Glycan-Directed Grafting-from Polymerization of Immunoglobulin G: Site-Selectively Modified IgG-Polymer Conjugates with Preserved Biological Activity

Article abstract of DOI:10.1021/acs.biomac.8b00669

Antibody and related antibody drugs for the treatment of malignancies have led to progress in targeted cancer therapy. Preparation of diverse antibody conjugates is critical for preclinical and clinical applications. However, precise control in tagging molecules at specific locations on antibodies is essential to preserve their native function. In this study, a synthetic boronic acid (BA)-tosyl initiator was used to trigger a glycan-directed modification of IgGs, and the obtained IgG macroinitiators allowed a growth of the poly N-isopropylacrylamide (PNIPAAm) chains specifically at Fc-domains. Therefore, the PNIPAAm chains are located away from the critical antigen-binding domains (Fab), which could reasonably prevent the loss of biological activity after the attachment of polymer chains. According to the proposed strategy, a site-selectively modified anticoncanavalin A (Con A) antibody-PNIPAAm conjugate showed 6-times higher efficiency in the binding of targeted Con A antigen to a randomly conjugated anti-Con A antibody-PNIPAAm conjugate. In this study, we developed the first chemical strategy for the site-specific preparation of IgG-polymer conjugates with conserved biological activity as well as intact glycan structures.

Full text of DOI:10.1021/acs.biomac.8b00669

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Article
Glycan Directed Grafting-from Polymerization of
Immunoglobulin G: Site-selectively Modified IgG-
polymer Conjugates with Preserved Biological Activity
Chih-Hung Chou, and Po-Chiao Lin
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00669 • Publication Date (Web): 11 Jun 2018
Downloaded from http://pubs.acs.org on June 12, 2018
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Glycan Directed Grafting-from Polymerization of
Immunoglobulin G: Site-selectively Modified
IgG-polymer Conjugates with Preserved
Biological Activity
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Chih-Hung Chou, and Po-Chiao Lin*
Department of Chemistry, National Sun Yatꢀsen University 70, Lienhai Road, Kaohsiung
80424, Taiwan
E mail: pclin@mail.nsysu.edu.tw
KEYWORDS: Proteinꢀpolymer, ATRP, AntibodyꢀPNIPAAm conjugates
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ABSTRACT
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Antibody and related antibody drugs for the treatment of malignancies have led to the progress
of targeted cancer therapy. Preparation of diverse antibody conjugates is critical for preclinical
and clinical applications. However, precise control in tagging molecules at specific locations on
antibodies is essential to preserve their native function. In this study, a synthetic boronic acid
(BA)ꢀtosyl initiator was used to trigger a glycanꢀdirected modification of IgGs and the obtained
IgG macroinitiators allowed a growth of the poly Nꢀisopropylacrylamide (PNIPAAm) chains
specifically at Fcꢀdomains. . Therefore, the PNIPAAm chains are located away from the critical
antigenꢀbinding domains (Fab) which could reasonably prevent the loss of biological activity
after the attachment of polymer chains. According to the proposed strategy, a siteꢀselectively
modified antiꢀconcanavalin A (Con A) antibodyꢀPNIPAAm conjugates showed six times higher
efficiency in the binding of targeted Con A antigen to a randomly conjugated antiꢀCon A
antibodyꢀPNIPAAm conjugates. In this study, we developed the first chemical strategy for the
siteꢀspecific preparation of IgGꢀpolymer conjugates with conserved biological activity as well as
intact glycan structures.
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INTRODUCTION
In the postꢀgenome era, understanding the function, modification, and regulation of
proteins remains an important but daunting task.1 Numerous proteins, such as enzymes, effectors,
and antibody (Ab), are known to regulate important biomolecular interactions and have potential
as powerful treatments in many diseases. In recent decades, proteinꢀpolymer conjugates have
been successfully applied in diverse biomedical applications, specifically for improving
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pharmacokinetic properties, affinity separation, as microfluidic devices, and as switches in
enzymatic reactions.2ꢀ6 For instance, the PEGylation of therapeutic proteins, especially Abs, has
been demonstrated to significantly improve both physical and thermal stability as well as reduce
immunogenicity. For these reasons, PEGylated proteins have been approved by the U.S. Food
and Drug Administration (FDA) for therapeutic use in diabetes, cancer, and hepatitis C.7ꢀ8
However, nonꢀselective polymer conjugation results in multiple polymer attachments at random
grafting sites. The heterogeneous mixtures of polymerized positional isomers could considerably
reduce their native bioactivity.9
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Recent advances in siteꢀselective proteinꢀpolymer ligation have rapidly grown to attain
homogeneity of proteinꢀpolymer conjugates.10ꢀ12
Two methods, graftingꢀto and graftingꢀfrom, depending on polymerization occurring before or
after the protein conjugation are major synthetic routes for the preparation of homogenous
proteinꢀpolymer conjugates. In the concept of graftingꢀto, the pendant polymers attach to the
proteins of interest via specific chemical reactions. With the high reactivity of the sulfhydryl
group and relatively sparse occurrence of cysteine residues in proteins, cysteine modification
provides a useful method in chemoselective protein modification. An incorporation of a cysteine
residue to maleimide or divinyl sulfoneꢀconjugated polymers via Michael addition showed a
convenient way in the preparation of proteinꢀpolymer conjugates.13ꢀ16 Using a pyridine disulfideꢀ
conjugated polymerization initiator, the Maynard group has successfully developed a cysteineꢀ
specific strategy in the preparation of BSA and T4 lysozymeꢀpolymer conjugates with conserved
bioactivity.17 Moreover, an activityꢀdirected graftingꢀto synthesis of proteinꢀpolymer conjugates
was developed using biotinꢀended poly(Nꢀisopropylacrylamide) to nonꢀcovalently associate with
streptavidin.18 The graftingꢀto method have also contributed in the homodimeric19 and
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heterodimeric20 proteinꢀpolymer conjugations. The prefabricated polymers can be synthesized in
harsh or bioincompatible conditions, and are easier to be characterized. However, the steric
hinderance and aqueous solubility of polymers can lead to low conjugation efficiency.
Subsequently, the difficulty for separating proteinꢀpolymer conjugates from unreacted synthetic
polymers can considerably limit its practical uses. In contrast, graftingꢀfrom method directly
elongates polymer chains from proteins can prevent aboveꢀmentioned drawbacks, but a well
characterization of attached polymers could be very challenging. To a similar concept, using a
pyridine disulfide functionalized initiator reacted with targeted T4 lysozyme and the formed
macroinitiator can then trigger the growth of polymer chains. The obtained T4 lysozymeꢀ
polymer conjugates showed no statistical differences in bioactivity.21 Similarly, Davis and coꢀ
workers used a pyridine disulfideꢀconjugated chainꢀtransfer agent (CTA) to prepare BSAꢀ
polymer conjugates via reversible addition fragmentation transfer polymerization.22 Moreover,
the sulfhydryl group of cysteine can proceed via Michael addition to prepare the macroinitiator
and macroꢀCTA, leading to direct polymer growth from the surface of the protein.23ꢀ24 A siteꢀ
specific (Cꢀterminal) preparation of green fluorescence protein (GFP)ꢀPEG conjugates by inteinꢀ
mediated protein ligation provides a general methodology for improving pharmacokinetic
profiles.25 Besides those chemoreactivity directed conjugation methods, affinityꢀdirected protein
macroinitiator preparation provided an alternative to achive siteꢀselective conjugation.
Biotinylated initiator was demonstrated to specifically bind streptavidin, forming the streptavidin
initiator, and subsequently triggering polymer chain growth to finally produce streptavidinꢀ
polymer conjugates.26 Although the active sites were blocked and could be at risk of dissociation,
no need of genetic engineering techniques allows this strategy to target endogenous proteins and
forms homogeneous proteinꢀpolymer conjugates.
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In this research, we aimed to propose an affinityꢀdirected strategy in the preparation of Abꢀ
polymer conjugates which can conserve the Ab’s native structure as well as bioactivity.
Immunoglobulin G (IgG) is the most abundant class of antibodies, constituting about 80 % of the
total serum immunoglobulin, and dominating the recognition, neutralization, and elimination of
foreign antigens and pathogens. IgGꢀbased drugs are believed to be the most rapidly expanding
class of pharmaceuticals for treating a variety of human diseases, especially cancers.27ꢀ28 The IgG
molecule consists of two identical heavy chains and two light chains which is endowed with a
variable region, comprising the antigen recognition site (Fab domain, pink part of IgG in Figure
1) and a constant region (Fc domain, cyan part of IgG in Figure 1) which bind to effector
molecules. The Fc domain, composed of two paired Ig domains, mediates the interaction of Ab
molecules and Fcγ receptors, and thereby facilitates the ingestion and destruction of potentially
harmful antigens recognized by IgG. Two oligosaccharide chains attached at Asn297 of the Fc
domain were found to be closely related to antibody’s function. Difference in glycosylation
might considerably affect antibody function in the studies of the remission of rheumatoid
arthritis,29 and inflammatory response.30ꢀ31 The integrity of Fab domains and of Fc domain with
glycan chains is crucial for the complete activity of IgG Ab. Therefore, a good control of
polymer conjugation sites would allow for the production of homogeneous, modified, but
completely functional Ab conjugates.
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Conventionally, the preparation of Abꢀpolymer conjugates is conducted by random conjugation
at reactive amino acid residues, including amino (lysine and histidine), thiol (cysteine), or
hydroxyl (serine, threonine, and tyrosine) groups. The formation of subsequent peptide bonds,
disulfides, and carboxylic esters is highly efficient; however, conjugation at multiple and random
sites is known to considerably reduce the affinity of binding to target proteins. When conjugation
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sites are located very close to antigenꢀbinding domains, the biological function of the Ab is
reduced due to steric hindrance. Further, the conventional uncontrolled conjugation strategy may
have considerable batchꢀtoꢀbatch variation in the preparation of Abꢀpolymer conjugates, thereby
resulting in inconsistent and heterogeneous products. To reduce the loss of Ab function, siteꢀ
selective modifications of Abs are usually targeted at the Fc domain, which is distant from the
antigen binding sites. Methods involving the specific reduction of interchain disulfides, followed
by alkylation, are popularly used for siteꢀspecific Ab modification.32ꢀ35 However, native disulfide
bonds of a protein are usually crucial to its structure and function, and cannot, therefore, be
easily modified.36ꢀ37 To avoid altering the native structure of Ab, recombinant techniques provide
an ideal tool for the production of Abs fused with a protein, such as green fluorescent protein.38
Schultz and coꢀworkers genetically incorporated unnatural amino acids (UAAs) into Abs,
placing ketone functional groups on their residues. The ketone groups can then selectively react
with aminooxyꢀderived ligands to achieve siteꢀspecific Ab modification.39 Moreover, enzymatic
reactions have also been used to introduce molecules of interest at a specific position in Abs. For
instance, an acylꢀtransfer reaction between the γꢀcarboxamide group of glutamine and a primary
εꢀamino group, mediated by transglutaminase, has been successfully used in siteꢀspecific and
stoichiometric modification of Abs.40 Francis and coꢀworkers developed a pyridoxal 5’ꢀ
phosphateꢀcatalyzed biomimetic transamination reaction that introduces a ketone group on the
Nꢀtermini of monoclonal antiꢀFLAG IgG.41
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Alternatively, carbohydrate residues of Ab Fc domains offer a unique chemical reactivity for
Ab siteꢀspecific modification. The diol groupꢀrich carbohydrates allow specific oxidative
cleavage by periodate; resultant aldehyde groups can then be used as sites for attachment of
amino or hydrazine derivatives to the Ab.42ꢀ48 Recently, Lin and coꢀworkers proposed a glycanꢀ
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mediated approach combined with photoꢀcrosslinking to conduct a siteꢀspecific Ab
immobilization.49
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All above mentioned methods can specifically introduce tag molecules at the Fc domains
of Abs of interest. However, the necessity of genetic engineering limited their use in native Abꢀ
polymer conjugate synthesis. Although glycanꢀdirected modification can prevent the loss of
antigen binding ability, the glycan chain function could be considerably affected. To overcome
this limitation, a Fcꢀspecific strategy conserving its glycan structures is critical to the
preparation of diverse Abꢀpolymer conjugates.50 To explore the general concept of activityꢀ
directed synthesis of proteinꢀpolymer conjugates, in this research, ligandꢀdirected tosyl
chemistry51 was applied in the study of Abꢀpolymer conjugates. Very recently, we successfully
applied this traceless labeling strategy to glycoproteins of interest with synthetic boronic acid
(BA)ꢀtosyl chemical probes.52 According to the well established synthetic method,53 a new
synthetic probes is proposed to selectively introduce the initiators at the Fc domain of IgG as the
macroinitiator and subsequently used in the preparation of Abꢀpolymer conjugates. Probe
molecules were composed of three general elements: an affinity ligand as a targeting head, a
reactive benzenesulfonyl group located behind the head part as an ideal leaving group, and a
terminal initiator for the growth of polymer chains. The general principle of this strategy is
illustrated in Figure 1. The affinity ligand, BA, specifically binds to glycosylated IgG, triggers
specific binding to the glycan chain, and thereby draws the probe molecule very close to the
molecular surface of the Fc domain in IgG (Figure 1, step a). The formation of the boronate ring
induces a proximity effect, facilitating an SN2 substitution reaction with a nucleophilic residue on
the labeled IgG, and subsequently releasing the benzenesulfonyl part (Figure 1, step b).
Concurrently, the initiator is covalently transferred onto the surface of the target protein to be the
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desired protein macroinitiator for further polymer chain growth. Notably, the boronateꢀblocked
glycans can be treated with polyol molecules, such as glycerol or sorbitol, to render native
glycans with the release of BAꢀbenzenesulfonate.
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Figure 1. General principal of BAꢀtosyl initiator directed Fcꢀselective polymerization of
antibodies. (a) IgG with treatment of BAꢀtosyl initiator, (b) covalent transfer of initiator, (c) the
release of BAꢀbenzenesulfonate and subsequent grafting from polymerization.
In this research, we proposed an intrinsic natureꢀdirected traceless synthesis of proteinꢀpolymer
conjugates in a siteꢀselective manner. A model using biotin and streptavidin was carefully
studied to validate the labeling process with conserved biological activity. Then, human IgG
were used to prepare corresponding Abꢀpolymer conjugates followed by careful characterization
of the conjugates. Very importantly, a comparison of siteꢀspecific antiꢀCon A antibodyꢀpolymers
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with randomly conjugated antiꢀCon A antibodyꢀpolymers in the Con Aꢀbinding assay is
discussed.
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EXPERIMENTAL SECTION
General methods and materials
All chemicals were purchased from SigmaꢀAldrich and Acros. AntiꢀCon A antibody was
purchased from Vector laboratories (CA, USA). The monomeric acrylamide/bisacrylamide
solution (40%, 29:1) was purchased from BioꢀRad. The BCA protein assay reagent kit was
obtained from Pierce. Sodium dodecyl sulfate (SDS) was purchased from GE Healthcare. IgG
from human serum (reagent grade, >95%) was purchased from SigmaꢀAldrich. Monoclonal antiꢀ
biotin antibody (peroxidase conjugated) for western blotting was purchased from SigmaꢀAldrich.
Water was obtained using a Barnstead™ Easypure™ RoDi Water Purification System (Thermo
Scientific, US).
Biotin-tosyl initiator directed synthesis of streptavidin synthesis.
Streptavidin was dissolved in 10% dimethyl sulfoxide (DMSO)(666 ꢁL, 10ꢀ5 M) aqueous
solution. Then, 13 ꢁL of a biotinꢀtosyl initiator stock solution (5.0 mg of biotinꢀtosyl initiator in
680 ꢁL of dimethyl sulfoxide, 10ꢀ3 M) was added to the streptavidin solution. After incubating at
37°C for 24 hours, excess biotinꢀtosyl initiators were removed by column chromatography
(Sephadex® Gꢀ25). The collected streptavidin macroinitiators were then concentrated by
centrifugal filters (Amicon® Ultra Centrifugal Filters，Merck Millipore, Germany) to obtain the
purified streptavidin macroinitiator.
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Atom transfer radical polymerization from the streptavidin macroinitiator.
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The obtained streptavidin macroinitiator (50 ꢁg, 9.4 mmol) and NIPAAm (5.7 mg, 50 ꢁmol)
were dissolved in 48 ꢁL of degassed ddH2O in an argon atmosphere. An oxygenꢀfree catalyst
solution (11 ꢁL, obtained by dissolving 1.0 mg of CuBr and 4.18 mg of 2,2’ꢀbipyridine in 78 ꢁL
degassed water) was added to the reaction mixture and initiated the polymerization. The reaction
was monitored by reverse phase HPLC with the consumption of monomers.(Figure S5). After
1.5 h, the monomer showed nonꢀdistinguishable change and the reaction was then stopped by
deactivating Cu (I) catalyst in open air. The formed streptavidinꢀPNIPAAm conjugates were
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heated to 45 C, it will be precipitated and separated from reaction mixtures by centrifugation.
Then, the precipitate was redissolved in chilled ddH2O followed by a heating precipitation
process to remove the reactants and catalysts. The washing step was repeated three times to
purify streptavidinꢀPNIPAAm conjugates.
Competition of streptavidin-PNIPAAm conjugates by amino biotin.
Streptavidin macroinitiator (300 ꢁg, 5.66 mmol) was prepared by treating streptavidin with
biotinꢀtosyl initiator 1 and succinidimyl biotin were respectively dissolved in 20 ꢁL deionized
distilled water. Then, 50 ꢁL of an amino biotin stock solution (100 mM in ddH2O) was added to
the two streptavidin macroinitiator solutions. After incubating at room temperature for 2 hours,
excess amino biotins were separated by column chromatography (Sephadex® Gꢀ25). The
collected streptavidin macroinitiators were then concentrated by centrifugal filters to obtain the
purified macroinitiators. These two obtained streptavidin macroinitiators were then subjected to
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polymerization conditions (90 mM CuBr, 180 mM 2,2ꢀbipyridine and 50 ꢁmole NIPAAm) The
streptavidinꢀPPNIPAAm conjugates were then analyzed by SDSꢀPAGE.
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BA-tosyl initiator directed labeling of anti-Con A antibody.
AntiꢀCon A antibody was dissolved in PBS (pH 8.0, 0.1M) buffer containing 10% dimethyl
sulfoxide (DMSO)(400 ꢁL, 5x10ꢀ6 M). Then, 20 ꢁL of a BAꢀtosyl initiator stock solution (1.0
mg of BAꢀtosyl initiator in 8.3 mL of DMSO, 2x10ꢀ4 M) was added to antiꢀCon A Ab solution.
After incubating at room temperature for 24 hours, the excess BAꢀtosyl initiator was removed by
column chromatography (Sephadex® Gꢀ25). The collected antiꢀCon A antibody macroinitiators
were then concentrated by centrifugal filters to obtain the purified macroinitiator.
Atom transfer radical polymerization in anti-Con A antibody.
The obtained antiꢀCon A macroinitiator (50 ꢁg, 3.33 mmol) and NIPAAm (17 mg, 150 ꢁmol)
were dissolved in 63 ꢁL of degassed water in an argon atmosphere. An oxygenꢀfree catalyst
solution (20 ꢁL obtained by dissolving 1.0 mg of CuBr and 4.2 mg of 2,2’ꢀbipyridine in 78 ꢁL
degassed water) was added to start the polymerization. The reaction was stopped after 1.5 hours
by exposure to air. The reverse phase HPLC was used to monitor the progress of polymerization
o
as shown in Figure S5. The formed antibodyꢀPNIPAAm conjugates were heated to 45 C, it will
be precipitated and separated from reaction mixtures by centrifugation. The precipitates were
then dissolved in chilled ddH2O followed by a heating precipitation process to remove the
reactants and catalysts. The washing step was repeated three times to obtain purified antibodyꢀ
PNIPAAm conjugates. The protein conversion yield is determined by bicinchoninic acid
(BCA) assay as 87%.
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Enrichment of biotinylated Con A by anti-Con A Ab-PNIPAAm conjugates.
The obtained antiꢀCon A Abꢀpolymer conjugates were then incubated with biotinylated Con
A solution (17 ꢁg in 15 ꢁL aqueous solution). After incubating at room temperature for 2 hours,
excess biotinylated Con A was then separated by centrifugation at 45°C to collect the precipitates
as the biotinylated Con Aꢀanti ConA AbꢀPNIPAAm complex. Then, 15 ꢁL stripping buffer
(glycineꢀHCl, pH 2.5, 0.1M) was used to dissolve the precipitates and release biotinylated Con A
from antiꢀCon A AbꢀPNIPAAm conjugates. The eluted biotinylated Con A was neutralized by
Tris buffer (pH 10.0, 1M) and directly analyzed by western blotting with antiꢀbiotin monoclonal
Ab.
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RESULTS and DISCUSSION
Figure 2. Chemical structures of the biotinꢀtosyl initiator (1), biotin initiator (2) and BAꢀtosyl
initiator (3).
Synthesis of Ligand-Tosyl Initiators. To demonstrate this concept, two polymer initiator
transfer molecules (biotinꢀtosyl initiator and BAꢀtosyl initiator) and control molecule (biotin
initiator) were designed (Figure 2); the details of the syntheses of biotinꢀtosyl initiator and BAꢀ
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tosyl initiator are described in Scheme 1. To model the biotinꢀstreptavidin interaction, biotinꢀ
tosyl initiator was synthesized from the highly reactive 3ꢀ(chlorosulfonyl)benzoyl chloride
(CSBC). A oneꢀpot twoꢀstep procedure was engaged to sequentially conjugate CSBC with spacer
5 and ATRP initiator 6.53 The formed compound 7 was then treated with trifluoroacetic acid
(TFA) to expose the reactive amine group. This compound was further reacted with activated
succimidinyl biotin 9 to obtain the biotinꢀtosyl initiator. Simultaneously, BAꢀtosyl initiator 3 was
designed and synthesized as a initiator transfer for Abs of interest. 3ꢀAminophenyl boronic acid
(BA) was first protected with pinacol to facilitate purification. Because of the strong binding
affinity to silica gel, the BAꢀcontaining molecules are difficult to purify by silica gel column
chromatography. The protected amino BApin 10 and initiator 6 were then sequentially added to
the starting material CSBC in the presence of diisopropylethyl amine (DIEA) and N,N-
dimethylaminopyridine (DMAP). The obtained pinacolꢀprotected BApinꢀtosyl initiator was then
deprotected with NaIO4 to furnish the BAꢀtosyl initiator 3 for advanced study in siteꢀselective
IgG modification. To demonstrate the importance of the key components (the targeting head and
the reactive tosyl group) in BAꢀtosyl initiator 3, control molecule (the biotinꢀinitiator) was
synthesized through conventional amide formation. The details of these corresponding syntheses
are included in the supporting information.
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Scheme 1. Syntheses of (A) biotinꢀtosyl initiator and (B) BAꢀtosyl initiator
Reaction Conditions for the Preparation of Thermoresponsive Protein-Polymer
Conjugates. To grow uniform lengths of polymer chains, we used controlled radical
10,54
polymerizations (CRPs);
CRPs have emerged as powerful methods in the preparation of
wellꢀdefined proteinꢀpolymer and peptideꢀpolymer conjugates. Atom transfer radical
polymerization (ATRP)55ꢀ56 is an ideal CRP method for the preparation of bioreactive polymeric
scaffolds due to its userꢀfriendly operation and potential to work under physiological conditions.
In a low concentration of Cu(I), the obtained protein macroinitiator can directly serve to extend
the polymer chain on the molecular surface of proteins. Here, Nꢀisopropylacrylamide (NIPAAm)
was used as the monomer in ATRP to assemble the thermoꢀresponsive smart polymer
(PNIPAAm)57 growing from the protein of interest. The PNIPAAm chain allows for instant
precipitation when the temperature rises above the low critical solution temperature (LCST). The
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unique property makes the PNIPAAmꢀconjugates be easily separated from the unreacted
mixtures and resuspends in buffer while raising temperature up to LCST. In this research,
reaction conditions for the PNIPAAm grafting from the protein of interest by ATRP was
optimized with a model protein, bovine serum albumin (BSA). BSA was first conjugated with
the ATRP initiator by random amide bond formation which experimental procedures are shown
in supporting information. To optimize the graftingꢀfrom ATRP in biomacromolecules, different
Cu catalysts and ligands have been carefully studied. In 2005, Maynard and coꢀworkers were
used streptavidinꢀbiotin initiator to graftingꢀfrom PNIPAAm through ATRP on streptavidin26.
And they also tried to utilize sacrificial initiator to improve polymerization process. The resultant
BSA macroinitiator was then used in the optimization of the ATRP reaction under physiological
condition. Different Cu(I) catalysts and ligands such as Tris(2ꢀdimethylaminoethyl)amine,
1,1,4,7,10,10ꢀHexamethyltriethylenetetramine, and 2,2’ꢀbipyridine have been examined by
screening reactant equivalents and reaction time. A sacrificial initiator was also used and
evaluated while screening conditions. The conditions to degas the protein of interest was defined
as 15 ꢁM protein in PBS buffer at pH 8.0, three times, and then treated with 2,2’ꢀbipyridine (180
mM) and CuBr (90 mM) at 25°C for 12 hours. This condition would be generally used in the
following studies of proteinꢀpolymer preparation.
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Biotin-tosyl initiator-directed synthesis of Streptavidin-PNIPAAm Conjugates.
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Scheme 2. Illustration of biotinꢀtosyl initiator used in the preparation of StreptavidinꢀPNIPAAm
conjugates with conserved biotin binding affinity. The lanes iꢀvii of the insert figure represent
the streptavidin conjugates iꢀvii in SDSꢀPAGE. (a) 2, 10% DMSO, 37oC, 24h; (b) amino biotin
(25), rt, 2h; (c) CuBr, 2,2’ꢀbipyridine, Nꢀisopropylacrylamide; (d) 1, 10% DMSO, 37oC, 24h; (e)
amino biotin (25), rt, 2h; (f) CuBr, 2,2’ꢀbipyridine, Nꢀisopropylacrylamide.
To demonstrate this siteꢀselective strategy in the preparation of proteinꢀpolymer conjugates, a
binding pair of biotin and streptavidin was selected as a model to evaluate this affinityꢀdirected
chemical strategy in the preparation of proteinꢀpolymer conjugates with conserved protein
function. A SDSꢀPAGE inserted in the Scheme 2 clearly indicates the formation of all
intermediates during this affinityꢀdirected protein modification. As illustrated in Scheme 2, the
biotinꢀtosyl initiator and its control molecule, biotinꢀinitiator, were treated with streptavidin
individually. The biotinꢀtosyl initiator can covalently transfers the initiator group to streptavidin
and leave biotin tosylate which nonꢀcovalently associated with streptavidin as the intermediate
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(ii). The negatively charged biotin tosylate may make the protein band be shifted to a lower
position in SDSꢀPAGE. In contrast, a nonꢀcovalently associated streptavidin/biotin initiator
intermediate (v) can be found by treating streptavidin with the control molecule biotinꢀinitiator.
Without the tosyl group as the initiator transferring agent, although intermediate (v) can still
grow polymer chains by terminal initiator, the nonꢀcovalent association is in a risk of
dissociation. More importantly, the resultant streptavidinꢀPNIPAAm conjugates would certainly
lose the biotinꢀbinding activity if trying to keep the proteinꢀpolymer conjugate’s integrity. . To
demonstrate the importance of the developed ligandꢀtosyl proteinꢀpolymer synthesis,
intermediates (ii) and (v) were subjected to the dissociation of streptavidinꢀbiotin complexes.
Trying to remove the bound biotin from streptavidin, a high concentration of biotin solution is
necessary due to the high affinity (KD = 10ꢀ15 M). However, no observed dissociation can be
observed while incubated the mixture with high concentrations of free biotin (1M in DMSO)
which cannot be prepared in neutral aqueous solution neither at 25°C nor heating at 70 °C. An
acidic condition (pH 1.5) has also been adopted to release the bound biotins, but still worked
poorly. Then, a water soluble amino biotin was synthesized and successfully used to remove
biotin form the complexes. The biotin benzenesulfonate was released by competition
(intermediate iii) and regained the biotin binding site. The initiator attached on the surface of the
streptavidin initiated ATRP by treatment with CuBr, 2,2’ꢀbipyridine and NIPAAm to grow
PNIPAAm chains. The resultant thermoꢀresponsive streptavidinꢀPNIPAAm conjugate (iv)
considerably increased in molecular weight as well as be endowed the thermoꢀresponsive
property. After heating and denaturing, no distinguished band can be observed by SDSꢀPAGE,
which strongly supports the success in the formation of this streptavidinꢀPNIPAAm conjugates
(lane iv, Scheme 2). In contrast, amino biotins successfully displaced the biotin from the biotin
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initiatorꢀtreated streptavidin (v) and therefore release the biotinꢀinitiator from streptavidin. A
clear streptavidin band of product (vii) strongly supports no or very few streptavidinꢀPNIPAAm
conjugates formed in the same polymerization condition (lane vii, Scheme 2).
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The results of this model study fully support the importance of this initiatorꢀtransferring
chemistry in the preparation of proteinꢀpolymer conjugates. The simple activityꢀbased synthesis
of a protein macroinitiator, such as biotinꢀstreptavidin, may suffer ligand dissociation. The
biotinꢀtosyl initiator directed specific recognition with streptavidin by nonꢀcovalent interaction,
then the proximity effect triggered the second covalent conjugation. The resultant stable
streptavidin macroinitiator was successfully used in the preparation of streptavidinꢀPNIPAAm
conjugates, which highly conserved affinity to biotin.
Preparation and Characterization of Human IgG-PNIPAAm Conjugates with Boronic
Acid (BA)-Tosyl Initiator.
With the success of streptavidinꢀPNIPAAm conjugates, this activityꢀdirected synthesis of
proteinꢀpolymer conjugates was extended to the application of the preparation of IgGꢀPNIPAAm
conjugates. BAꢀtosyl initiator 3 was used to selectively recognize glycan chains of IgG antibody
specifically on the Fc domain (Figure 1). The resultant mixture was then purified by Sephadex®
Gꢀ25 column chromatography to remove excess BAꢀtosyl initiator 3, generating the Fcꢀspecific
IgG macroinitiator. To optimize conditions for ATRP, different amounts of NIPAAm were
treated with hIgG macroinitiator (15 ꢁM, human immunoglobulin G from serum, Sigmaꢀ
Aldrich) in the presence of CuBr (90 mM) and 2,2’ꢀbipyridine (180 mM). To the treatment of
NIPAAm monomers 50 ꢁmole, the obtained IgGꢀPNIPAAm conjugates were analyzed by SDSꢀ
PAGE electrophoresis. Because all samples were denatured in 95 °C, those polymerꢀattached
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fragments would be precipitated and therefore cannot be observed in SDSꢀPAGE. In Figure 3A,
a gradual reduction of heavy chain bands in SDSꢀPAGE with nearly consistent bands of light
chains strongly supports the selective growth of PNIPAAm chains on heavy chains of IgG. The
conjugated heavy chains and unreacted light chains could be dissociated by treatment the
reducing agent, βꢀmercaptomethanol, and the thermal sensitive IgG heavy chainꢀPNIPAAm
conjugates would be precipitated in heating condition which led to the reduction of heavy chain
bands in SDSꢀPAGE. This result reveals that this BAꢀtosyl initiator strategy can be efficiently
used in Fc domainꢀspecific IgGꢀpolymer conjugation.
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To compare with conventional random proteinꢀpolymer conjugates, human IgG was added
with initiator succinimidyl ester to prepare randomlyꢀconjugated IgGꢀinitiators conjugates. The
multiple and randomly located initiators on IgG initiated the growth of PNIPAAm chains at both
light chains and heavy chains. As shown in Figure 3B (lane 4), only very weak bands of both
light chains and heavy chains can be observed after random polymer growth, indicating
uncontrollable grafting on the whole IgG molecule.
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Figure 3. (A) lane 1: BAꢀtosyl initiator; lane 2: lane 1 after polymerization. (B) lane 1: BAꢀtosyl
initiator; lane 2: lane 1 after polymerization; lane 3: OSuꢀinitiator; lane 4: lane 3 after
polymerization.
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The obtained IgG conjugates exhibited a temperature responsive phase change and the
corresponding values of LCST were determined by UV illumination at 280 nm. In the screening
range of 20ꢀ65°C, the LCST was at 38°C. (Figure 4A). To further characterize properties of
these obtained IgGꢀPNIPAAm conjugates, the obtained IgGꢀpolymer conjugates have been
further analyzed by fast protein liquid chromatography (FPLC) (Figure 4B). While the IgGꢀ
initiator molecule gives the retention time at 60 mins, the formed IgGꢀPNIPAAm conjugate’s
retention time was shifted to 50 minutes. Subsequently, dynamic light scattering (DLS) was used
to determine the corresponding hydrodynamic diameters of IgGꢀPNIPAAm conjugates prepared
by this method. The diameter of IgG was determined to be 4.9 ± 0.4 nm. Then, polymerization of
IgG macroinitiator with NIPAAm monomers formed IgGꢀPNIPAAm conjugates with diameter
increasing to 59.2 ± 9.7 nm (Figure S4B).
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Figure 4. (A) Determination of LCST of IgGꢀPNIPAAm conjugates by UVꢀVis spectrometer. (B)
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FPLC diagram of IgGꢀinitiator, IgGꢀPNIPAAm conjugates.
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Figure 5. Polyacrylamide gel electrophoresis (SDSPAGE, left) and Western blotting (right) of
hIgG after treatment with BAꢀmꢀtosylꢀalkyne followed by biotinylation. Coomassie blue staining
of the SDSꢀPAGE gel (left) shows that heavy chain and light chain cleavage by βꢀME. The
signal was revealed by Western blotting with an antiꢀbiotin monoclonal antibody.
To further demonstrate the siteꢀspecifically modification on Fc domain, a BAꢀtosylꢀalkyne
was synthesized and treated with IgG to transfer a terminal acetylene group on Fc domain.
Subsequently, in the presence of Cu (I) catalyst and azido biotin, the IgG molecule would be
further tagged a biotin. Monoclonal antiꢀbiotin antibody was then used in Western Blot
experiment to indicate the existing of biotins. As shown in Figure 5, only a band located at heavy
chains (~55 kDa) can be observed which strongly supports the specific modification site at heavy
chains by BAꢀtosyl chemistry. Furthermore, an ester bond, BAꢀtosylꢀesterꢀinitiator 29, was
introduced to displace the amide bond in the initiator linkage which allows the dissociation of
polymer chains from conjugated IgG by basic hydrolysis. As shown in Figure S6, the intensity of
IgG’s heavy chain on SDSꢀPAGE electrophoresis was considerably reduced after polymerization
as usual. By contrast, light chain showed no distinguishable change. Under the basic condition,
the ester bond could be cleaved and released the NIPAAm chains from IgG.58 As the result, the
intensity of IgG’s heavy chain was recovered which strongly supports the specific
polymerization site on heavy chains. (Figure S6)
Site-Specific Conjugated anti-Con A antibody-PNIPAAm Conjugates in the Enrichment of
Con A.
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Through the characterization of IgGꢀPNIPAAm conjugates, the BAꢀtosyl strategy was
demonstrated to specifically attach the PNIPAAm chains of interest on IgG at the Fc domain.
Accordingly, the antigen binding (Fab) domains of modified IgG should be completely exposed
to environment and preserve its native function. To further evaluate this, an antiꢀCon A
(Concanavalin A) antibody was treated with BAꢀtosyl initiator followed by ATRP with NIPAAm,
2,2ꢀbipyridine and CuBr; meanwhile, a control experiment was carried out by treating the antiꢀ
Con A Ab with succinimidyl initiator (OSu initiator) to generate a randomly conjugated antiꢀCon
A AbꢀPNIPAAm conjugate.
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The siteꢀselective anti ConꢀA AbꢀPNIPAAm and random antiꢀCon A AbꢀPNIPAAm
conjugates (50 ꢁg) were mixed with biotinylated Con A (17 ꢁg, 0.64 nmol) respectively. After
Incubation for 30 mins, the reaction mixtures were then heated to 40 °C to precipitate the anti
ConꢀA Ab ꢀPNIPAAmꢀbiotinylated Con A complexes. Then, the bound biotinylated Con A
precipitates were separated from unbound ones by centrifuging. The biotinylated Con A were
dissociated from antiꢀCon A Ab in acidic conditions (stripping buffer, glycineꢀHCl, pH 2.5) and
collected for subsequent analysis by western blotting with antiꢀbiotin monoclonal Ab. In Figure
6, a strong band (lane 1) represented the extraction of Con A by siteꢀselective antiꢀCon A Ab but
a much weaker biotinylated Con A band (lane 2) can be observed using random antiꢀCon A Ab.
The quantitative analysis of this western blotting result showed about six times higher intensity
of Con A being biotinylated by BAꢀtosyl initiator rather than the use of random conjugated antiꢀ
Con A antibodyꢀPNIPAAm conjugates. These experiments were repeated three times and gave
the consistent results. Using the same amount of antiꢀCon A Ab, the randomly conjugated Ab
considerably lost its native function in binding its substrate Con A, which can be attributed to the
block of Fab domain by randomly located PNIPAAm chains. Therefore, siteꢀselective grafting in
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the preparation of IgGꢀpolymer conjugates is certainly an important issue for biomedical
applications.
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Figure 6. Enrichment of biotinylated Con A by (A) BAꢀtosyl initiator mediated antiꢀCon A Abꢀ
PNIPAAm conjugates and (B) OSuꢀinitiator mediated antiꢀCon A AbꢀPNIPAAm conjugates
(lane 1, site selective) (lane 2, random). (C) The results of western blotting and its quantitative
analysis of eluted biotinylated Con A.
CONCLUSION
Considering the importance of proteinꢀpolymer drugs in modern therapeutics, a need of new
methods in the preparation of proteinꢀpolymer conjugates is emerging to provide wellꢀdefined
architectures of proteinꢀpolymer conjugates instead of heterogeneous structures. In this research,
a new chemical strategy was proposed to prepare the protein macroinitiator in a siteꢀselective
manner for use in the preparation of antibodyꢀpolymer conjugates. This activityꢀbased strategy
can effectively preserve the native function of modified proteins. To the best of our knowledge,
we have conducted the first study in the preparation of Fcꢀdirected IgGꢀpolymer conjugates with
native glycans, preserving the inherent structure of IgG molecules. A comparison in the
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enrichment of biotinylated Con A by antiꢀCon A AbꢀpNIPAAm conjugates strongly supported
the importance of siteꢀselectivity when attaching polymers to antibodies of interest.
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ASSOCIATED CONTENT
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1
Supplementary information (Figure S1 to S6) include all, H NMR, 12C NMR, Mass and UVꢀ
Vis spectra, as well as reverse phase HPLC. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
Prof. PoꢀChiao Lin
Email : pclin@mail.nsysu.edu.tw
ACKNOWLEDGMENT
This work is supported by grants from Ministry of Science and Technology (MOST 103ꢀ2113ꢀ
Mꢀ110ꢀ010ꢀ and MOST 103ꢀ2627ꢀMꢀ007ꢀ004ꢀ) and National Sun Yatꢀsen University
(01C030703 and 01A06802). We also thank the support from National Sun Yatꢀsen Universityꢀ
Kaohsiung Medical University Joint Research Center, Kaohsiung, Taiwan.
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