Virus-glycopolymer conjugates by copper(I) catalysis of atom transfer radical polymerization and azide-alkyne cycloadditiont

Article abstract of DOI:10.1039/b502444g

The Cu^I-catalyzed ATRP and azide-alkyne cycloaddition reactions together provide a versatile method for the synthesis of end-functionalized glycopolymers and their attachment to a suitably modified viral protein scaffold. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b502444g

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Virus–glycopolymer conjugates by copper(I) catalysis of atom transfer
radical polymerization and azide–alkyne cycloaddition{
Sayam Sen Gupta,{ Krishnaswami S. Raja,{ Eiton Kaltgrad, Erica Strable and M. G. Finn*
Received (in Cambridge, MA, USA) 16th February 2005, Accepted 25th May 2005
First published as an Advance Article on the web 19th July 2005
DOI: 10.1039/b502444g
The CuI-catalyzed ATRP and azide–alkyne cycloaddition
comprised a broad exploration of virus particles as chemical
building blocks, focused on cowpea mosaic virus (CPMV) as a
prototype. This plant virus can be produced and purified in large
quantities, is structurally characterized to near-atomic resolution, is
stable to a variety of conditions compatible with both hydrophobic
and hydrophilic molecules, and can be manipulated at the genetic
level to introduce mutations at desired positions. One of our goals
is to bring new functions to virus particles by attaching functional
molecules to the capsid protein, thereby generating novel
species with diagnostic and therapeutic applications. Attachment
of single carbohydrate compounds to CPMV residues produces a
dendrimer-like display with polyvalent lectin-binding properties.15
We have also derivatized CPMV with PEG to give well-controlled
loadings of polymer on the outer surface of the coat protein
assembly.16 The resulting conjugates displayed altered physical
properties and reduced immunogenicities, consistent with previous
reports of PEGylated adenovirus vectors.17
reactions together provide a versatile method for the synthesis
of end-functionalized glycopolymers and their attachment to a
suitably modified viral protein scaffold.
The polyvalent clustering of carbohydrate derivatives based on
linear polymers1 and dendrimers2 has proven to be an effective
tool in the study of carbohydrate-based cellular processes and is a
useful strategy in the development of therapeutic agents.3 Dense
clusters of carbohydrates can be formed by arraying an end-
functionalized glycopolymer to a biocompatible scaffold such as a
protein. Such polymers have been recently prepared by cyanoxyl-
mediated free radical polymerization (employing initiators bearing
amine, carboxylic acid, hydrazide, or biotin moieties, with
subsequent protein attachment by biotin–avidin binding4) and
atom transfer radical polymerization (ATRP; side-chain poly-
(ethylene glycol) (PEG) or poly(2-hydroxyethyl methacrylate)
polymers containing N-hydroxysuccinimide or pyridyl disulfide
end groups, with protein attachment to lysozyme and bovine
serum albumin).5
The strength and selectivity of binding interactions between
polyvalently displayed carbohydrates and target cells are likely to
depend on the number and flexibility of the arrayed sugars. We
desired to explore one extreme by covering a virion as densely as
possible with carbohydrate groups. Increasing the degree of virus
coverage requires the reactive polymer end group to be compatible
with polymer synthesis and/or elaboration and yet reactive enough
to accomplish a demanding subsequent connection to the virus
coat protein—a union of two large molecules present in low
concentrations.
This worthy strategy can be further augmented by the use of the
most active and selective bioconjugation reactions. Organic azides
have achieved wide application due to their inert nature towards
biological molecules and their participation in the Staudinger
ligation with phosphine-esters6 and the 1,3-dipolar cycloaddition
reactions with alkynes.7 We have found the latter process to be
extraordinarily fast and versatile in demanding bioconjugation
applications under dilute conditions.7a,8 It has also been used in a
wide variety of other applications, including the creation of small
dendrimer-style polyvalent carbohydrate assemblies.9 Since Cu(I)
complexes catalyze both the ATRP10 and azide–alkyne cycloaddi-
tion (AAC)11 reactions, their combination is logical. We describe
here the construction of azide-terminated glycopolymers by
ATRP, their end-labeling with fluorophores, and the subsequent
conjugation of these compounds to virus particles in high yield for
purposes of polyvalent binding to cell-surface lectins.
The side-chain neoglycopolymer 3 was prepared by ATRP of
methacryloxyethyl glucoside (2) using azide-containing initiator 1
(Scheme 1).18 The presence of the azide chain end in the polymer
was confirmed by colorimetric testing19 and by the presence of the
characteristic peak at 2100 cm21 in the infrared spectrum. GPC
analysis established the clean nature of the material and an average
molecular weight (Mn) of 13 000 with a polydispersity of 1.3,
consistent with the initiator : monomer ratio used and within
expectations for ATRP of acrylates in water.18b,20,21
Viruses are intriguing scaffolds for the polyvalent presentation
of functional structures. Chemistry-based studies have included the
organization of inorganic materials in or around virus cages,12 the
organization of viruses on surfaces,13 and the chemical conjugation
of organic compounds to virus coat proteins.14 Our work has
Azide-terminated polymer 3 was elaborated to the alkyne-
terminated form 5 by reaction with fluorescein dialkyne 4
(Scheme 1). The excess dye was removed by filtration and the
polymer products were further purified by size-exclusion chroma-
tography (Sephadex G-15). The complete conversion of the azide
to the alkyne end group was confirmed by the observation of a
negative colorimetric test and by the disappearance of the azide IR
resonance (the corresponding alkyne resonance is much less
intense and therefore not visible). The chromophore thus installed
serves as a spectroscopic reporter for subsequent manipulations.
The dimeric polymer, formed as a minor byproduct from the
Department of Chemistry and The Skaggs Institute for Chemical
Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La
Jolla, CA, USA. E-mail: mgfinn@scripps.edu; Fax: 01-858-784-8850;
Tel: 01-858-784-8845
{ Electronic supplementary information (ESI) available: detailed synthetic
procedures and analytical data. See http://dx.doi.org/10.1039/b502444g
{ These authors contributed equally to this work.
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Chem. Commun., 2005, 4315–4317 | 4315

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Fig. 1 (A) Size-exclusion FPLC (Superose 6) of wild-type CPMV and
glycopolymer conjugate 9. Protein from disassembled particles would
appear at longer retention times than the peaks observed here, and the
A260 : A280 ratios are characteristic of intact, RNA-containing capsids for
Scheme 1 Synthesis of glycopolymers and virus–polymer conjugates.
both samples. The more rapid elution of 9 is indicative of a substantial
increase in the size of the particle, as 10 mL is the void volume of the
column. Dye absorbance at 495 nm appears only for 9. (B) FPLC on
concanavalin-A Sepharose column of wild-type CPMV and virus–polymer
conjugate 9. The elution buffer was the indicated gradient mixture of
20 mM Tris-HCl, pH 7.4, with 0.15 M NaCl, 0.1 mM Ca2+, and
0.1 mM Mn2+ (solution A) and 1 M glucose (solution B). (C) SDS-PAGE
of 9 (lane 1) and WT-CPMV (lane 2). On the right (light background) is
the gel visualized after Coumassie blue staining; note that almost all of the
protein is converted to a slower-eluting form, expected for protein–
glycopolymer conjugation. On the left (dark background) is the gel
illuminated by ultraviolet light before staining (lane 2 shows no emission
and is omitted). The arrows mark the center of the bands derived from the
small (S) and large (L) subunits; their broad nature derives from the
polydispersity of the polymer and the possibility for more than one
attachment of polymer per protein subunit. (D) (Left) Negative-stained
TEM of 9. (Right) Enlarged TEM image of a WT-CPMV particle
surrounded by 9.
reaction of two molecules of 3 and one of 4, was not separated
from 5 as it cannot participate in bioconjugation.
CPMV was derivatized with N-hydroxysuccinimide 6 to install
azide groups at lysine side chains of the coat protein (Scheme 1).
NHS esters have been previously established to acylate lysine
residues over the external surface of the capsid, with loadings
controlled by overall concentration, pH, and reaction time.14b In
this case, conditions were employed which result in the derivatiza-
tion of a substantial fraction of the approximately 240 solvent-
accessible lysine side chains (m 5 approximately 150 in Scheme 1).
The resulting azide-labeled virus (7) was then condensed with
20 equivalents of polymer–alkyne 5 in the presence of copper(I)
triflate and sulfonated bathophenanthroline ligand 88a under inert
atmosphere to produce the glycopolymer–virus conjugate 9 in
excellent yield after purification by sucrose-gradient sedimentation
to remove unattached polymer. By virtue of the calibrated dye
absorbance, the number of covalently bound polymer chains was
found to be 125 ¡ 12 per particle, representing the addition of
approximately 1.6 million daltons of mass to the 5.6 million Da
virion. This procedure, the general application of which will
be described elsewhere, is far more efficient than the previous
CuI-mediated method,7a which required 100 equivalents of 5 with
respect to azide to achieve similar results.
The glycosylated particles interacted strongly with both an
immobilized form of the glucose-binding protein concanavalin A
(Fig. 1B) and with tetrameric conA in solution. The latter process
resulted in the formation of large aggregates, the rate of which was
monitored by light scattering at 490 nm. At a concentration of
0.7 mg mL21 in 9 (approximately 0.1 mM in virions) and
0.3 mg mL21 in conA, aggregation occurred within seconds (see
Supporting Information{), as expected for the efficient formation
of a network by a large and polyvalent particle.
Covalent labeling of the vast majority of CPMV protein
subunits with glycopolymer was confirmed by denaturing gel
electrophoresis (Fig. 1C). The intact nature of the particle assembly
and its larger size was verified by size-exclusion fast protein liquid
chromatography (FPLC, Fig. 1A) as well as transmission electron
microscopy (TEM, Fig. 1D). TEM images revealed the virus
conjugates to be more rounded in shape, to take on uranyl acetate
stain differently, and to be 12–15% larger in diameter than the
wild-type particle. The hydrodynamic radius and molecular
In conclusion, we have demonstrated an efficient strategy for
end-functionalization of glycopolymers, using an azide-containing
initiator for a living polymerization process followed by click
chemistry elaboration of the unique azide end group. Azide–
alkyne cycloaddition with a chromophoric dialkyne served to label
the polymer with a single dye molecule, allowing for convenient
monitoring of further manipulations. The copper-catalyzed
cycloaddition reaction provides very efficient coupling of such
polymers to a functionalized viral coat protein. In our hands, this
method outperforms bioconjugation procedures previously used
for polymer attachment to proteins such as acylation of lysine
amine groups by activated esters and reaction of cysteine thiols
with 2-thiopyridyl disulfides.22 To the best of our knowledge, this
weight of
9 were found by multi-angle dynamic light
scattering (DLS) to be dramatically larger as well: 30.3 ¡ 3.4 nm
and 1.4 ¡ 0.4 6 107 Da, compared to 13.4 ¡ 1.3 nm and 6.1 ¡
0.3 6 106 Da for wild-type CPMV. That both radius and
molecular weight values are substantially greater than expected
reflects the uncertainties of calibration and interpretation of light
scattering data for these unique polymer–virus hybrid species.
4316 | Chem. Commun., 2005, 4315–4317
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We have found particles such as 9 to have extraordinarily high
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We acknowledge The David and Lucille Packard Foundation
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Institutes of Health (EB00432), and The Skaggs Institute
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Chem. Commun., 2005, 4315–4317 | 4317