Hetero-Diels–Alder Cycloaddition with RAFT Polymers as Bioconjugation Platform

Article abstract of DOI:10.1002/anie.202005747

We introduce the bioconjugation of polymers synthesized by RAFT polymerization, bearing no specific functional end group, by means of hetero-Diels–Alder cycloaddition through their inherent terminal thiocarbonylthio moiety with a diene-modified model protein. Quantitative conjugation occurs over the course of a few hours, at ambient temperature and neutral pH, and in the absence of any catalyst. Our technology platform affords thermoresponsive bioconjugates, whose aggregation is solely controlled by the polymer chains.

Full text of DOI:10.1002/anie.202005747

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: Hetero-Diels–Alder Cycloaddition with RAFT Polymers as
Bioconjugation Platform
Authors: Ana Beloqui, Shivshankar R Mane, Marcel Langer, Mathias
Glassner, Dennis M Bauer, Ljiljana Fruk, Christopher Barner-
Kowollik, and Guillaume Delaittre
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202005747
Link to VoR: https://doi.org/10.1002/anie.202005747

10.1002/anie.202005747
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Hetero-Diels–Alder Cycloaddition with RAFT Polymers as
Bioconjugation Platform
Ana Beloqui,[a,b,c,d] Shivshankar R. Mane,[a,b] Marcel Langer,[b] Mathias Glassner,[b] Dennis M. Bauer,[e]
Ljiljana Fruk,[e,f] Christopher Barner-Kowollik,*[b,g,h] and Guillaume Delaittre*[a,b,i]
[a]
[b]
Dr. A. Beloqui, Dr. S. R. Mane, Prof. Dr. G. Delaittre
Institute of Biological and Chemical Systems (IBCS)
Karlsruhe Institute of Technology (KIT)
Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany
Dr. A. Beloqui, Dr. S. R. Mane, Dr. M. Langer, Dr. M. Glassner, Prof. Dr. C. Barner-Kowollik, Prof. Dr. G. Delaittre
Macromolecular Architectures
Institute for Chemical Technology and Polymer Chemistry (ITCP)
Karlsruhe Institute of Technology (KIT)
Engesserstr. 18, 76131 Karlsruhe, Germany
Dr. A. Beloqui
Department of Applied Chemistry (UPV/EHU)
Avda. Manuel de Lardizabal 3, E-20018 Donostia - San Sebastian, Spain
Dr. A. Beloqui
IKERBASQUE, Basque Foundation for Science
Maria Diaz de Haro 3, E-48013 Bilbao, Spain
Dr. D. M: Bauer, Dr. L. Fruk
[c]
[d]
[e]
Center for Functional Nanostructures (CFN)
Karlsruhe Institute of Technology (KIT)
Wolfgang-Gaede-Straße 1a, 76131 Karlsruhe, Germany
Dr. L. Fruk
[f]
Department of Chemical Engineering and Biotechnology
University of Cambridge
West Cambridge Site, Philippa Fawcett Drive, Cambridge CB3 0AS, United Kingdom
Prof. Dr. C. Barner-Kowollik
Centre of Materials Science, Physics and Mechanical Engineering
Queensland University of Technology (QUT)
Brisbane, QLD 4000, Australia
[g]
[g]
Prof. Dr. C. Barner-Kowollik
School of Chemistry, Physics and Mechanical Engineering
Queensland University of Technology (QUT)
Brisbane, QLD 4000, Australia
E-mail: christopher.barnerkowollik@qut.edu.au
Prof. Dr. G. Delaittre
[i]
Organic Functional Molecules, Organic Chemistry
University of Wuppertal
Gaußstrasse 20, 42119 Wuppertal, Germany
E-mail: delaittre@uni-wuppertal.de
Supporting information for this article is given via a link at the end of the document.
Abstract: We introduce the bioconjugation of polymers synthesized
far most employed polymer for PPCs is polyethylene glycol
by RAFT polymerization, and bearing no specific functional end group, (PEG). Yet, utilizing other polymers than PEG gives access to
by means of hetero-Diels–Alder cycloaddition through their inherent
terminal thiocarbonylthio moiety with a diene-modified model protein.
Quantitative conjugation occurs over the course of a few hours, at
ambient temperature and neutral pH, and in the absence of any
catalyst. Our technology platform affords thermoresponsive
bioconjugates, whose aggregation is solely controlled by the polymer
chains.
a wider range of properties and may elude some shortcomings
of PEG,7–12 notably its immunogenicity.13,14 In this context,
reversible
addition-fragmentation
transfer
(RAFT)
polymerization is one the most powerful synthetic techniques
to access macromolecules with defined chain length and (end-
group) functionality.15–17 One of the methods to achieve
RAFT-based PPCs involves reacting one end of the synthetic
polymer with one or several residues on the protein surface.
The reactive end of the RAFT polymer is typically introduced
through the reinitiating fragment – the so-called R group – of
a specifically designed chain transfer agent (CTA).12 The
Proteins are essential compounds in modern medicine and
biotechnology.
However,
their
physicochemical
characteristics brings about critical limitations, particularly in
terms of solubility and stability.1 One of the most important
ways of addressing these issues relies on the attachment of
synthetic polymer chains, in order to produce so-called
protein–polymer conjugates (PPCs).2–6 The pioneering and so
RAFT-hetero-Diels–Alder
cycloaddition
(RAFT-HDA)
emerged about a decade ago as a complementary and highly
efficient method for chain-end conjugation of RAFT polymers
without the need for introducing functional R groups.18–22
.
1
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10.1002/anie.202005747
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useful temperatures. Responsive PPCs31,32 are particularly
interesting for control of biomolecular activity,33 triggered
reversible self-assembly into biohybrid nanostructures34–38
and fast removal/recovery of proteins from solution.39,40
Interestingly, similar polymers with short oligoethylene glycol
side chains were shown not to exhibit the non-desired
antigenicity
of
PEG.41
2-cyanoprop-2-yl
diethoxyphosphoryldithioformate (CPDPDT) was employed
as CTA because it simultaneously enables the controlled
polymerization of acrylates and the synthesis of polymers with
a terminal C=S bond sufficiently electron-deficient for a rapid
HDA cycloaddition to occur.23 As seen in Figure 1, PmTEGAs
with number-average molar masses Mn of 2000 and 6000 g
mol–1 and narrow dispersities (Đ = 1.1–1.2) were obtained
(noted as PmTEGA2000 and PmTEGA6000, respectively).
Similarly, P(eDEGA-co-mOEGA)s CoP15000 and CoP18000,
with Mn of 15000 and 18000 g mol–1 (Đ = 1.3), respectively,
were
produced
by
CPDPDT-mediated
RAFT
copolymerization. All polymers displayed the classic
maximum of absorption at 327 nm (Figure S8), characteristic
of
the
π
→
π*
transition
in
the
diethoxyphosphoryldithioformate end group.
For conjugation reactions not based on natural amino
acids, reactive proteins can be obtained either by genetic
engineering42 or simple post-translational chemical
Scheme 1. Synthesis of bovin serum albumin polymer conjugates by RAFT-
HDA, as described in the current contribution.
RAFT-HDA relies on RAFT agents possessing a C=S
double bond with a specifically adjusted electron deficiency.
The latter should be sufficiently high to enable HDA with a
range of dienes, yet not too high in order for a well-controlled
RAFT polymerization to take place. While RAFT-HDA in
organic solvents requires heat, catalysts, or highly active
diene
partners
(e.g.,
cyclopentadiene
or
o-
quinodimethanes),18–22 we have previously demonstrated that
a fast RAFT-HDA is achieved in aqueous solutions simply by
mixing the components at ambient temperature and in the
absence of a catalyst, even with less reactive dienes.23 Such
mild conditions seem ideal for the functionalization of proteins,
which are generally sensitive to heat or additives. Importantly,
most if not all cycloadditions are biorthogonal, thus offer an
ideal platform to specifically conjugate polymers to
biomolecules.24 In the present contribution, we report the first
protein–polymer conjugates obtained through the RAFT-HDA
pathway (Scheme 1).
A range of water-soluble acrylic polymers based on
ethylene glycol side chains were first synthesized: (i)
homopolymers of triethylene glycol methyl ether acrylate
(PmTEGA) and (ii) copolymers of diethylene glycol ethyl ether
acrylate and oligoethylene glycol methyl ether acrylate
(P(eDEGA-co-mOEGA)). PmTEGA is water-soluble in the
useful temperature range of PPCs (< 55–70 °C)25–28 and may
impart a stealth character and improved solubility to proteins.
Figure 1. (Top) Synthetic route for oligoethylene glycol-based polyacrylates
by RAFT polymerization in the presence of 2-cyanoprop-2-yl
diethoxyphosphoryldithioformate (CPDPDT). (i) mTEGA, AIBN, ethanol,
60 °C. (ii) eDEGA:mOEGA 80/20 mol/mol, AIBN, ethanol, 60 °C. (Bottom)
Corresponding SEC traces.
P(eDEGA-co-mOEGA)s exhibit
a lower critical solution
temperature (LCST), which depends on the exact comonomer
ratio,29,30 and will lead to thermoresponsive PPCs at possibly
2
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10.1002/anie.202005747
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showed the incorporation of an average of 6–7 diene tags per
protein molecule, that is, x = 6–7 in Scheme 1 (Figure S10).
Protein–polymer conjugation was performed by simple
incubation of dBSA with the RAFT polymers in aqueous
buffers, in the absence of catalyst and at room temperature.
During initial experiments, it was observed that the
characteristic purple color of the RAFT polymer stock
solutions in aqueous media faded with time. It is known that
RAFT end groups are sensitive to a range of reagents, mostly
primary amines and basic conditions44 and that discoloration
of RAFT polymers implies end-group loss. For this reason, we
monitored this phenomenon by UV-Vis spectroscopic
measurements of PmTEGA6000 solutions in various aqueous
buffers. As expected, basic conditions led to the fastest
decrease in absorption at 327 nm and consequently the
fastest deactivation (Figure S11). Particularly, incubation in
bicarbonate buffer at pH 9.15 or Tris buffer at pH 8.1 led to
instantaneous full degradation. Phosphate buffer at the same
pH was less damaging. Decreasing pH further led to
significantly slower degradation. Eventually, sodium
phosphate buffer (50 mM at pH 6.0) was chosen for
bioconjugation as it offered the best compromise between the
slow degradation and close-to-neutral conditions. Before
protein–polymer conjugation was addressed, model HDA
conjugation reactions with DSS were performed. It was
observed that absorption at 327 nm decreased significantly
faster in reaction mixtures of PmTEGA6000 and DSS
compared to the polymer alone (Figure S12). Higher amounts
of DSS (2 and 3 equivalents) led to faster disappearance of
the characteristic RAFT moiety absorption, a clear sign of the
HDA reaction occurring at the C=S double bond.23
A range of conditions were assessed in order to
determine the optimum conditions for polymer bioconjugation.
Mixtures with various PmTEGA6000:dBSA molar ratios were
prepared in sodium phosphate buffer at pH 6.0 for overnight
reactions. As observed in gels obtained by SDS-PAGE and
the corresponding intensity plots (Figure 2A), higher
polymer:protein ratios generated species with higher molar
masses. With 80 eq. and above, conjugates with an average
of 6 to 7 grafted polymer chains were obtained, as deduced
from the  40 kDa shift. It can thus be assumed that in these
conditions all accessible diene groups within the protein have
been coupled via HDA reaction. Moreover, the kinetics of the
reaction was monitored using a 100-fold molar excess of
polymer (Figure 2B). The reaction was arrested at various
incubation times by removing the non-reacted PmTEGA6000
by size-exclusion centrifugation. We observed that the
conjugation of the first two polymer chains to dBSA occurred
within just 1–2 h, with the fully conjugated protein obtained
after 6 h. The first easily reachable diene moities might react
fast, while the attachment of further polymer chains is certainly
slowed down for more buried dienes and due the increasing
steric constraints imposed by already grafted polymer chains.
Figure 2. Coomassie-stained SDS-PAGE gels of BSA conjugates obtained by
RAFT-HDA with PmTEGA6000 (up) and corresponding electrophoretograms
(bottom). (A) Variation of the [PmTEGA6000]:[dBSA] ratio for a fixed reaction
time of 12 h: 25 (c), 50 (d), 80 (e), and 100 (f). (B) Variation of the reaction time
for a fixed [PmTEGA6000]:[dBSA] ratio of 100: 1 h (g), 2 h (h), 4 h (i), 6 h (j),
and 8 h (k). Molecular weight protein ladder (a) and control sample (b, dBSA)
are added as references.
modification.43 Here, we have chosen the latter for its ease of
implementation. To introduce diene moieties, the difunctional
linker 2,5-dioxopyrrolidin-1-yl (hexa-2,4-dien-1-yl)succinate
DSS (see Supporting Information), consisting of a sorbyl
group and a succinimidyl ester on either side, was readily
synthesized in two steps and reacted with the lysine residues
of the model protein bovin serum albumin (BSA). The diene-
functionalized BSA (dBSA) remained fully soluble in aqueous
medium and did not show any significant change in circular
dichroism (CD) (Figure S14). Mass spectrometry analysis
Moreover,
the
successful
preparation
of
dBSA-
PmTEGA2000 and dBSA-PmTEGA6000 conjugates was
also confirmed by a shift in the hydrodynamic diameter
distribution compared to that of the free protein, as measured
by dynamic light scattering (DLS) (Figure S13). Again, the
protein secondary structure was not affected by the
modification, as demonstrated by CD (Figure S14). Albeit not
an enzyme, BSA possesses an esterase activity that can be
Figure 3. Number-based hydrodynamic diameter distributions for BSA
conjugates obtained by RAFT-HDA with P(eDEGA-co-mOEGA) copolymers
CoP15000 and CoP18000. dBSA is shown as reference.
3
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10.1002/anie.202005747
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heating ramp from 40 to 55 C to aqueous solutions of
CoP15000 and CoP18000 polymers and monitored the
absorbance at 670 nm, a wavelength at which no interference
with possible chromophores may occur. We observed that
while CoP18000 showed turbidity at temperatures higher than
45 C, the turbidity of CoP15000 sample was detected only
above 48 C. We measured cloud points (temperature at 50%
of the maximal absorbance) of 49.5 and 46.5 C for CoP15000
and CoP18000 polymers, respectively.
Finally, we assessed how the thermoresponsive
properties of P(eDEGA-co-mOEGA) copolymers transferred
to their corresponding BSA conjugates. Note that BSA was
previously shown to be stable in the considered temperature
range (i.e., below 60 °C)[45,46] and that one could in any case
modulate the transition temperatures by varying the
compositions of the copolymers. As the turbidity of the media
implied the temperature-induced aggregation of the polymeric
components, the overall size of the PPCs is expected to
increase at temperatures higher than LCST, with the protein
component stabilizing the aggregates.34 Purified PPCs were
thus subjected to a heating ramp and the hydrodynamic
diameter of the particles was simultaneously measured. As
shown in Figure 4B, the average hydrodynamic diameter
increased from 7.9  1.2 and 9.1  1.5 nm at 40 C to 69  5
and 77  4 nm at 55 C for dBSA-CoP15000 and dBSA-
CoP18000 conjugates, respectively. Interestingly, the onsets
of aggregation of the PPCs match well those measured for the
polymers alone, and the cloud points only slightly decrease:
48.6 and 45.8 C for dBSA-CoP15000 and dBSA-CoP18000,
respectively, versus 49.5 and 46.5 C for their corresponding
free polymers. The polymers are physically bound to the
protein counterpart and the protein does not seem to
significantly interfere in the temperature-induced physical
aggregation of the polymers.
Figure 4. (A) Evolution of the turbidity measured at 670 nm with temperature
for aqueous solutions of CoP15000 and CoP18000 (15 µM in PBS). (B)
Evolution of number-average hydrodynamic diameters of dBSA-CoP15000
and dBSA-CoP18000 conjugates with temperature as measured by DLS. In
both cases, measurements were carried out with 1 °C increments and a
stabilitization time of 5 min before acquisition.
In conclusion, we introduce the application of the RAFT-
HDA chemistry for catalyst-free protein–polymer conjugation
under mild conditions (ambient temperature, neutral pH).
Using this method, we have decorated the surface of BSA with
up to 7 hydrophilic polymer chains, as well as with
thermoresponsive polymers. The reaction of the diene-
exploited to further assess modifications. In a colorimetric
glyceryl acetate-based esterase assay (Figure S16), no
significant difference between BSA and dBSA-PmTEGA6000
were observed, which confirms the conservation of the protein
structure and stability.
functionalized
protein
with
the
RAFT-derived
diethoxyphosphoryldithioester polymer is relatively fast, pH-
dependent, and can be monitored by UV-Vis spectroscopy
and SDS-PAGE. Finally, we demonstrate that the
thermoresponsive properties of the polymers are transferred
to the protein–polymer conjugates, showing similar phase-
separation temperatures. The current procedure entails pre-
conditioning of the protein through covalent anchoring of
reactive diene tags. Further control of the bioconjugation
degree and location is certainly achievable by the introduction
of genetically encoded unnatural aminoacid containing the
diene group.42,45
The conjugation of functional polymers to proteins leads
to PPCs with specific properties. Here, the RAFT-HDA
conjugation was carried out with the thermoresponsive
P(eDEGA-co-mOEGA)
copolymers
CoP15000
and
CoP18000 (see Figure 1). DLS measurements revealed a
significant increase of the average hydrodynamic diameter
from 6.5  0.8 nm for dBSA to 7.9  1.2 and 9.1  1.5 nm after
the conjugation reaction with CoP15000 and CoP18000,
respectively (Figure 3), confirming successful conjugation. As
for PmTEGA6000, CD measurements revealed no alteration
in the secondary structure of BSA through the grafting of
CoP15000 and CoP18000 (Figure S15).
The thermoresponsive behavior of the newly generated BSA
conjugates was subsequently evaluated. P(eDEGA-co-
Acknowledgements
mOEGA)
copolymers
typically
exhibit
a
tunable
thermoresponsive behavior over the 25–75 C range,
depending on their comonomer composition and molar
mass.29,30 The thermal transition is readily detectable via an
increase in the turbidity of the sample, which can be measured
within a UV-Vis spectrophotometer (Figure 4A). We applied a
A. B. thanks the Spanish Research Agency (AEI) for the funds
within Ramón y Cajal program (RYC2018-025923-I) and
Retos call (PID2019-110239RB-I00). C.B.-K. acknowledges
funding by the Australian Research Council (ARC) in the
context of a Laureate Fellowship and continued key support
4
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