Polymeric dibromomaleimides as extremely efficient disulfide bridging bioconjugation and pegylation agents

Article abstract of DOI:10.1021/ja210335f

A series of dibromomaleimides have been shown to be very efficacious at insertion into peptidic disulfide bonds. This conjugation proceeds with a stoichiometric balance of reagents in buffered solutions in less than 15 min to give discrete products while

Full text of DOI:10.1021/ja210335f

Article
pubs.acs.org/JACS
Polymeric Dibromomaleimides As Extremely Efficient Disulfide
Bridging Bioconjugation and Pegylation Agents
Mathew W. Jones,† Rachel A. Strickland,† Felix F. Schumacher,‡ Stephen Caddick,‡ James. R. Baker,‡
Matthew I. Gibson,† and David M. Haddleton*,†
†Department of Chemistry, University of Warwick, Coventry CV4 7AL, U.K.
‡Department of Chemistry, University College London, London WC1H 0AJ, U.K.
S
* Supporting Information
ABSTRACT: A series of dibromomaleimides have been
shown to be very efficacious at insertion into peptidic disulfide
bonds. This conjugation proceeds with a stoichiometric
balance of reagents in buffered solutions in less than 15 min
to give discrete products while maintaining the disulfide bridge
and thus peptide conformation. The insertion is initiated by
disulfide reduction using a water-soluble phosphine, tris(2-
carboxyethyl)phosphine (TCEP) which allows for subsequent
substitution of the two maleimide bromides by the generated thiols. Reaction of salmon calcitonin (sCT) with 2,3-
dibromomaleimide (1.1 excess) in the presence of TCEP (1.1 equiv) in aqueous solution at pH 6.2 gives complete production of
a single conjugate which requires no workup. A linear methoxy poly(ethylene glycol) (PEG) was functionalized via a Mitsunobu
reaction and used for the successful site-specific and rapid pegylation of sCT. This reaction occurs in 15 min with a small
stoichiometry excess of the pegylating agent to give insertion at the disulfide with HPLC showing a single product and MALDI-
ToF confirming conjugation. Attempts to use the group in a functional ATRP polymerization initiator led to polymerization
inhibition. Thus, in order to prepare a range of functional polymers an indirect route was chosen via both azide and aniline
functional initiators which were converted to 2,3-dibromomaleimides via appropriate reactions. For example, the azide functional
polymer was reacted via a Huisgen CuAAC click reaction to an alkyne functional 2,3-dibromomaleimide. This new reagent
allowed for the synthesis of conjugates of sCT with comb polymers derived from PEG methacrylic monomers which in addition
gave appropriate cloud points. This reaction represents a highly efficient polymer conjugation method which circumvents
problems of purification which normally arise from having to use large excesses of the conjugate. In addition, the tertiary
structure of the peptide is efficiently maintained.
reducible disulfides are essential for maintaining protein
structure, shape, stability, and bioactivity are lost. Brocchini et al.
developed an elegant but rather elaborate approach to
overcome this whereby reduced disulfides are rebridged using
a three-carbon linker.35,36 This has the obvious advantage that
the tertiary structure of the protein is largely maintained as
well as the site-selective incorporation of functionality, such
as PEG.37
Baker et al. recently reported the development of a range of
different functional maleimides for cysteine modification and
conjugation.38,39 Dibromomaleimides were shown to be
efficacious and fast-reacting disulfide rebridging agents,
installing a rigid two-carbon linker. In addition, dibromomalei-
mides have an added advantage that the formed linker is
potentially cleavable in a reversible manner under intracellular-
like conditions to rejuvenate the native polypeptide in its
reduced state. This same team reported the development of
new substituted maleimides, dithiomaleimides, which are less
susceptible to side reactions with reducing agents, such as
INTRODUCTION
■
Conjugation of synthetic polymers to proteins and peptides is a
well established method to enhance properties of native
biomolecules and aid the introduction of polymer-based
therapeutics.1,2 The covalent attachment of poly(ethylene
glycol) (PEG) chains, pegylation, in particular has been well
documented to yield conjugates often with improved solubility,
stability, and plasma half-lives.3−19 The early reports, and
probably still the majority of reports, describe the targeting of
lysine residues due to their relative abundance, with a range of
simple chemistries, including activated esters, which are now
commonly employed for bioconjugation.20−22 This approach
often results in isolation of a statistical mixture of conjugated
species. Targeting of cysteine residues has been explored as an
alternative, and a number of reagents have been developed to
selectively target this thiol-containing residue. N-Substituted
maleimide,23−27 pyridyl disulfide,28−30 and acrylic31,32 func-
tional polymers have been explored for conjugation at cysteine.
However, free cysteine residues are relatively rare in proteins
and other biomolecules, although they can be introduced by
genetic engineering or by the reduction of disulfide bridges.33,34
The latter approach has the inherent problem that if the
Received: November 3, 2011
Published: December 21, 2011
© 2011 American Chemical Society
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TCEP, and have been shown to have potential for in situ
bridging protocols.40 The development of controlled radical
polymerization (CRP) techniques such as ATRP,41,42 RAFT,43
and NMP44 have enabled the ready synthesis of well-defined
α-functional polymers for bioconjugation under undemanding
experimental conditions. Herein, we report on the study of
dibromomaleimide functional polymers for applications in
protein disulfide bridging, that are synthesized by ATRP.
Analysis of the reaction mixture revealed the complete
consumption of the reduced sCT and the appearance of a new
single peak (blue trace), ascribed as the bridged product,
moving to longer retention time due to consumption of the two
cysteine residues and conjugation of the maleimide. MALDI-
ToF-MS showed the expected mass gain of 95.04 Da due to
disulfide bridging of the maleimide (Figure 3). No traces of
RESULTS AND DISCUSSION
■
We focused on the modification of salmon calcitonin (sCT), a
32-amino acid hormone secreted by C-cells of the thyroid in
mammals and by the ultimobranchial glands in submammals.
This is currently used for the treatment of a range of bone
conditions including postmenopausal osteoporosis, Paget’s
disease, and hypercalcaemia.45 This polypeptide contains a
disulfide bridge (Cys1-Cys7) that can be reduced to give two
sulfhydryl functionalities as targets for rebridging conjugation
with dibromomaleimides. Our initial experiments involved
disulfide bridging using the commercially available 2,3-
dibromomaleimide (1) (Figure 1).
Figure 3. MALDI-ToF-MS analysis of native sCT (red trace) and
maleimide-bridged sCT following sampling after 10 min.
either native or reduced sCT were observed by MALDI-ToF-
MS, in agreement with RP-HPLC, evidence of highly efficient
bridging with no observable side reactions. Trypsin digest of
(2) and analysis by MALDI-ToF-MS revealed that the
disulfide-containing fragment was the only modified species,
and circular dichroism revealed that the structural integrity of
the peptide had been maintained following modification (see
Supporting Information).
In order to demonstrate the viability of this reaction for
polymer bioconjugation, a linear PEG-chain with α-functional
dibromomaleimide functionality was synthesized40 using a
modified Mitsunobu reaction.46 This functionalized PEG5000
chain (3) was conjugated to reduced sCT under identi-
cal reaction conditions as with 2,3-dibromomaleimide (1),
(Figure 4).
Figure 1. One-pot reduction of the disulfide bridge of sCT, followed
by rebridging using dibromomaleimide.
The disulfide bridge of sCT was reduced in the presence of
tris(2-carboxyethyl)phosphine (TCEP) and the reaction
monitored by RP-HPLC; ring-opening was observed by a
large shift in retention time due to the formation of two
relatively polar thiols, giving a very convenient analytical handle
(Figure 2). Following complete reduction of the disulfide
Figure 2. RP-HPLC analysis of sCT disulfide-bridging with 2,3-
dibromomaleimide following reduction of the disulfide bridge of sCT.
bridge the reaction pH was adjusted to 6.2, and 2,3-
dibromomaleimide (1.1 equiv) was added. Immediately upon
addition, the solution turned pale yellow, in accordance with
previous observations for dithio-bridged maleimides,40 and after
10 min an aliquot was removed for RP-HPLC analysis.
Figure 4. sCT disulfide rebridging using the dibromomaleimide-
functional linear PEG5000 chain.
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As before, addition of the dibromomaleimide-functional
PEG5000 chain gives an immediate pale-yellow color, indicative
of the bis-thio-bridged maleimide. Analysis after 15 min by
RP-HPLC revealed the complete disappearance of the reduced
polypeptide as well as the formation of a new single species
with retention time at approximately 18 min (Figure 5).
are well documented as highly reactive substrates in radical
polymerizations,47−49 the dibromo derivative was analyzed as a
viable functionality for the synthesis of functional polymers.
Polymerization using standard ATRP conditions of PEG-
methacrylate using ethyl 2-bromoisobutyrate was carried out in
the presence of dibromomaleimide alongside a control reaction
conducted as a reference (Figure 7).
Figure 7. ATRP polymerization of PEGMA in the presence and
absence of the dibromomaleimide functionality.
Large differences in the polymerization rates were observed
(Figure 8). Polymerization in the absence of 3,4-dibromomaleimide
Figure 5. RP-HPLC analysis of the disulfide-bridging of sCT using a
dibromomaleimide-functional PEG chain following reduction with
TCEP.
MALDI-ToF-MS analysis showed a distribution of peaks
around the expected mass (∼8.5 kDa) upon conjugation of the
PEG5000 chain to sCT (3431 Da) (Figure 6). A number of
Figure 8. First-order kinetic plot for the polymerizations of PEGMA475
using [ethyl 2-bromoisobutyrate]/[Cu(I)Br]/[Lig]/[PEGMA475] =
1:1:3:20, toluene/PEGMA475 2:1 (v/w), 60 °C in the absence (blue
data) and presence of 3,4-dibromomaleimide 1 (1 equiv with respect
to initiator) (red data).
Figure 6. MALDI-ToF-MS of the reaction mixture after 15 min,
showing the formation of the monoconjugated sCT-PEG5000 species,
around 8.5 kDa as expected.
proceeded as expected, with linear first-order kinetics observed.
However, polymerization in the presence of 3,4-dibromo-
maleimide was retarded in comparison with the blank
polymerization.
PEG-derived distributions were observed, with the predom-
inant peaks corresponding to the sodiated conjugate and
smaller peaks attributed to the protonated conjugate (Figure 6
and ESI).
Thus, the direct polymerization using dibromomaleimide-
functional initiators was abandoned as a viable route, and
alternative strategies were investigated. Two postpolymeriza-
tion modification50 routes were investigated for the introduc-
tion of the dibromomaleimide functionality; first, an azido-
functional initiator (5, Figure 9) was synthesized for further
transformation using CuAAC, and a second strategy was
devised whereby aniline-functional polymers were synthesized
in order to introduce the maleimide functionality using a
condensation reaction with dibromomaleic anhydride, followed
by ring closure via acid-promoted dehydration (6, Figure 9).
Despite successful functionalization of PEG with dibromo-
maleimide using modified Mitsunobu conditions, both the
efficiency and isolated yield were relatively low, with the
requirement of column chromatography for purification of the
final product. Thus, a strategy was devised to synthesize
α-functional polymers by atom transfer radical polymerization
(ATRP). Since each chain is grown from the initiator in this
case, the synthesized polymer inherently contains the initiator-
derived functionality. The dibromomaleimide functionality was
investigated as a novel end-group for the synthesis of male-
imide-functional polymers. Since unfunctionalized maleimides
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investigated (ESI), with the cloud points of showing little or no
change following end-group modification.55
A second strategy was also investigated as a route to
dibromomaleimide functional polymers using Boc-protected
aniline initiator 6. Oligo(ethylene glycol) methacrylate was
homopolymerized and copolymerized with DEGMEMA by
ATRP using the protected aniline initiator 6 in order to
synthesize α-functional polymers 16 and 17 (Figure 12).
Figure 9. ATRP initiators employed in this study for the
postpolymerization incorporation of dibromomaleimide functionality.
The azide-functional initiator 5 was synthesized as described
previously51 and was employed for the polymerization of oligo-
(ethylene glycol) methacrylates by ATRP. Polymers synthe-
sized using this initiator have been shown to maintain an
apparent 100% of the azido chain end functionality,51 an
essential requirement for efficient post polymerization
modification.
Polymerization of oligo(ethylene glycol) methacrylates has
emerged as a popular route for the synthesis of thermores-
ponsive polymers52,53 and protein conjugates.32,54 Tri(ethylene
glycol) methacrylate (TEGMEMA) was homopolymerized and
copolymerized in a 1:1 molar ratio with di(ethylene glycol)
methacrylate (DEGMEMA) using initiator 5 by ATRP (Figure 10)
to synthesize azide-functional polymers 7 and 8. Following
Figure 12. Polymerization of oligo(ethylene glycol) methacrylates
using initiator 10 as a route to α-Boc-protected polymers.
Following purification and isolation, the Boc-protecting
group was removed by treatment with TFA and the obtained
aniline converted to a dibromomaleimide-functional polymer
following heating in the presence of dibromomaleic anhydride
(Figure 13). Each of the postpolymerization modification steps
Figure 10. Polymerization of oligo(ethylene glycol) methacrylates
using azide-functional initiator 7.
purification, the polymers were isolated as colorless oils and the
presence of the α-azido functionality confirmed by IR-
spectroscopy (ESI). A dibromomaleimide-functional terminal
alkyne was synthesized and the functionality installed to the
polymers by CuAAC (Figure 11) and the presence of the new
Figure 13. Postpolymerization modification of poly(oligo(ethylene
glycol)) methacrylate by deprotection of the Boc-protecting group to
yield an α-aniline functionality, followed by treatment with
dibromomaleic anhydride to furnish the dibromomaleimide end group.
1
was monitored by H and 13C NMR, with deprotection of the
Boc-group and functionalization with dibromomaleic anhydride
monitored by the shift of the aromatic protons and compared
to synthesized model compounds (ESI).
Following successful incorporation of the dibromomaleimide
functionalities to the aniline- and azide-functional pre-
polymers, conjugation to sCT was investigated. As previously,
the disulfide bridge of sCT was reduced in the presence of
TCEP and monitored by RP-HPLC. Following complete
reduction of the disulfide, polymer 10 was added in a buffered
solution at pH 6.2 (Figure 14).
Figure 11. Installation of dibromomaleimide functionality to α-azido
functional polymers using CuAAC.
end-group confirmed by 13C NMR, along with the loss of the
characteristic signal from the azide observed by IR-spectroscopy.
The thermoresponsive nature of polymers 7 and 8 were
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Figure 14. sCT disulfide rebridging using dibromomaleimide functional polymer 10.
As previously, an immediate change in solution color to pale-
yellow was observed,40 and analysis by RP-HPLC 20 min
following addition of the polymer showed a complete loss of
the signal corresponding to reduced sCT, along with the
formation of a broad peak from the conjugate (Figure 15).
minutes. Both routes serve as viable approaches to disulfide-
bridging agents for polypeptides and proteins, although the
N-phenyl maleimide derivatives have further advantages as both
stable and cleavable linkers following controlled maleimide
hydrolysis.56 This approach has clear benefits over currently
employed techniques since little or no purification of
macromolecular species is required due to the stoichiometric
nature of the reaction.
ASSOCIATED CONTENT
■
S
* Supporting Information
1
Experimental details, H and 13C NMR, IR, MALDI-ToF-MS,
and RP-HPLC data. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
D.M.Haddleton@warwick.ac.uk
Figure 15. RP-HPLC analysis of the disulfide-bridging of sCT using
polymer 10 following reduction of the disulfide bridge with TCEP.
ACKNOWLEDGMENTS
Similarly, the conjugation of the dibromomaleimide-func-
tional polymer synthesized via the aniline route was
investigated. Following reduction of sCT using TCEP, polymer
16 was added in a buffered solution at pH 6.2, and the
characteristic yellow color was observed immediately. RP-
HPLC analysis of the reaction mixture showed the loss of the
signal corresponding to the reduced polypeptide, as well as the
formation of the broad conjugate peak (ESI). These
bioconjugation reactions demonstrate the power of this
approach for the rebridging of generated cysteine residues
derived from reduced disulfides. The ability to rapidly (<30 min)
and quantitatively modify polypeptides with equimolar
amounts of dibromomaleimide functional polymers holds
great promise for the development of a new class of biocon-
jugates. Time scales for conjugation have been dramatically
reduced relative to commonly employed protocols and
purification of products have been simplified due to a reduction
in the required amounts of conjugating polymer.
■
We thank the University of Warwick, and Warwick Effect
Polymers Limited for funding (M.W.J.). Equipment used in this
research was funded in part through support from Advantage
West Midlands (A.W.M.) Science City Initiative and funded in
part by the ERDF. D.M.H. is a Wolfson/Royal Society Fellow
and M.I.G. is a HEFCE Science City Fellow.
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