Site-directed conjugation of "clicked" glycopolymers to form glycoprotein mimics: Binding to mammalian lectin and induction of immunological function

Article abstract of DOI:10.1021/ja072999x

Synthesis of well-defined neoglycopolymer-protein biohybrid materials and a preliminary study focused on their ability of binding mammalian lectins and inducing immunological function is reported. Crucial intermediates for their preparation are well-defined maleimide-terminated neoglycopolymers (M _n = 8-30 kDa; M_w/M_n = 1.20-1.28) presenting multiple copies of mannose epitope units, obtained by combination of transition-metal-mediated living radical polymerization (TMM LRP) and Huisgen [2+3] cycloaddition. Bovine serum albumin (BSA) was employed as single thiol-containing model protein, and the resulting bioconjugates were purified following two independent protocols and characterized by circular dichroism (CD) spectroscopy, SDS PAGE, and SEC HPLC. The versatility of the synthetic strategy presented in this work was demonstrated by preparing a small library of conjugating glycopolymers that only differ from each other for their relative epitope density were prepared by coclicking of appropriate mixtures of mannopyranoside and galactopyranoside azides to the same polyalkyne scaffold intermediate. Surface plasmon resonance binding studies carried out using recombinant rat mannose-binding lectin (MBL) showed clear and dose-dependent MBL binding to glycopolymer-conjugated BSA. In addition, enzyme-linked immunosorbent assay (ELISA) revealed that the neoglycopolymer-protein materials described in this work possess significantly enhanced capacity to activate complement via the lectin pathway when compared with native unmodified BSA.

Full text of DOI:10.1021/ja072999x

Published on Web 11/17/2007
Site-Directed Conjugation of “Clicked” Glycopolymers To
Form Glycoprotein Mimics: Binding to Mammalian Lectin and
Induction of Immunological Function
Jin Geng,† Giuseppe Mantovani,*,† Lei Tao,† Julien Nicolas,†, Gaojian Chen,†
Russell Wallis,§ Daniel A. Mitchell,‡ Benjamin R. G. Johnson,|
Stephen D. Evans,| and David M. Haddleton*,†
Contribution from the Department of Chemistry, UniVersity of Warwick, CV4 7AL, CoVentry,
United Kingdom; Clinical Sciences Research Institute, Warwick Medical School, UniVersity of
Warwick, CV2 2DX, CoVentry, United Kingdom; Department of Infection, Immunity and
Inflammation, UniVersity of Leicester, Leicester, United Kingdom; and School of Physics and
Astronomy, UniVersity of Leeds, Leeds, United Kingdom
Received April 29, 2007; E-mail: D.M.Haddleton@warwick.ac.uk
Abstract: Synthesis of well-defined neoglycopolymer-protein biohybrid materials and a preliminary study
focused on their ability of binding mammalian lectins and inducing immunological function is reported. Crucial
intermediates for their preparation are well-defined maleimide-terminated neoglycopolymers (Mn ) 8-30
kDa; Mw/Mn ) 1.20-1.28) presenting multiple copies of mannose epitope units, obtained by combination
of transition-metal-mediated living radical polymerization (TMM LRP) and Huisgen [2+3] cycloaddition.
Bovine serum albumin (BSA) was employed as single thiol-containing model protein, and the resulting
bioconjugates were purified following two independent protocols and characterized by circular dichroism
(CD) spectroscopy, SDS PAGE, and SEC HPLC. The versatility of the synthetic strategy presented in this
work was demonstrated by preparing a small library of conjugating glycopolymers that only differ from
each other for their relative epitope density were prepared by coclicking of appropriate mixtures of
mannopyranoside and galactopyranoside azides to the same polyalkyne scaffold intermediate. Surface
plasmon resonance binding studies carried out using recombinant rat mannose-binding lectin (MBL) showed
clear and dose-dependent MBL binding to glycopolymer-conjugated BSA. In addition, enzyme-linked
immunosorbent assay (ELISA) revealed that the neoglycopolymer-protein materials described in this work
possess significantly enhanced capacity to activate complement via the lectin pathway when compared
with native unmodified BSA.
Introduction
microbial mannose-rich glycans by lectins of the immune system
mediates important host defense mechanisms such as pathogen
The post-genome era is one of the most challenging and
exciting periods in the history of chemical and biological
research. The study of oligosaccharides and lectins in the context
of whole organismssfunctional glycomicssis one of the most
difficult yet crucial fields of post-genome analysis, with impact
upon many biochemical and physiological processes such as
pathogen-host interactions, cell adhesion, tissue integrity and
homeostasis, and cell signaling.1-7 For example, recognition of
neutralization and phagocytosis.8 A major obstacle to rapid
progress in the field of glycobiology and functional glycomics
is the availability of purified oligosaccharides in sufficient
quantities for inclusion in biological and biophysical analysis
strategies.
Synthetic neoglycopolymers having multiple copies of mono-
dentate sugar moieties are a class of macromolecular displays
that have shown very promising results when employed as
natural oligosaccharide mimics.9-13 The ability of some of these
materials to specifically recognize lectins and cell surfaces has
† Department of Chemistry, University of Warwick.
‡ Warwick Medical School, University of Warwick.
§ University of Leicester.
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| University of Leeds.
Present address: Laboratoire de Physico-Chimie, Pharmacotechnie et
Biopharmacie, UMR CNRS 8612, Univ Paris-Sud, 92296 Chatenay-Malabry
Cedex, France.
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10.1021/ja072999x CCC: $37.00 © 2007 American Chemical Society

Site-Directed Conjugation of “Clicked” Glycopolymers
A R T I C L E S
been the subject of extensive research, and now that the
development of novel therapeutic strategies can be based on
multivalent interactions a better understanding of these factors
is required.13-16
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galactose-containing neoglycopolymers by combination of TMM
LRP and Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition,61,62
a “click chemistry”63 process. Our approach involved the
grafting of sugar azides onto a polyalkyne “clickable” scaffold
prepared by TMM LRP. Several azide-containing molecules
could be simultaneously grafted in different proportions onto
the same clickable scaffold, yielding libraries of different
copolymers in a process that we named a “coclicking” approach.
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cells is strongly dependent on their size, shape, structure, and
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A R T I C L E S
Geng et al.
Scheme 1. Synthesis of the Glycoprotein Mimic Employed in This Study
Scheme 2 a
a Reagents and conditions: (a) CuSO4, sodium ascorbate, methanol/H2O (1:1 v/v), ambient temperature; (b) N-(ethyl)-2-pyridylmethanimide, Cu(I) Br,
rhodamine B methacrylate 9, initiator 8, CH3OH/H2O 5:2 (v/v), ambient temperature; (c) toluene reflux, 18 h; (d) N-(ethyl)-2-pyridylmethanimide, Cu(I) Br,
initiator 8, rhodamine B methacrylate 9, toluene, 30 °C; (e) TBAF‚3H2O, THF, 0 °C to ambient temperature, 12 h; (f) (i) 1, (PPh3)3CuBr, Et3N, DMSO,
ambient temperature, 2 days; (ii) toluene reflux, 18 h.
ployed for polymer multiple functionalization, can undergo
important side reactions during the final conjugation step that
could potentially limit their applicability for the synthesis of
multivalent therapeutics.65
Alternatively, controlled radical polymerization techniques
allow for simple synthesis of well-defined glycopolymers by
simply polymerization of appropriate sugar monomers. The main
advantage of this approach is its simplicity, and the two synthetic
strategies, postfunctionalization and direct polymerization of
carbohydrate monomers, can now be regarded as complementary
tools for the preparation of neoglycopolymers.
Another key advantage in using TMM LRP is that R-func-
tional polymers can be easily prepared by simply choosing
appropriate polymerization initiators; this has been exploited
in the past for the synthesis of biohybrid materials by (poly)-
peptide-polymer conjugation.46,66-70
containing glycoprotein mimics suitable for probing interactions
with mannose-binding lectin (MBL), a key mammalian lectin
of the immune system. Mannoside structures are prevalent on
the cell surfaces of many bacterial and fungal pathogens, their
arrangements distinct from human mannoside glycans, thus
being distinguishable from the human host. MBL is able to
recognize these non-self-arrangements of mannoside residues
and through binding in this way to pathogen surfaces is able to
trigger the complement cascade, a powerful arm of the circulat-
ing innate immune system that subsequently opsonises the
pathogens for phagocytosis as well as driving cellular pore
formation that leads to pathogen cell destruction.
Essential polymeric starting materials are novel maleimide-
terminated glycopolymers able to selectively react with the free-
cysteine (Cys34) residue of bovine serum albumin (BSA)san
abundant model protein molecule.
In the present work we aimed to modify our “click” approach
to glycopolymers for the synthesis of R-mannopyranoside-
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Site-Directed Conjugation of “Clicked” Glycopolymers
A R T I C L E S
Table 1. Polymers Prepared in This Study
Free cysteine residues are rare in native polypeptides, and
when present, they constitute an excellent attachment site for
protein modification as they are present in very discrete number
(typically one) and their location is often identifiable. When
necessary, the protein of interest can be genetically modified
in order to obtain free cysteine residues in specific positions of
the protein surface. Development of protein-glycopolymer
conjugates carries potential for the generation of therapeutic
agents and biological probes. Through site-directed conjugate
formation, defined biological molecules with novel dual proper-
ties can be formed very easily. Protein components can
potentially confer functions such as highly defined ligand
binding, enzyme activity, and sources of specific amino acid
sequences in tertiary structural motifs or in the case of
immunological antigen presentation as digested peptides loaded
onto major histocompatibility complex molecules. Glycopoly-
mers can confer selective lectin binding and thus engagement
with the counter-glycome in plasma, cells, and tissues. Mannose
polymers are of particular interest due to the fact that a number
of mammalian lectins associated with immune function have
been shown to bind mannose-containing structures expressed
on the surface of pathogens such as Gram-positive and Gram-
negative bacteria, fungal pathogens such as Candida albicans,
and lethal viruses such as HIV, Ebola, and Hepatitis C.71-74 In
this study we focused upon modifying BSA, a protein that is
inert with regard to the innate immune system, via conjugation
of mannose glycopolymer in order to investigate binding to
MBL, an acute phase C-type lectin of the immune system that
triggers activation of the complement cascadesthe fundamental
and archetypal fluid-phase innate immune effector mechanism
present in plasma, cerebrospinal fluid, and tissue mucosa.75,76
polymer
DP (NMR)
M
n (NMR) (kDa)
Mw/Mn (SEC)
3a
3b
4aa
4ba
6
70
20
74
22
41
40
44
26.1
7.5
27.6
8.2
8.0
5.0
1.20c
1.22c
1.20d
1.23d
1.22c
1.25c
1.28d
7
4cb
16.4
a Prepared via path A. b Prepared via path B. c Determined by SEC using
CHCl3/Et3N 95:5 as the mobile phase. d Determined by aqueous SEC.
Table 2. Maleimide-Terminated Polymers Featuring Different
Mannose Density Prepared in This Study
polymer
% mannose
% galactose
DP (NMR)
M
n (NMR) (kDa)
Mw/Mn(SEC)a
23
24
25
26
27
100
75
50
25
0
0
25
50
75
100
139
141
139
136
138
52.3
53.1
52.3
51.1
51.9
1.26
1.25
1.25
1.26
1.25
a Obtained by SEC analysis using DMF as the mobile phase and RI
detection.
Results and Discussion
Synthesis of the R-Maleimide Neoglycopolymers. The
required maleimide-terminated neoglycopolymers were prepared
following two independent synthetic pathways (Scheme 2).
Visibly fluorescent tag based on rhodamine B dye was
introduced onto the polymers backbone in order to facilitate
characterization of the relative protein conjugates and improve
their traceability during the in vitro tests carried out in the
present study.
In the first protocol (Path A) the mannose-containing
monomer 2 was readily prepared by Huisgen 1,3-dipolar
cycloaddition of the mannose azide 1 and propargyl methacrylate
using (PPh3)3CuBr as the catalyst. Polymerization of 2 in the
presence of the maleimide-protected initiator 8 and fluorescent
rhodamine B comonomer 9,77 using iminopyridine/Cu(I) Br as
the catalytic system, afforded the macromolecular intermediate
3. This was then suspended in refluxing toluene for 18 h in
order to remove the furan protecting group by retro-Diels-Alder
reaction, leading to the maleimide-terminated neoglycopolymer
4. Alternatively, we found that the latter reaction occurred even
Figure 1. SDS-PAGE [Coomassie staining (left) and UV excitation at λ
) 302 nm (right)] for the conjugation reaction of BSA with 4a: (a) MW
markers; (b) native BSA; (c) glycopolymer 4a; (d) BSA with 10 equiv of
4a: conjugation mixture; (e) 10 purified by SEC followed by affinity
chromatography.
in the solid state by simply leaving a powder sample of 3 in a
vacuum oven at 80 °C overnight, yielding directly pure 4 and
avoiding use of organic solvents and the need for a further final
precipitation step.
The second synthetic strategy relies on the ability of poly-
(propargyl methacrylate) to act as a versatile “clickable”
polymeric scaffold able to efficiently react with sugar azides
(Path B). The first step involves polymerization of the trimeth-
ylsilyl-protected propargyl methacrylate 5 in toluene, using
iminopyridine/Cu(I) Br as the catalyst, in the presence of the
initiator 8. Removal of the trimethylsilyl groups with TBAF in
THF followed by clicking of the R-mannopyranoside azide 1
in DMSO and retro-Diels-Alder deprotection of the maleimide
moiety afforded the expected maleimide-terminated mannose-
containing polymer 4.
In the polymerization steps the first-order kinetic plots showed
some deviation from linearity, yet the linear increase of Mn with
monomer conversion and the narrow molecular weight distribu-
tions indicate that polymerization occurred in a controlled
fashion (see Supporting Information).
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(74) Brown, K. S.; Ryder, S. D.; Irving, W. L.; Sim, R. B.; Hickling, T. P.
Immunol. Lett. 2007, 108 (1), 34-44.
(75) Walport, M. J. N. Engl. J. Med. 2001, 344 (14), 1058-1066.
(76) Walport, M. J. N. Engl. J. Med. 2001, 344 (15), 1140-1144.
(77) Nicolas, J.; San Miguel, V.; Mantovani, G.; Haddleton, D. M. Chem.
Commun. 2006, 4697-4699.
Both synthetic approaches developed led to the desired
maleimide-terminated polymers (Tables 1 and 2). Particular
9
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A R T I C L E S
Geng et al.
Figure 2. BSA + maleimide glycopolymer 4a (10 equiv). (Left) Purification of conjugate 10 through an immobilized Concanavalin A column before
eluting with 1.0 M mannose solution. (Right) SEC analysis of the conjugation reaction mixture (purple) and the final purified product 10. UV detection (λ
) 280 nm) was employed.
Scheme 3. Synthesis of the Glycoprotein Mimic 10 Obtained from Neoglycopolymer 4a
effort was spent in order to further simplify the synthetic
protocol presented in this study. For example, mannose azide
building block 1 was originally prepared following a well-
established four-step procedure (see Supporting Information).78
Subsequently, we employed an alternative two-step procedure
starting from unprotected D-mannose by first reacting the sugar
with 2-bromoethanol at 90 °C in the presence of Amberlite IR-
120 acid catalyst and then converting the resulting R-2′-
bromoethyl-D-mannopyranoside into the desired mannose azide
1 with sodium azide in refluxing water/acetone mixture.79
Synthesis of BSA Glycoprotein Mimics. Bovine serum
albumin (BSA) is a commercially available 66 kDa protein that
is often chosen as a model single-thiol-containing substrate. Its
free Cys34 residue is generally regarded as the attachment site
of choice for BSA site-specific conjugation. In the past both
we68 and Maynard67 have shown that BSA can be conjugated
with functional polymers obtained by controlled radical polym-
erization bearing chain ends able to react selectively with free
thiol-containing (poly)peptides. In addition, BSA had been
employed as a protein carrier for the chemoenzymatic synthesis
of N-glycan-containing neoglycoproteins,80 and it appeared,
therefore, to be a suitable substrate to be employed in our study.
In the present work, glycoconjugate 10 was obtained by
reaction of maleimide-terminated display 4a with BSA in 1:1
DMSO:PBS (50 mM, pH 7.0) (Scheme 3).
Several reaction conditions and purification protocols were
explored for the conjugation reaction. In particular, an excess
of both glycopolymer 4a and BSA protein, 10:1 and 1:10 mol/
mol, respectively, was successfully employed.
In the former case, the excess of unreacted maleimide polymer
4a was removed by repeated SEC purifications, while unreacted
BSA and BSA dimer were separated by affinity chromatography
using an agarose-immobilized Concanavalin A (Con A) column.
Con A is a 26 kDa lectin (normally forming dimers or tetramers
depending on the pH) extracted from Jack Beans that specifically
recognize R-mannopyranoside sugar moieties. When the mixture
obtained from SEC purification was passed through the affinity
chromatography column, only the bioconjugate 10 was able to
selectively interact with the Con A while the unreacted BSA
and BSA dimer were rapidly eluted (Figure 1). 10 was then
released from the column by simply washing the latter with a
1.0 M solution of D-mannose, a monovalent competitive ligand
for Con A.
This protocol allowed for isolation of the pure 10 biohybrid
material as confirmed by SDS-PAGE and SEC analysis (Figure
1). It was evident from SDS-PAGE analysis of the crude product
that some unreacted BSA was still present at the end of the
conjugation reaction. This result agreed well with previous
(78) Kleinert, M.; Roeckendorf, N.; Lindhorst, T. K. Eur. J. Org. Chem. 2004,
18, 3931-3940.
(79) To the best of our knowledge, two examples of direct conversion of
D-mannose into its corresponding 2′-bromoethyl-glycoside have been
previously reported: Smith, E. A.; Thomas, W. D.; Kiessling, L. L.; Corn,
R. M. J. Am. Chem. Soc. 2003, 125 (20), 6140-6148 (acid catalyst,
camphorsulfonic acid). Mukhopadhyay, B.; Maurer, S. V.; Rudolph, N.;
Van Well, R. M.; Russell, D. A.; Field, R. A. J. Org. Chem. 2005, 70
(22), 9059-9062 (catalyst, HClO4 immobilized on silica)
(80) Andre, S.; Unverzagt, C.; Kojima, S.; Dong, X.; Fink, C.; Kayser, K.;
Gabius, H.-J. Bioconjugate Chem. 1997, 8 (6), 845-855.
9
15160 J. AM. CHEM. SOC. VOL. 129, NO. 49, 2007

Site-Directed Conjugation of “Clicked” Glycopolymers
A R T I C L E S
Figure 3. BSA (10 equiv) + maleimide glycopolymer 4a. FPLC anion-
exchange chromatography purification of the conjugate 10 using Source Q
column in 20 mM TRIS buffer at pH 9.0 as the mobile phase. A gradient
of NaCl (pink dotted line) was used in order to elute the protein-based
products.
reports that indicated that only 36-69% of the BSA molecules
present in commercially available samples have the free cysteine
thiol required for protein conjugation.68,81-83 SDS-PAGE analy-
sis also showed the absence of both BSA and BSA dimer in
the final purified sample. SEC chromatography analysis of the
purified conjugate 10 confirmed the presence of a fluorescent
product with a molecular mass higher than BSA, in full
agreement with the SDS-PAGE results (Figure 2).
The ability of 10 to interact strongly with the Con A lectin
during affinity chromatography was in itself a preliminary direct
evidence of the ability of this biohybrid material to act as a
multivalent ligand for lectin recognition.
Figure 4. Surface plasmon resonance analysis of immobilized BSA-
glycopolymer conjugate 10 with fluid phase mannose binding lectin (MBL)
(a), and immobilized MBL with fluid phase BSA-glycopolymer conjugate
(b). Due to the naturally heterogeneous nature of MBL oligomers,
concentrations of fluid-phase MBL on immobilized BSA-conjugate are
indicated assuming a molecular weight of 75 kD. (b) An average molecular
weight of 100 kDa is assumed for indications of fluid-phase BSA-
conjugate.
Ion-exchange chromatography was used for the case in which
a 1:10 glycopolymer 4a:BSA molar ratio was employed in an
attempt to simplify the purification process of the glycoprotein
mimic 10. Optimum results were obtained by anion-exchange
chromatography using a Source Q column in 20 mM TRIS
buffer at pH 9.0. Under these experimental conditions the
unreacted maleimide-terminated polymer 4a was not retained,
while 10 showed a retention time between that of free polymer
and BSA and BSA dimer (Figure 3). SEC analysis of the
conjugate 10 isolated using this method confirmed that the two
purification protocols developed were equivalent in terms of
purity of the desired glycoprotein mimic 10 (see Supporting
Information), although use of ion-exchange chromatography
appeared to be more suitable for large-scale purification
processes. The two purification processes are suitable for
purification of crude mixtures obtained from both conjugation
protocols with the SEC + affinity chromatography protocol that
appeared to be less indicated for the case in which an excess of
conjugating glycopolymer was employed due to the rather
tedious SEC purification of the excess of unreacted glycopoly-
mer from the polymer-protein conjugate 10.
the length of the polydentate ligand counterpart21 as well as
the density of the binding epitopes present in the neoglyco-
polymer.26,28 This makes the development of efficient and
reliable strategies for the synthesis of polyvalent ligands bearing
multiple binding epitopes of particular importance.
Once the necessary synthetic pathways and purification
processes for the glycoconjugates were developed, a library of
conjugating neoglycopolymers differing from each other for the
relative density of mannose epitopes was prepared following a
coclicking approach28 (Scheme 4) in order to further demonstrate
the efficiency of the strategy presented in this work. As reported
previously, libraries of glycopolymers identical in terms of
macromolecular features (Mn, PDi, macromolecular architecture)
can be obtain by simply introducing appropriate mixtures of
different sugar azides in the reaction feed. BSA glycoconjugates
23-27 were then prepared using a 10:1 BSA to glycopolymer
molar ratio and purified by anion-exchange chromatography.
Circular dichroism (CD) analysis of the conjugates 23-27
was performed in order to evaluate the influence of the
conjugated glycopolymer onto the secondary structure of BSA.
R-Helix and â-sheet contents were found to be 51-65% and
0-2%, respectively, values that do not significantly differ from
54% R-helix and 1% â-sheet that was found for native BSA
under identical conditions.
Glycoprotein Mimics with Different Mannose Binding
Unit Density. Recognition of lectin receptors is dependent on
(81) Ellman, G. L. Arch. Biochem. Biophys. 1959, 82 (1), 70-77.
(82) Grassetti, D. R.; Murray, J. F., Jr. Arch. Biochem. Biophys. 1967, 119,
41-49.
(83) Riener, C. K.; Kada, G.; Gruber, H. J. Anal. Bioanal. Chem. 2002, 373
(4-5), 266-276.
Pseudo-esterase activity of the protein part of our conjugate
was assessed using p-nitrophenol acetate as model ester substrate
9
J. AM. CHEM. SOC. VOL. 129, NO. 49, 2007 15161

A R T I C L E S
Scheme 4 a
Geng et al.
a Reagents and conditions: (a) R-2′-azidoethyl-D-mannopyranoside 1/â-2′-azidoethyl-D-galactopyranoside 28, (PPh3)3CuBr, Et3N, DMSO, ambient
temperature, 2 days; (b) reduced pressure, 80 °C, solid state, 12 h; (c) BSA (10 equiv), DMSO:PBS (50 mM, pH 7.0).
Scheme 5 a
a Reagents and conditions: (a) toluene reflux, 18 h; (b) BSA (10 equiv) DMSO:PBS (50 mM, pH 7.0).
in 50 mM PBS, pH 8.5. Since this assay relies on the
spectophotometric detection (λ ) 405 nm) of the p-nitrophe-
nolate ion produced by hydrolysis of the ester substrate, our
rhodamine B-tagged conjugates appeared to be unsuitable for
such a test due to their non-negligible absorbance at 405 nm.
A non-rhodamine-tagged biohybrid material, 31, was therefore
prepared by direct polymerization of the mannose methacrylate
monomer 2 in methanol/water 5:2 v/v at ambient temperature
in the presence of the initiator 8 (Scheme 5). Retro-Diels-Alder
removal of the furan protecting group followed by protein
conjugation using an excess of BSA afforded the desired
conjugate 31.
Pseudo-esterase assay84-86 revealed that 31 had an activity
comparable with that of the BSA starting material (see Sup-
porting Information), which further confirmed that the experi-
mental conditions employed for both conjugation and purifi-
cation of our biohybrid materials did not damage the protein
part of the conjugates.
Conjugates and unmodified BSA were immobilized in equal
quantities on parallel flow cells within a CM5 sensor chip and
sensorgrams recorded with MBL in the fluid-phase at concen-
trations indicated in Figure 4. Correction for background binding
was achieved through subtraction of parallel sensorgram read-
ings from hemeoglobin-coated flow cells. Clear and dose-
dependent MBL binding to glycopolymer-conjugated BSA was
observed within the physiological range, which is typically 20
nM, in stark contrast to plain BSA where no signal was observed
(data not shown). Although MBL exists in vitro and in vivo as
a heterogeneous collection of oligomers, average kinetic values
were calculated for MBL binding to immobilized polymer giving
a kon rate of 1.2 ( 0.45 × 105 M-1 s-1 and a koff rate of 7.3 (
2.3 × 10-4 s-1 with a KD of 7.9 ( 4.5 × 10-9. Binding of
fluid-phase glycopolymer-conjugated BSA to immobilized MBL
was positive but gave lower absolute signal and showed different
curve characteristics with a substantially faster dissociation curve
(Figure 4b). This indicates the likely effects of multiple protein-
sugar contacts required to increase avidity, but we acknowledge
that immobilization of MBL on the CM5 surface may substan-
tially effect its binding properties. Binding was not observed
in either case when D-mannose was introduced into the analyte
at 10 mM (data not shown).
Binding studies using surface plasmon resonance (BIAcore)
were carried out to examine glycopolymer-BSA conjugate
interactions with recombinant rat mannose-binding lectin (MBL).
(84) Tildon, J. T.; Ogilvie, J. W. J. Biol. Chem. 1972, 247 (4), 1265-1271.
(85) Boyer, C.; Bulmus, V.; Liu, J.; Davis Thomas, P.; Stenzel Martina, H.;
Barner-Kowollik, C. J. Am. Chem. Soc. 2007, 129 (22), 7145-7154.
(86) Liu, J.; Bulmus, V.; Herlambang David, L.; Barner-Kowollik, C.; Stenzel
Martina, H.; Davis Thomas, P. Angew. Chem., Int. Ed. 2007, 46 (17), 3099-
103.
Functional analysis of the glycopolymer-BSA conjugates
was performed using a protocol designed to measure comple-
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Site-Directed Conjugation of “Clicked” Glycopolymers
A R T I C L E S
as source of specific aminoacid sequences, with lectin binding
properties, with consequent engagement with the counter-
glycome in plasma, cells, and tissues, typical of its neoglyco-
polymer component. The versatility of the approach was further
demonstrated by preparing a library of BSA-neoglycopolymer
hybrid materials featuring different relative amounts of D-
mannose and D-galactose binding epitopes following a coclick-
ing approach.
Surface plasmon resonance (BIAcore) tests carried out in the
presence of the model mammalian lectin, recombinant rat
mannose-binding lectin (MBL), revealed a significant and dose-
dependent binding of the latter to the BSA-neoglycopolymer
conjugates. ELISA tests also revealed that our biohybrid
materials were also able to activate the complement system via
the lectin pathway.
Figure 5. Detection of complement system activation. BSA-conjugate
10, BSA, and yeast mannan were immobilized on microwell plates and
exposed to diluted human serum. Values indicate the level of activated
complement C9 deposition resulting from positive cascade triggering, as
determined by a subsequent ELISA-style assay involving p-nitrophenol
detection at λ ) 405 nm. ELISA units obtained as direct absorbance at 405
nm values with reagent blank values deducted
In summary, through a simple and efficient conjugation step,
a non-glycosylated model protein, BSA, has been site-specifi-
cally modified with a mannoside neoglycopolymer leading to
a substantial addition of function with regard to engagement of
a critical arm of mammalian innate immunity. This indicates
the principle of how proteins can be effectively modified with
neoglycopolymers in a highly defined fashion, providing a
gateway for rapid, diverse, and cost-effective neoglycoprotein
developmentsa phenomenon that could have profound value
in the deconvolution and exploitation of the functional glycome
in a broad range of biological species.
ment system activation. Compared with BSA, immobilized
glycopolymer-BSA conjugate showed significantly enhanced
capacity to activate complement via the lectin pathway as
assessed by quantitative measurement of pathway-dependent
complement protein deposition by enzyme-linked immunosor-
bent assay (Student’s t test, P < 2 × 10-6; Figure 5). Thus,
incorporation of the glycopolymer-modified BSA imparts the
inert protein with innate immune system interaction properties.
Conclusions
Acknowledgment. The authors thank the University of
Warwick (J.G. and L.T.) for funding. D.A.M. is a Research
Councils UK Academic Fellow with additional support from
the Warwick Research Development Fund. R.W. is a Research
Councils Academic Fellow and supported by grant 077400 from
the Wellcome Trust. Dr. Gianfranco de Pascale and Rajan
Kumar Randev are also thanked for their help in the FPLC
purification of the conjugates and providing an analytically pure
sample of p-nitrophenol acetate.
Glycoproteins are potent biological molecules active through
the union of structural and chemical properties imparted by both
specific protein and carbohydrate components. Neoglycopoly-
mers represent very attractive molecules through which defined
macrocarbohydrate properties can be generated rapidly and with
high yield, providing a realistic alternative in many cases to
scarce and prohibitively expensive natural oligosaccharides.
In this work we introduced a simple and general method for
the synthesis of glycoprotein mimics. Well-defined maleimide-
terminated neoglicopolymers (Mn ) 8-30 kDa; Mw/Mn ) 1.20-
1.28) having multiple copies of D-mannose epitopes have been
obtained following two independent synthetic pathways that
involve a combination of copper-catalyzed living radical po-
lymerization and alkyne-azide [2+3] cycloaddition (“click”).
These coupled with model thiol-containing protein BSA and
the resulting bioconjugates were first purified following two
independent purification protocols and then characterized by
SEC HPLC, SDS PAGE, and CD analysis. These biohybrid
materials appear to be particularly intriguing as they could
potentially combine features proper of their protein component,
such as enzymatic activity, ligand affinity, or the ability of acting
Note Added after ASAP Publication: There was a produc-
tion error in the version published on the Internet on November
17, 2007. In the print version and in the version published on
the Internet on November 19, 2007, the correct version of
Scheme 4 is present.
Supporting Information Available: Experimental section,
including characterization of maleimide polymers and all
intermediates, bioconjugation experiments, and in vitro test
procedures. This material is available free of charge via the
Internet at http://pubs.acs.org.
JA072999X
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