RAFT polymers for protein recognition

Article abstract of DOI:10.3762/bjoc.6.66

A new family of linear polymers with pronounced affinity for arginine- and lysine-rich proteins has been created. To this end, N-isopropylacrylamide (NIPAM) was copolymerized in water with a binding monomer and a hydrophobic comonomer using a living radical polymerization (RAFT). The resulting copolymers were water-soluble and displayed narrow polydispersities. They formed tight complexes with basic proteins depending on the nature and amount of the binding monomer as well as on the choice of the added hydrophobic comonomer.

Full text of DOI:10.3762/bjoc.6.66

RAFT polymers for protein recognition
Alan F. Tominey1, Julia Liese1, Sun Wei2, Klaus Kowski2,
Thomas Schrader*2 and Arno Kraft*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2010, 6, No. 66.
1Chemistry, School of Engineering & Physical Sciences, Heriot-Watt
University, Riccarton, Edinburgh EH14 4AS, United Kingdom and
2Universität Duisburg-Essen, Fachbereich Chemie, Institut für
Organische Chemie, Universitätsstraße 5, 45117 Essen, Germany
doi:10.3762/bjoc.6.66
Received: 02 February 2010
Accepted: 28 May 2010
Published: 17 June 2010
Email:
Thomas Schrader* - Thomas.Schrader@uni-due.de; Arno Kraft* -
A.Kraft@hw.ac.uk
Guest Editor: C. A. Schalley
© 2010 Tominey et al; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
Keywords:
electrostatic interactions; hydrophobic effect; isothermal calorimetry;
protein recognition; RAFT polymers
Abstract
A new family of linear polymers with pronounced affinity for arginine- and lysine-rich proteins has been created. To this end,
N-isopropylacrylamide (NIPAM) was copolymerized in water with a binding monomer and a hydrophobic comonomer using a
living radical polymerization (RAFT). The resulting copolymers were water-soluble and displayed narrow polydispersities. They
formed tight complexes with basic proteins depending on the nature and amount of the binding monomer as well as on the choice of
the added hydrophobic comonomer.
Introduction
The ability of biological receptors to bind strongly and specific- characteristic shape, size and highly complex functionalized
ally to a particular molecular target is an essential part of bio- surface. Artificial protein receptors are desired for protein
logical machinery. The best example is the immune system enrichment and purification, sensing and diagnostics applica-
where antibodies are generated in response to minute amounts tions, as well as therapeutic uses involving interference with
of foreign antigens. A continual challenge in nanoscale chem- critical protein–protein interactions.
istry is to mimic the biological molecular recognition functions
by synthetic chemistry with the aim of producing systems of Multivalency represents the key to generate high-affinity ma-
lower complexity. When successful, this will enable the manu- terials for biomacromolecules with a sufficient number of
facturing of robust and specific synthetic receptors for a given binding sites for Coulomb attraction and hydrophobic interac-
protein target [1]. Proteins are a formidable challenge in this tions [2]. A statistical evaluation of crystal structures led to the
respect because they represent large macromolecules with a discovery that hot spots in protein–protein contact areas are
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Beilstein J. Org. Chem. 2010, 6, No. 66.
enriched in aromatic amino acids and in arginine. These are the overall emission intensity change resulting from protein
often surrounded by energetically less important residues that addition will reflect only a virtual averaged value, because short
most likely serve to occlude bulk solvent from the hot spot and and long chains will bind simultaneously, most likely with
lower the local dielectric constant [3,4].
different affinities and stoichiometries. A quantitative descrip-
tion must inherently suffer from this averaging effect.
With this principle in mind, several groups have designed rela-
tively simple linear polymeric structures with branched ionic Results and Discussion
comonomers and thus achieved remarkable affinities and bio- Reversible addition–fragmentation chain transfer (RAFT) poly-
logical properties. In their elegant work, Kulkarni et al. reported merization [10] and atom-transfer radical polymerization
the use of NIPAM-based copolymers for lysozyme recovery by (ATRP) have become extremely useful tools for the controlled
affinity thermoprecipitation. These polymers contained multiple synthesis of a wide range of polymers and could solve both
acetamido groups in a hydrophilic environment for maximum problems by formation of monodisperse functionalized polymer
interaction with the catalytic cleft and achieved high affinities chains of equal length, without the need for final polymer-
[5]. Rotello and Thayumanavan have described amphiphilic analogous deprotection. So far, there have been no reports of
polymer scaffolds, which nonspecifically bound to the successful use of ATRP with acrylamides. In contrast,
chymotrypsin, inhibited its peptidase activity and modulated RAFT can be used in a variety of solvents and, most import-
substrate specificity; very high ionic strengths again released antly, it is compatible with NIPAM [11,12]. For this reason,
the protein from the polymer [6,7].
RAFT was chosen in this paper as the preferred method for
controlled synthesis of linear polymers.
Protein recognition by multifunctional polymeric hosts features
two prominent advantages. On one hand, it simplifies the For initial screenings we selected a combination of anionic and
complex recognition interface to isolated 1:1 complexes hydrophobic binding monomers (Figure 1) that were well suited
between monomeric binding sites and single complementary for simultaneous recognition of basic amino acids (Lys/Arg) as
amino acid residues, while simultaneously allowing for an well as nonpolar residues (Val, Leu, Ile, Phe). NIPAM was
extensive induced-fit process of the linear polymer on the chosen as the main comonomer because it forms polymers
protein surface – in other words they encourage polymer/protein which are water-soluble at room temperature and even allow
self-assembly in order to maximize attractive noncovalent inter- thermoprecipitation with a bound protein guest. NIPAM-based
actions.
polymers are also reminiscent of peptides since both contain an
amide group in the repeat unit. RAFT makes use of a chain
A second major advantage of multivalent polymeric hosts is transfer agent (CTA) for which we selected the water-soluble
their rapid and efficient synthesis at low cost as well as the high trithiocarbonate 8 [13,14] which efficiently caps the growing
proteolytic stabilities of most polymer backbones. They also polymer chain, but can be completely removed from the final
pose fewer racemization problems which often accompany polymer by reaction with an excess of AIBN and selective
proteinogenic amino acids in peptidic environments.
polymer precipitation into hexane [11].
In recent years, our group has developed water-soluble linear Three anionic comonomers suitable for binding lysine and
polymeric protein binders which contained one or more arginine were chosen from earlier work with linear polymers
different binding monomers and displayed micromolar protein and microgels [9,15,16]: Sodium methacrylate (2) (S), polymer-
affinities [8], accompanied in a number of cases with prom- izable tetrazolate 3 (T) and bisphosphonate 4 (B). These anionic
ising protein selectivities [9]. These linear polymers were all comonomers were directly copolymerized with NIPAM and a
prepared by free radical copolymerization in DMF followed by hydrophobic acrylamide. The latter carried cyclohexyl (CH),
deprotection of the binding monomers in polymer-analogous benzyl (BN) or octyl (OC) moieties as hydrophobic residues. In
transformations. Thus, a polymerized bisphosphonate tetra- the polymer designation code, the first letter indicates the
methyl ester was subjected to LiBr-assisted nucleophilic anionic comonomer used (S, T or B), the subsequent number its
cleavage to furnish the free bisphosphonate dianion binding mol % in the monomer mixture; the two-letter abbreviation
site. This procedure has two major drawbacks. First, if the func- (CH, BN or OC) stands for the hydrophobic comonomer used,
tional groups on the polymer backbone become restricted in again followed by the mol %; the balance to 100 mol % was
their accessability, the final deprotection step will suffer from made up by NIPAM. For example, S10CH10 means that this
low conversion rates. Second, the resulting material is polydis- RAFT copolymer was made from sodium methacrylate
perse, rendering the characterization of the protein binding (10 mol %), N-cyclohexylacrylamide (10 mol %), and NIPAM
event problematic. Even with incorporated fluorescence labels, (80 mol %).
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Figure 1: Structures of monomers 1–7 and chain transfer agent 8 used in the RAFT polymerizations.
RAFT polymerizations were carried out in methanol at 60 °C dissociation constant of 1.6 × 103 M−1 could be fitted to the
for 48 hours in the presence of CTA 8 and azo initiator V-50. binding isotherm when the second derivative values of the
The monomer concentration was 0.75 M, the molar ratio of protein at 415 nm were plotted against the RAFT polymer
[V-50]/[CTA] was 3, and the concentration of CTA and V-50 concentration (Figure 2b). Cytochrome C already showed notic-
were adjusted to target polymers with a molecular weight of able and selective binding to microgels [16] containing
3000, 7000 or 17000 g mol −1 at full conversion. This is 10 mol % sodium methacrylate and RAFT copolymers of
possible since the degree of polymerization under RAFT condi- similar composition. Unlike microgels whose molecular weight
tions is equal to the ratio between monomer and chain transfer is very high (typically 106–108 g mol−1), cytochrome C pos-
reagent concentration. Conversion was almost 100%, and sesses a relatively small molecular weight similar to the RAFT
copolymers were isolated by precipitation in hexane. The copolymers. As a result, the RAFT copolymers and cyto-
absence of low-molecular weight impurities such as monomers chrome C favor 1:1 binding. The incorporation of a hydro-
was ascertained by 1H NMR spectroscopy. Molecular weights phobic comonomer further improved binding. The maximum
were determined by gel-permeation chromatography (GPC) binding strength was observed for polymers containing
analysis of the copolymers. Narrow polydispersities (≤1.3) were 15 mol % of N-cyclohexylacrylamide and 20 mol % of sodium
observed for the shorter copolymers, although the highest methacrylate (Table 1).
molecular weights (targeted at 17000 g mol−1) reached only
experimental values of 11,000–12,000 g mol−1 and also For an independent comparison, the same protein–polymer pairs
produced slightly higher polydispersities (1.56). For compar- were subsequently subjected to microcalorimetric titrations
ison, some copolymers such as S20CH15 were also prepared (Figure 3), which confirmed the major trends gained from spec-
with a molecular weight of ~3000 g mol−1.
Table 1: UV–vis titrations of cytochrome C with selected RAFT copoly-
mers.
Titrations were first carried out by UV–vis spectroscopy with
cytochrome C, a protein carrying a chromophore. Second
derivative spectra were calculated using the Savitzky–Golay
algorithm [17-19]. The second derivative is a useful method of
refining the spectra to reveal subtle changes in the UV–vis
absorption plot. The UV titration of a typical RAFT copolymer
into a solution of cytochrome C in a phosphate buffer (pH 7,
0.15 M KCl) showed characteristic second derivative spectra,
similar to those observed in the titrations of microgels into
protein solutions [16]. Isosbestic points are clearly visible along
with a bathochromic shift of the absorbance peak (Figure 2a). A
Macroscopic
Ka / M−1
Polymer : Protein
Stoichiometry
RAFT Copolymera
S10CH10
S10BN10
S10OC10
S10CH15
S20CH15
400
n.d.
1:1
n.d.
1:1
1:1
n.d.
20
1600
>2000
aS = sodium methacrylate, CH = N-cyclohexylacrylamide, BN =
N-benzylacrylamide, OC = N-octylacrylamide.
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Beilstein J. Org. Chem. 2010, 6, No. 66.
mers, which also corresponded to previous results with micro-
gels [16].
By contrast, tetrazolate copolymer T20CH15 and bisphos-
phonate copolymer B20CH15 showed large enthalpy changes
and hence much higher Ka values (>106 M−1) which were about
two orders of magnitude higher than those achieved with
sodium methacrylate copolymer S20CH15 (~104 M−1). This is
not surprising for the bisphosphonate, which carries twice the
amount of negative charges. However, the monoanionic
tetrazolate anion is very similar in acidity and hydrogen bond
pattern to a carboxylate, so that similar affinities would have
been expected. Most likely, the difference is explained by inter-
actions with the π-face of the tetrazolate anion, which are not
possible with a carboxylate.
In all cases, protein complexation by RAFT polymers was
endothermic, i.e., entropy-driven. Hence, unspecific electro-
static attraction in combination with solvophobic forces contrib-
uted the most towards protein binding.
To quantify the contribution of nonpolar comonomers, hemo-
globin was also titrated with pure tetrazolate and bisphos-
phonate copolymers. Intriguingly, Ka values dropped substan-
tially by 1–2 orders of magnitude (see Table 1: T20 vs
T20CH15). In other words, the random incorporation of cyclo-
hexyl comonomers into the polymer was beneficial for the
protein recognition event. Close inspection of thermodynamic
data revealed that the entropy term was responsible for this
increased affinity. We therefore tentatively explain the gain in
free energy by an increased classical hydrophobic effect due to
the presence of additional nonpolar cyclohexyl residues
throughout the polymer chain.
Figure 2: a) Second derivative UV–vis spectra [17-19] observed
during a full titration of a stock solution of RAFT copolymer S10CH15
(6.3 × 10−3 mol L−1) into a solution of cyt C (9.9 × 10−6 mol L−1) in
phosphate buffer at pH 7 and ionic strength of 0.15 mol L−1 KCl. The
arrows indicate increasing amounts of RAFT copolymer added. b) Plot
of second derivative values at 416 nm as a function of volume (in mL)
of RAFT copolymer solution added. The filled diamonds are experi-
mental values, whereas the drawn curve represents the calculated
isotherm for a Ka of 1.6 × 103 M−1 assuming 1:1 binding [20].
For biological applications, it is desirable to keep the polymer
troscopic detection but differed in several details (Table 2). size close to the size of the protein, so that specific 1:1 com-
Specifically, RAFT copolymers S10CH10, S10BN10, plexation is favored (Figure 4). In order to investigate this
S10OC10, S10CH15 and S20CH15 were examined in their assumption, the sodium methacrylate polymer S20CH15 was
complex formation with cytochrome C (MW 14 kD, pI 9.2) and titrated as a short oligomer (MW 3000 g mol−1) and an average-
hemoglobin (MW 68 kD, pI 7.0). Negligible heat changes were size polymer (MW 12000 g mol−1). Direct comparison
observed for all titrations with sodium methacrylate-containing produced a drastic difference: No binding could be detected for
polymers, consistent with the small Ka values already deter- the short version, indicating that size matters and promotes
mined by UV–vis titrations (20–1600 M−1); obviously, the multivalent or cooperative binding.
methacrylate anion is a weak binder for lysines and arginines on
these protein surfaces. Moderate binding (3 × 104 M−1) was Finally, the protein series was extended to lysine-rich histone
only detected with S20CH15, which carries twice the amount of (pI 10), lysozyme (pI 9), proteinase K (pI 8) and bovine serum
carboxylate groups. Association constants were initially calcu- albumin or BSA (pI 6). Again, the strong binders B20CH15 and
lated for each 1:1 complexation event of a single protein by the T20CH15 were examined concerning their affinities towards
copolymer [20]. However, even with S20CH15, no binding was proteins of varying pI (Table 2). In direct comparison, the
detectable with hemoglobin, confirming an interesting cyto- bisphosphonate seems to be superior to the tetrazolate. While
chrome C preference of all sodium methacrylate-carrying poly- B20CH15 stayed well below micromolar Kd values even with
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Beilstein J. Org. Chem. 2010, 6, No. 66.
Figure 3: Isothermal calorimetric binding curves for selected polymer/protein host–guest pairs. a) Typical binding curves with representative proteins
for the major polymers based on the three anionic binding sites. Note the marked affinity increase from sodium methacrylate over tetrazolate to
bisphosphonate dianion. b) Binding curves of two bisphosphonate RAFT copolymers, one without and one with the hydrophobic N-cyclohexylacryl-
amide comonomer (15 mol %). The contribution of the nonpolar cyclohexyl monomer towards hemoglobin binding is evident from the steeper slope of
the binding curve.
BSA, T20CH15 hardly ever reached the micromolar regime. chiometry, curve fitting is greatly improved with a sequential
Obviously, the bisphosphonate’s high negative charge density is binding or 2-sites model [21]. In all these cases, the first
especially effective for protein surfaces with a high density of polymer binds very tightly to the protein surface, but leaves
basic amino acids such as the DNA-binding histones or for significant room for a second polymer forming an – admittedly
those offering distinct clusters of cationic amino acid residues much weaker – 2:1 complex. Histone association with
(e.g. BSA). Interestingly, although in most cases nonlinear B20CH15 is an illustrative example. The first Kd value is
regression converges with an assumed 1:1 complex stoi- 16 nM, followed by very weak binding at a second site with a
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Beilstein J. Org. Chem. 2010, 6, No. 66.
Table 2: Microcalorimetric protein titrations with RAFT polymers.
RAFT
copolymera
Polymer :
protein
Ka per residue /
Monomer : ΔG / kcal ΔH / kcal TΔS / kcal
Proteinb Macroscopic Ka / M−1
M–1
protein
mol−1
mol−1
mol−1
S10CH10
S10BN10
S10OC10
S10CH15
S10CH10
S20CH15b
S20CH15c
S20CH15c
T20
Cyt C
Cyt C
Cyt C
Cyt C
Hem
Cyt C
Cyt C
Hem
Hem
His
NA
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
NA
NA
–
–
NA
–
NA
–
–
–
–
–
–
–
NA
–
3 × 104
7:1
–
9 × 102
–
15:1
–
NA
~2 × 104
–
~9 × 103
2 × 104
1 × 104
3 × 103
1 × 104
6 × 103
7 × 104
2 × 105
4 × 104
–
–
T20CH15
8 × 105 → 5 × 103
8 × 105 → 5 × 103
4 × 105 → 3 × 103
4 × 106
2 sites
2 sites
2 sites
3:1
10:1
2:1
2 sites
2:1
–
–
Lys
7:1
13:1
78:1
6:1
20:1
18:1
15:1
–
−5.5
−4.6
−5.7
−5.2
−6.6
−7.4
−6.3
–
+21.2
+17.7
+4.2
+4.4
+1.2
+2.4
+0.7
–
+26.7
+22.3
+9.9
+9.6
+7.8
+9.8
7.0
Prot K
Hem
BSA
Hem
His
4 × 105 → 3 × 103
7 × 105
B20
B20CH15
6 × 107 → 7 × 102
1 × 106 → 3 × 103
NA
Lys
Prot K
Hem
BSA
–
4 × 106
1:1
3:1
2 × 105
9 × 104
15:1
5:1
−7.2
−6.7
+5.1
+15.4
+12.3
+22.1
2 × 106
aS = sodium methacrylate, T = tetrazolate 3, B = bisphosphonate 4, CH = N-cyclohexylacrylamide, BN = N-benzylacrylamide, OC = N-octylacryl-
amide.
bCyt C = cytochrome C; Hem = hemoglobin; His = histone; Lys = lysozyme; Prot K = proteinase K; BSA = bovine serum albumin.
cMW ~3000 g mol−1.
dMW ~17000 g mol−1. NA indicates that no binding constant and thermodynamic data were obtained from microcalorimetry titrations, because heat
changes were too small.
Kd of 1 mM. With respect to varying pI values, both RAFT
polymers display little selectivity: From lysozyme (pI > 9)
down to BSA (pI < 6) protein affinities vary by less than one
order of magnitude.
Conclusion
In summary, RAFT copolymerization of NIPAM with
monomers containing anionic binding sites for basic amino
acids led to polymers of low polydispersities which were
effective protein binders in buffered aqueous solution, with
tunable stoichiometries close to the ideal 1:1 ratio. Although
molecular recognition is based on unspecific electrostatic attrac-
tion and hydrophobic forces, those proteins which feature a high
density of positive charges on their surfaces are bound espe-
cially well by the bisphosphonate site, in some cases reaching
micromolar or sub-micromolar Kd values. Copolymerization
with N-cyclohexylacrylamide introduced additional nonpolar
groups beneficial for protein binding, leading to a substantial
entropy gain and significantly improving protein affinities. The
best pair was a bisphosphonate-containing RAFT copolymer
and lysine–rich histone (Kd = 16 nM). In the future, we intend
Figure 4: Graphical illustration of the potential binding mode on hemo-
globin tetramer (represented as electrostatic potential surface, lysines
= blue). The RAFT copolymer T20CH15 (tetrazole rings = red) under-
goes an extensive induced fit procedure on the protein surface maxim-
izing unspecific electrostatic and hydrophobic contacts. Some NIPAM
sidechains were omitted for clarity.
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Full experimental procedures, characterization details,
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[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-6-66-S1.pdf]
Acknowledgements
License and Terms
We gratefully thank the School of Engineering and Physical
Sciences at Heriot-Watt University for support and Dr Steven
Rimmer (Sheffield University) for help with the aqueous GPC
of the RAFT copolymers.
This is an Open Access article under the terms of the
Creative Commons Attribution License
(http://creativecommons.org/licenses/by/2.0), which
permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
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