack of facile methods for direct
characterization of probe–lectin interactions.
Hairy micellar nanoparticles, with coronas comprised of hy-
drophilic linear polymers and hydrophobic cores, can be assem-
bled from amphiphilic diblock copolymers in aqueous solutions.
We envisioned that the incorporation of glycan ligands onto
hydrophilic polymer chains located at the corona could lead
to novel glyco-probes that combine the advantages of both
linear polymers and nanoparticles. In hairy glyco-nanoparticles,
the flexibility of the polymer chains together with the tunable
size of the nanoparticles will allow simultaneous binding of
the hairy nanoparticle surface glycans with multimeric lectins
or multiple lectin molecules on host cell surfaces. The efficient
multivalent glycan–lectin interaction is essential for enhanced
binding affinity. Furthermore, the hydrophobic inner cores of
micellar nanoparticles have been utilized to entrap hydrophobic
Poly{styrene-co-[(maleic acid)-alt-styrene]}, a well-known am-
phiphilic diblock copolymer capable of forming micellar nanopar-
ticles in aqueous solutions, was employed as the scaffold to display
mannose. Mannose–lectin interactions are vital in a broad spec-
trum of physiological processes including human fertilization,13
and pathogen–host cell recognition.14,15 Hence, mannose is used
as a model ligand to incorporate into poly{styrene-co-[(maleic
anhydride)-alt-styrene]} for evaluation of the efficacy of the new
scaffold in multivalent lectin–glycan interactions.
Results and discussion
Low affinity cell surface glycan receptors that function through
multivalent interactions are intrinsically poor targets for the
rational design of ligands that will bind to the cell. The molecular
size of a lectin is generally about several nanometers. The spacing
between glycan ligands displayed on traditional scaffolds, like
symmetrical dendrimers, is too small to bridge lectin subunits
or multiple lectins on the cell surface. The flexible hydrophilic
chains radiating from size-tunable hydrophobic cores render hairy
micellar glyco-nanoparticles constructed from poly{styrene-co-
[(maleic acid)-alt-styrene]} suitable for simultaneous binding with
a large number of lectins on cell surfaces. Additional advantageous
features of poly{styrene-co-[(maleic acid)-alt-styrene]} as glycan-
displaying scaffolds include: (1) the anhydride moieties on the
poly(maleic anhydride-alt-styrene) fragment can be easily ami-
dated by amine-containing glycan ligands in high yields; (2) the
number and density of glycan ligands on the nanoparticle surface
can be controlled by variation of the degree of polymerization of
the poly(maleic anhydride-alt-styrene) fragment; (3) amphiphilic
diblock copolymers self-assemble in water and form nanoparticles
with hydrophobic cores that can trap fluorescence quenchers
or fluorophores for direct monitoring of lectin–glycan probe
interactions or cell surface labeling.
aDepartment of Chemistry, College of Chemistry and Chemical Engineering,
and The Key Laboratory for Chemical Biology of Fujian Province, Xiamen
University, 361005, P. R. China. E-mail: shoufa@xmu.edu.cn
Poly{styrene-co-[(D-mannopyranosyl-a-1-ethylamidomaleic
acid)-alt-styrene] was successfully prepared by amidation of
poly{styrene-co-[(maleic anhydride)-alt-styrene]} with a-1-O-(2¢-
aminoethyl)-D-mannopyranoside (compound 1) (Scheme 1).
As compared with that of the non-glycosylated polymer
bCollege of Materials, Xiamen University, 361005, P. R. China
cCollege of Life Science, Xiamen University, 361005, P. R. China
† Electronic supplementary information (ESI) available: Further experi-
mental methods and results. See DOI: 10.1039/b817823b
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Scheme 1 Synthesis and assembling of hairy glyco-nanoparticles from poly{styrene-co-[(maleic anhydride)-alt-styrene]}.
1
(NP-COONa) (Figure 6S, ESI†), the H-NMR spectrum of the
lectin molecules or a large number of lectins on cell surfaces
simultaneously. To evaluate the efficacy of the hairy nanoparticles
as multivalent glycan probes, the interaction of NP-mannose with
concanavalin-A (Con A) was investigated. Con A lectin from Jack
bean exists as a homotetramer at physiological conditions. Each
monomeric subunit of Con A has one binding site for D-mannose.
Con A has been used extensively as a model lectin to evaluate
the efficacy of various scaffolds displaying multivalent mannose,
mainly via hemagglutination assays.9 Although hemagglutination
assays have been frequently used to give the relative activity of vari-
ous glyco-probes by their inhibition of lectin induced red blood cell
agglutination, they do not provide information regarding binding
affinity.9 In contrast, in our system lectin–glycan interactions could
be directly monitored via fluorescence resonance energy transfer
(FRET) between the fluorescein isothiocyanate-labeled Con A
(FITC-Con A) (donor) with the ethyl ester of methyl red trapped
in the NP-mannose cores (quencher) (Scheme 2). Correlation of
the fluorescence signals of the glyco-probes with added inhibitory
NP-mannose showed extra signals (e.g. d 4.5) which originated
from incorporated mannose (Figure 5S, ESI†). The exact amount
of mannose incorporated in the polymer, titrated using a reported
colorimetric assay,16 was 350 mg per mg of polymer (Table 1S,
ESI†), suggesting that the anhydride moiety was amidated in
roughly quantitative yield. Assembly of the resultant mannosy-
lated polymer together with 2-{[4-(dimethylamino)phenyl]azo}-
benzoic acid, ethyl ester (ethyl ester of methyl red, fluores-
cence quencher) or rhodamine 6G (fluorophore) afforded hairy
nanoparticles with mannose on the surface (designated as NP-
mannose) doped with fluorescence quencher or fluorophores. The
statistical mean diameter size of NP-mannose was 89.3 nm as
determined by dynamic light scattering (Fig. 1).
Generally, the size of a protein molecule is in the range of
several nanometers. Thus, more than one order of magnitude
of difference in the diameters of NP-mannose relative to lectin
molecules renders NP-mannose spatially accessible to multimeric
Fig. 1 Diameter size of NP-mannose measured by dynamic light scattering. The statistical mean size of NP-mannose was 86.9 nm.
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Scheme 2 Theoretical representation of FRET based competitive binding assay between FITC-Con A and NP-mannose with trapped methyl red in the
presence of D-mannose.
species allows direct evaluation of their relative binding affinity to
a lectin. The new assay is simple and straightforward, offering
a valuable alternative to currently employed hemagglutination
assays in the study of protein–glycan interaction.
using the equation Keq = [mannose/Con A][NP-mannose]/[NP-
mannose/Con A][mannose] (Scheme 2), favored the formation
of the NP-mannose/Con A complex by roughly 5 orders of
magnitude relative to the D-mannose/Con A complex, indicating
that multivalent binding to the tetrameric subunits of Con A
was occurring (Scheme 2). The FRET between fluorescently
labeled lectins and the dye-doped glyco-nanoparticles allows
spectrofluorimetric monitoring of the protein–glycan interactions,
offering a facile method of directly comparing the relative binding
affinity of different glyco-probes towards selected lectins.
To verify the hypothesis that the affinity between NP-mannose
and tetrameric Con A was due to simultaneous binding of the four
ligand binding sites by mannose on the nanoparticle surface, we
sought to study the interaction of NP-mannose with dimeric Con
A. Con A dissociates into dimers at pH 6.5 or below.17 The FRET
based competitive binding assay was performed in sodium acetate
buffer (50 mM, pH 5.5). The emission spectra of the solutions are
shown in Fig. 2 (B). The inhibition efficiency of the binding of
NP-mannose with dimeric FITC-Con A to the concentrations of
exogenous mannose was much more sensitive than was the binding
to tetrameric Con A. The recovery of fluorescence reached 30% in
the presence of 0.1M of D-mannose, indicating that the binding
affinity between dimeric Con A and the mannose-nanoparticles
was at least 150 times weaker than compared with tetrameric Con
A. The observed significant differences in affinity on the basis
of Con A valency are supportive of multiple binding with ligand
binding sites by NP-mannose.
The fluorescence intensity of FITC-Con A was greatly sup-
pressed in the presence of NP-mannose owing to quenching of the
fluorescence by methyl red (Fig. 2, A). In an analogous experiment,
no obvious FRET was observed between FITC-Con A and NP-
galactose with ethyl ester of methyl red doped in the hydrophobic
cores (Scheme 1) (Figure 10S, ESI†). The dfference in FRET
efficiency between NP-mannose and NP-galactose in the presence
of FITC-Con A indicates that the formation of FITC-Con
A/NP-mannose complexes was mannose dependent. To probe the
multivalent effects between NP-mannose and tetrameric Con A,
D-mannose monosaccharide was added to the FITC-Con A/NP-
mannose complex at various concentrations. The fluorescence
emission spectra, recorded as a function of the concentration of
D-mannose (Fig. 2, A), showed that fluorescence intensity of the
FITC-Con A/NP-mannose complex increased with addition of
D-mannose. The FRET efficiency of the donor–quencher pair
was distance-dependant. Thus in the presence of free mannose
that competed with NP-mannose for the lectinic binding sites,
Con A was stripped off the NP-mannose leading to enhanced
detectable fluorescence (Scheme 2). The recovery of fluorescence
relative to free FITC-Con A was about 20% in presence of 1.5M
of D-mannose (Figure 10S, ESI†). In the control experiments,
the fluorescence of FITC-Con A was not obviously affected
by the mannose monosaccharide at concentrations employed in
the inhibitory assays. Based on the inhibitory FRET studies,
the half maximal inhibition concentration (IC50) of mannose
on the formation of NP-mannose/Con A complex was about
1.5M. Based on the reported colorimetric assay,16 the concentra-
tion of the nanoparticle-bound mannose in the assay conditions
was determined to be 3 mM. The binding equilibrium, calculated
Mannose-binding lectin expressed on sperm cell surfaces is
vital for sperm–oocyte fusion.18–20 Demonstrated to bind Con A
with high affinity, NP-mannose was further tested for its ability
to bind cell surface lectins. NP-COONa, the control assembled
from polystyrene-co-poly(maleic acid, sodium salt-alt-styrene)
(Scheme 1), and NP-mannose with rhodamine 6G trapped in
the hydrophobic cores (Scheme 2) were separately incubated with
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heads and tails could be identified probably due to interference by
NP-mannose binding on cell surface lectins. Investigation of the
interference of free mannose monosaccharide on the interaction
of NP-mannose with sperms was also performed using the same
procedure with the addition of various amounts of mannose. The
aggregation of sperms was gradually inhibited with increasing
amounts of mannose. At high concentrations of mannose (2M),
normal morphology of sperms was observed (Figure 12S, ESI†).
The inhibitory effects of high concentrations of mannose indicate
that the interaction between NP-mannose and sperm cell surface
lectins is much stronger than with monovalent mannose.
D-mannose derivatives and D-mannose monosaccharide have
been documented to block fertilization.19,13,22 Multivalent mannose
probes that could bind sperm with higher affinity might be
superior fertilization inhibitors than mannose monosaccharide.
Our preliminary results suggest that it is worth evaluating the
inhibition efficacy of NP-mannnose on sperm–oocyte fusion for
potential utility as novel topical contraceptive reagents.
Conclusions
Mannose displaying hairy nanoparticles were easily assem-
bled from polystyrene-co-poly(maleic anhydride-alt-styrene) and
bound lectins in a multivalent manner. Due to the high lectin
binding affinity and other advantageous features mentioned above,
hairy micellar glyco-nanoparticles have broad application in the
study of protein–glycan interactions.
Experimental
Materials and methods
a-1-O-(2¢-Aminoethyl)-D-mannopyranoside (1) and b-1-O-(2¢-
aminoethyl)-D-galactopyranoside (2) (Scheme 1) were synthesized
according to the reported literature.23 Poly{styrene-co-[(maleic
anhydride)-alt-styrene]} was prepared following a published
procedure.24 Unless otherwise noted, all reagents or lectins were
obtained commercially from either Alpha-Aesar or Sigma-Aldrich
and used without further purification. Column chromatography
was performed on silica gel (300–400 mesh). NMR spectra (1H
at 400 MHz and 13C at 100 MHz) were recorded on a Bruker
instrument using tetramethyl silane as the internal reference. The
fluorescence emissions were performed on a spectrofluorimeter
(Shimadzu, RF-5301, Japan) using the excitation wavelength (lex)
of 490 nm. The fluorescence microscopy of sperms was visualized
and recorded using a phase-contrast microscope (Olympus, Japan)
(¥100 magnification). Dynamic light scattering experiments were
performed on Malvern Nano-ZS & MPT-2 (U.K.).
Fig. 2 Inhibitory effects of D-mannose on the fluorescence emission of
tetrameric FITC-Con A doped with methyl red (0.13 mg/mL) (A) and
dimeric FITC-Con A doped with methyl red (0.13 mg/mL) (B) in the
presence of NP-mannose (0.13 mg/mL).
semen specimens in PBS buffer. Fluorescence microscopy revealed
normal morphology for the sperms treated with NP-COONa. The
fluorescence on the cell surface is probably due to ionic interactions
between sperms and polyanionic compounds.21 In the presence
of multivalent NP-mannose, the sperm cells tend to aggregate
as compared with sperms incubated with NP-COONa (Fig. 3)
presumably due to the binding of NP-mannose with multiple lectin
molecules on neighboring sperms. The cell morphology of NP-
mannose treated sperms was altered and no sperms with normal
Synthesis of 2-{[4-(dimethylamino)phenyl]azo}-benzoic acid, ethyl
ester (ethyl ester of methyl red)
To a 25 mL flask containing dry ethanol (10 mL) was added 4-
dimethylaminopyridine (DMAP, 4.8 mg, 0.04 mmol), 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDC, 1.0 g,
5 mmol) and 2-{[4-(dimethylamino)phenyl]azo}-benzoic acid
(methyl red, 0.59 g, 2 mmol) (Scheme 1S, ESI†). The reaction mix-
ture was stirred at room temperature overnight. The reaction was
monitored by TLC. When the reaction was complete, the solvent
was removed by rotary evaporation and the resulting residue was
Fig. 3 Fluorescence microscopy images of sperm cells treated with
NP-COONa doped with rhodamine 6G (A) (0.4 mg/mL) as compared with
cells treated with NP-mannose doped with rhodamine 6G (B) (0.4 mg/mL).
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suspended in dichloromethane (30 ml). The organic solution was
washed with aqueous hydrochloric acid (1M, 20 ml), dried with
Na2SO4, filtered, and concentrated by rotary evaporation. The
crude product was further purified by column chromatography
over silica gel to afford the desired product as an orange solid
(0.5 g) in 76% yield. Rf = 0.5 (1:5, ethyl acetate/petroleum ether);
1H-NMR (CDCl3): d 1.31 (t, 3H, J = 7.16 Hz), 3.10 (s, 6H), 4.36
(q, 2H, J = 7.13 Hz), 6.74 (d, 2H, J = 9.11 Hz), 7.40 (t, 1H, J =
6.42 Hz), 7.55 (t, 1H, J = 6.42 Hz), 7.62 (d, 1H, J = 7.07 Hz), 7.77
(d,1H, J = 6.46 Hz), 7.87 (d, 2H, J= 5.19 Hz) (Figure 3S, ESI†);
13C-NMR (CDCl3): 14.37, 40.26, 61.18, 111.38, 119.11, 125.39,
128.22, 128.36, 129.47, 131.57, 143.73, 152.46, 152.65, 168.32
(Figure 4S, ESI†); MS (ESI, C17H19N3O2): calculated [MH+]:
298.2, found: 298.2.
Preparation of NP-COONa nanoparticles doped with
rhodamine 6G
Aqueous NaOH solution (1M, 0.3 mL) was added to a 5 ml flask
containing DMF (1 mL) and poly{styrene-co-[(maleic anhydride)-
alt-styrene]} (20 mg). The solution was stirred at ambient temper-
ature for 4 hours. To the stirred solution was added rhodamine 6G
(1 mg) and then water (9 mL). The solution was neutralized with
hydrochloric acid (1M) to pH 7.0, ultrasonicated for 20 minutes,
and then extensively dialyzed against deionized water using a
dialysis tube (MWCO 3000), affording poly{styrene-co-[(maleic
acid, sodium salt)-alt-styrene]} nanoparticles (designated as NP-
COOH) (Scheme 1) doped with rhodamine 6G. The statistical
mean diameter size of NP-COONa was 35.2 as determined by
dynamic light scattering.25
FRET based competitive binding assays of tetrameric FITC-Con
A/NP-mannose complex
Tetrameric FITC-Con A/NP-mannose complex was formed via
incubation of solutions containing mannose-NP (0.4 mg), FITC-
labeled Con A (0.4 mg) in Tris-HCl buffer (50 mM, pH 7.2, 50 mL)
containing calcium chloride (2 mM) (buffer A) and manganese
chloride (2 mM) at room temperature for 15 minutes. Then various
volumes of D-mannose solution in buffer A (3M) and tetrameric
FITC-Con A/NP-mannose complex solution (50 mL) were added
to cuvettes. The final volume was adjusted to 3 mL with addition
of Buffer A. The fluorescence emission spectra were recorded as a
function of the concentrations of D-mannose monosaccharide.
Preparation of NP-mannose hairy nanoparticles doped with the
ethyl ester of methyl red or rhodamine 6G
a-1-O-(2¢-Aminoethyl)-D-mannopyranoside (30 mg), triethy-
lamine (0.1 mL) and poly{styrene-co-[(maleic anhydride)-alt-
styrene]} (20 mg) were added to a 5 ml flask containing DMF
(1 mL). The solution was stirred at ambient temperature overnight
followed by addition of ethyl ester of methyl red (1 mg) or
rhodamine 6G (1 mg) and then water (15 mL). The resultant
mixture was extensively dialyzed against deionized water using
a dialysis tube (MWCO 3000) to remove excess compound 1,
DMF, and triethylamine, and then was sonicated for 40 minutes
to afford a clear solution. The solution was filtered through a
0.22 mm filter to give poly{styrene-co-[(D-mannopyranosyl-a-1-
ethylamidomaleic acid)-alt-styrene]} nanoparticles (designated as
NP-mannose) (Scheme 1) doped with ethyl ester of methyl red or
rhodamine 6G. The statistical mean diameter size of NP-mannose
was 89.3 nm as determined by dynamic light scattering.
FRET based competitive binding assays of dimeric FITC-Con
A/NP-mannose complex
Dimeric FITC-Con A/NP-mannose complex was formed via
incubation of solutions containing NP–mannose (0.4 mg), FITC-
labeled Con A (0.4 mg) in sodium-acetate buffer (50 mM, pH 5.5,
50 mL) containing calcium chloride (2 mM) and manganese
chloride (2 mM) (buffer B) at room temperature for 15 minutes.
Then various volume of D-mannose solution in buffer B (3M) and
dimeric FITC-Con A/NP-mannose complex solution were added
to cuvettes containing buffer B for 20 minutes. The final volume
was adjusted to 3 mL with addition of buffer B. The fluorescence
emission spectra were recorded as a function of the concentrations
of D-mannose monosaccharide.
Preparation of NP-galactose hairy nanoparticles doped with the
ethyl ester of methyl red
b-1-O-(2¢-Aminoethyl)-D-galactopyranoside (30 mg), triethy-
lamine (0.1 mL) and poly{styrene-co-[(maleic anhydride)-alt-
styrene]} (20 mg) were added to a 5 ml flask containing
DMF (1 mL). The solution was stirred at ambient tempera-
ture overnight followed by addition of ethyl ester of methyl
red (1 mg) and then water (9 mL). The mixture was first
extensively dialyzed against deionized water using a dialysis
tube (MWCO 3000) to remove excess compound 2, DMF, and
triethylamine and then ultrasonicated for 20 minutes afford-
ing poly{styrene-co-[(D-galactopyranosyl-b-1-ethylamidomaleic
acid)-alt-styrene]} nanoparticles (NP-galactose) (Scheme 1)
doped with ethyl ester of methyl red. The statistical mean diameter
size of NP-galactose was 26 nm as determined by dynamic light
scattering (Figure 9S, ESI†).
Binding of NP-mannose with human spermatozoa
Human spermatozoa samples were stained using the procedure
described in the published literature.18 Briefly, NP-mannose
(0.4 mg/mL) and NP-COONa (0.4 mg/mL) doped with rodamine
6G were separately incubated with human spermatozoa for 20 min
at 37 ◦C in HEPES buffer (30 mM, pH 7.0) containing NaCl
(150 mM), MgCl2 (0.5 mM), CaCl2 (20 mM) and bovine serum
albumin (1%, w/v). Spermatozoa were then washed three times
with HEPES buffer without calcium chloride, pipetted onto glass
slides and fixed on cover glass. The morphology of the sperm cells
was visualized using a phase-contrast microscope.
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12 L. Y. Qiu and Y. H. Bae, Pharm. Res., 2006, 23, 1–30.
13 K. Mori, T. Daitoh, M. Irahara, M. Kamada and T. Aono, Am. J. Ob-
stet. Gynecol., 1989, 161, 207–11.
14 L. Heggelund, T. E. Mollnes, T. Ueland, B. Christophersen, P. Aukrust
and S. S. Froland, J. Acquir. Immune Defic. Syndr., 2003, 32, 354–61.
15 I. Ofek, A. Mosek and N. Sharon, Infect. Immun., 1981, 34, 708–
11.
Acknowledgements
This work was supported by Chinese 863 project (No.
2006AA02Z164) and National Natural Science Foundation of
China (No. 20842001 and 20802060).
16 M. G. K. A. Dubois, J. K. Hamilton, P. A. Rebers and F. Smith, Anal.
Chem., 1956, 28, 350–356.
17 T. W. Hamelryck, J. G. Moore, M. J. Chrispeels, R. Loris and L. Wyns,
J. Mol. Biol., 2000, 299, 875–83.
18 A. Gabriele, G. D’Andrea, G. Cordeschi, G. Properzi, M. Giammatteo,
C. De Stefano, R. Romano, F. Francavilla and S. Francavilla, Mol.
Hum. Reprod., 1998, 4, 543–53.
19 S. Tanghe, A. Van Soom, L. Duchateau and A. De Kruif, Mol. Reprod.
Dev., 2004, 67, 224–32.
20 H. M. Youssef, G. F. Doncel, B. A. Bassiouni and A. A. Acosta, Fertil.
Steril., 1996, 66, 640–5.
21 E. Howes, J. C. Pascall, W. Engel and R. Jones, J. Cell. Sci., 2001, 114,
4127–36.
References
1 B. E. Collins and J. C. Paulson, Curr. Opin. Chem. Biol., 2004, 8, 617–25.
2 J. E. Hanson, N. K. Sauter, J. J. Skehel and D. C. Wiley, Virology, 1992,
189, 525–33.
3 J. Rojo, V. Diaz, J. M. de la Fuente, I. Segura, A. G. Barrientos, H. H.
Riese, A. Bernad and S. Penades, ChemBioChem, 2004, 5, 291–7.
4 R. Stahn and R. Zeisig, Tumour Biol., 2000, 21, 176–86.
5 W. J. Lees, A. Spaltenstein, J. E. Kingery-Wood and G. M. Whitesides,
J. Med. Chem., 1994, 37, 3419–33.
6 E. Kaltgrad, M. K. O’Reilly, L. Liao, S. Han, J. C. Paulson and M. G.
Finn, J. Am. Chem. Soc., 2008, 130, 4578–9.
22 K. Mori, T. Daitoh, M. Kamada, N. Maeda, M. Maegawa, K. Hirano,
M. Irahara and T. Aono, Hum. Reprod., 1993, 8, 1729–32.
23 T. Hasegawa, T. Fujisawa, M. Numata, T. Matsumoto, M. Umeda, R.
Karinaga, M. Mizu, K. Koumoto, T. Kimura, S. Okumura, K. Sakurai
and S. Shinkai, Org. Biomol. Chem., 2004, 2, 3091–8.
24 M. Q. Zhu, L. H. Wei, M. Li, L. Jiang, F. S. Du, Z. C. LI and F. M. Li,
Chem. Commun., 2001, 365–366.
7 N. Sharon and H. Lis, Sci. Am, .1993, 268, 82–9.
8 L. L. Kiessling, J. E. Gestwicki and L. E. Strong, Curr. Opin. Chem.
Biol., 2000, 4, 696–703.
9 E. K. Woller and M. J. Cloninger, Org Lett, .2002, 4, 7–10.
10 Z. Dai, A. N. Kawde, Y. Xiang, J. T. La Belle, J. Gerlach, V. P.
Bhavanandan, L. Joshi and J. Wang, J. Am. Chem. Soc., 2006, 128,
10018–9.
25 W. Fang et al., Bioorg. Med. Chem. Lett., 2009,
DOI: 10.1016/j.bmcl.2009.02.059.
11 A. Robinson, J. M. Fang, P. T. Chou, K. W. Liao, R. M. Chu and S. J.
Lee, ChemBioChem, 2005, 6, 1899–905.
This journal is
The Royal Society of Chemistry 2009
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