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Chapter 12
Biocatalytic Polymerization, Bioinspired Surfactants, and Bioconjugates Using RAFT Polymerization Alex P. Daniselson,1 Melissa Lucius Dougherty,1 Rebecca Falatach,2 Thaiesha A. Wright,1 Emily E. Clark,1 Andrew Craig,1 Indra D. Sahu,1 Jason A. Berberich,2 Richard C. Page,1 Gary A. Lorigan,1 and Dominik Konkolewicz*,1 1Department
of Chemistry and Biochemistry, Miami University, 651 E. High Street, Oxford, Ohio 45056, United States 2Department of Chemical, Paper and Biomedical Engineering, Miami University, 650 E. High Street, Oxford, Ohio 45056, United States *E-mail:
[email protected].
Reversible addition-fragmentation chain transfer polymerization (RAFT) is a versatile reversible deactivation radical polymerization tool. RAFT is compatible with a wide range of chemical functionalities, making it well suited for aqueous polymerization. This chapter explores enzymatically initiated RAFT and biohybrid materials using RAFT. The biohybrids discussed in this chapter are well-defined protein polymer bioconjugates, and lipid polymer nanoscale discs. Protein-polymer conjugates are discussed with respect to the impact of the polymer on the activity of the protein is, and the lipid/polymer hybrids are evaluated in terms of the surfactant properties of the RAFT polymer.
Introduction Over the past 2 decades, reversible deactivation radical polymerization (RDRP) methods have lead to a vast range of new macromolecules with new structures and functionalities and powerful polymeric materials (1, 2). Control over the polymerization in RDRP reactions is achieved through a dynamic equilibrium between dormant and active forms of the polymer (2, 3). Using RDRP © 2018 American Chemical Society Matyjaszewski et al.; Reversible Deactivation Radical Polymerization: Materials and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
methods, polymers can be synthesized with control over polymer microstructure comparable to “living”-ionic polymerizations, with tolerance to monomer functionality similar to conventional radical polymerizations (4). The three most commonly used RDRP techniques are nitroxide mediated polymerization (NMP) (5, 6), atom transfer radical polymerization (ATRP) (7–9), and reversible addition-fragmentation chain transfer polymerization (RAFT) (10–13). RAFT polymerization is a metal-free degenerative transfer process, where a propagating radical is able to rapidly exchange among thiocarbonyl-thio groups. This is shown in Scheme 1. RAFT is a particularly versatile technique, since it is compatible with a wide range of monomers under relatively mild conditions, including aqueous conditions (14). This makes RAFT an excellent tool for biological applications, and RAFT polymerization to be performed under biorelevant conditions (15).
Scheme 1. Currently accepted mechanism of reversible addition-fragmentation chain transfer (RAFT) polymerization. The well-defined polymers that result from RAFT polymerization and the tolerance to aqueous media and functional groups enable RAFT to be employed in a range of biological and biomaterials applications. This chapter highlights three areas where the versatility of RAFT creates biorelevant structures with increased precision. Enzymatic polymerization is an important field of biocatalysis, where the high efficiency of enzymatic biocatalysis is used to create polymers under mild conditions (16, 17). Metalloprotein-catalyzed ATRP is an area of research, where proteins such as laccases, hemoglobin or horseradish peroxidase participate in the ATRP activation/deactivation equilibrium (18–23). Enzymes have also been used to remove oxygen from radical polymerization media (24, 25), which is especially important at low volumes where traditional deoxygenation is very challenging (26). Further, horseradish peroxidase (HRP) has been known as an effective enzyme to catalyze and initiate conventional free radical polymerization of water soluble monomers such as acrylamide (18, 27–33). This creates a stepping stone to controlled polymerization since HRP can catalytically generate radicals that can feed into the RAFT mechanism to give well-defined polymers. In part, this chapter highlights efficient biocatalytic RAFT polymerization, where HRP catalytically initiates RAFT polymerization, giving well-defined water-soluble polymers. 220 Matyjaszewski et al.; Reversible Deactivation Radical Polymerization: Materials and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Building off of the precision polymerization enabled by RAFT, well-defined biohybrid materials can be synthesized. This chapter will highlight two distinct types of biohybrid materials: protein-polymer bioconjugates (34–36), where a synthetic polymer and a structurally defined protein are covalently bonded together, as well as styrene maleic acid lipid particles (SMALPs) (37, 38), where a synthetic polymer of styrene-co-maleic acid and lipid molecules form well-defined nanoparticles by non-covalent interactions. These biohybrid materials have the potential to impact a range of biomedical applications including protein therapeutics with improved potency and circulation lifetime, enzymatic biocatalysts with enhanced activity and stability, as well as materials for structural biology studies. In all cases, the ability of RAFT to create well-defined polymer materials with excellent tolerance to functionalities enables these advanced applications. This chapter will describe enzymatic protein-polymer bioconjugates, as well as lipid-polymer hybrid nanoparticles with tunable size and structure.
Enzymatically Catalyzed RAFT Polymerization Despite the excellent tolerance to chemical functionality and ease of implementation, RAFT polymerization most often uses thermal radical initiators to generated the radicals needed to drive the polymerization forward (12). This can be a challenge for systems that are heat sensitive such as biological media and protein containing samples. Therefore, efficient biocatalytic radical generation could enable RAFT to be rapidly performed under ambient conditions. Already, metaloproteins of hemoglobin, laccase, and horseradish peroxidase (HRP) have shown significant potential as catalysts for ATRP (19, 20, 22). In the catalytic cycle of HRP, hydrogen peroxide is catalytically converted to OH radicals (39), and in the presence of an appropriate radical mediator such as acetylacetone (acac) carbon based radicals that can initiate radical polymerization are generated (29). Introducing an appropriate RAFT chain transfer agent in the presence of this HRP catalyzed radical generation is hypothesized to lead to an efficient and well-controlled polymerization. The proposed mechanism of HRP mediated RAFT polymerization is given in Scheme 2. Figure 1 shows efficient and well-controlled RAFT polymerization of dimethyl acrylamide (DMAm) using HRP to catalytically generate radicals. Reaction conditions were chosen to be biorelevant, in particular near ambient temperatures of 25 °C and less than 20% monomer by volume in an aqueous medium comprised of an acetate/acetic acid buffer at pH =5.5 (40). All experimental details may be found in the literature (40). High conversion (>95%) is achieved in less than 30 min, after a short induction period. This induction period, could be due to residual oxygen in the aliquot containing the enzyme stock solution. Although all other reaction components are thoroughly deoxygenated, to minimize the denaturation of the enzyme the enzyme solution is not sparged with nitrogen. Although the polymerization of DMAm is rapid, the data in Figure 1B and 1C indicates that well-defined polymers are created by this approach. Polymers with narrow molecular weight distributions, 221 Matyjaszewski et al.; Reversible Deactivation Radical Polymerization: Materials and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
linear evolution of number averaged molecular weight (Mn) with conversion that closely follows the theoretical line and low dispersities (Mw/Mn). The HRP catalyzed RAFT process is applicable to a range of water-soluble monomers including N-isopropylacrylamide (NiPAm) and oligo(ethylene oxide)methylether acrylate (OEOA). Figure 2 displays the well-defined polymers of each monomer can be synthesized using the HRP catalyzed RAFT method. This makes enzymatically initiated RAFT polymerization a useful tool for the synthesis of a wide range of macromolecules in aqueous media under benign conditions.
Scheme 2. Proposed mechanism of horseradish peroxidase catalyzed reversible addition-fragmentation chain transfer polymerization. Reprinted with permission from Ref. (40). Copyright 2016 John Wiley and Sons.
Figure 1. RAFT polymerization of DMAm initiated by HRP catalysis. (A) Semilogarithmic plot of DMAm polymerization, (B) Evolution of Mn (Solid points) and Mw/Mn (Hollow points) with conversion. Conditions: [DMAm]:[PAETC]:[acac]:[H2O2]= 100:1:9.5:1.7, [M]=170 mM, [HRP]= 0.71 mg/mL in 4.2 mL of pH=5.5, 20 mM acetate buffer at 25 °C. Reprinted with permission from Ref. (40). Copyright 2016 John Wiley and Sons. 222 Matyjaszewski et al.; Reversible Deactivation Radical Polymerization: Materials and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Figure 2. Molecular weight distribution of poly(DMAm), poly(OEOA) and poly(NiPAm) synthesized by HRP initiated RAFT. Conditions: [M]:[PAETC]:[acac]:[H2O2]=100:1:9.5:1.7, [M]=170 mM, [HRP]= 0.71 mg/mL in 4.2 mL of pH=5.5, 20 mM acetate buffer at 25 °C. Reprinted with permission from Ref. (40). Copyright 2016 John Wiley and Sons.
Enzyme Polymer Bioconjugates by gRAFT-to and gRAFT-from The excellent tolerance to monomer functionality and the compatibility with aqueous polymerization makes RAFT an excellent tool for bioconjugate synthesis (41). Generally, bioconjugates are synthesized either by a grafting to approach, where the polymer is first generated and subsequently attached to the biomolecule, or by grafting-from where an initiator or chain transfer agent is attached to the biomolecule, with subsequent polymerization directly from the biomolecule’s surface (36). Both approaches have been used to create well-defined bioconjugates by RAFT (36, 41). Each strategy has distinct advantages and disadvantages. Grafting-to has the advantage of synthesizing the polymer under conditions optimized for the polymer synthesis, although it can be difficult to separate the unreacted polymer from the bioconjugate, and high grafting densities are a challenge due to steric repulsion of one attached chain can prevent the attachment of a subsequent chain (36). Grafting-from leads to facile purification due to the major impurities being small molecules rather than polymer chain, although the polymerization must be performed under bio-friendly conditions and in certain cases the attached initiating group can alter the solubility of the biomolecule (36). To overcome these limitations, a hybrid grafting-to of a short water-soluble oligomer, followed by grafting-from strategy is implemented. This is demonstrated in Scheme 3. 223 Matyjaszewski et al.; Reversible Deactivation Radical Polymerization: Materials and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Scheme 3. Grafting to and grafting from biomolecules, with lysozyme shown in this example. Reprinted with permission from Ref. (42). Copyright 2016 American Chemical Society. The grafting-to followed by grafting-from approach was applied to lysozyme as a model enzyme, as outlined in the literature (42). As seen in Figure 3, there is a clear and positive shift of bioconjugate molecular weight upon the attachment of an oligomer of acrylamide (Am) containing a trithiocarbonate RAFT end group to lysozyme in poly(acrylamide) gel electrophoresis (PAGE). The subsequent chain extension of the RAFT end-group with a range of different monomers was achieved as evidenced by the major shift of conjugates to higher molecular weights in the SDS-PAGE. This indicates that the grafting-to and grafting from approach can be used to create complex bioconjugates from a wide range of functional monomers. Two series of conjugates were generated, the L series that had a low degree of polymerization of ca. 50 units, and the H series that had a relatively high degree of polymerization of ca. 250 units. In all cases the grafting from process was performed at 30 °C, using VA-044 as the radical initiator (42). The L-Am/AA* and L-Am/DMAEMA* had similar chain lengths to the L-Am/AA and L-Am/DMAEMA materials, except L-Am/AA* and L-Am/DMAEMA* had twice the content of the AA or DMAEMA monomer as L-Am/AA or L-Am/DMAEMA bioconjugates. The bioconjugates were assessed for enzymatic activity against two substrates, as shown in Figure 4. A small trimer of N-acetyl glucosamine(NAG) with a fluorescent tag (NAG3-MuF) and Micrococcus lysodeikticus, a large micron sized substrate that is a native substrate for lysozyme. As indicated in Figure 4, polymer conjugation reduces lysozyme activity against both the small fluorescent molecule substrate and the large Micrococcus substrate. In general the small molecule substrate has a small reduction of activity, presumably due to some steric occlusion of the active site by the attached polymer. In contrast, against the large Micrococcus substrate, the enzyme exhibits greatly reduced activity, particularly with the high molecular weight polymers attached. This is presumably due to the enhanced effect of steric interactions of the polymer against the large Micrococcus substrate. 224 Matyjaszewski et al.; Reversible Deactivation Radical Polymerization: Materials and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Figure 3. Poly(acrylamide) gel electrophoresis data of lysozyme and its bioconjugates. A shows the PAGE data for the majority of the lower molecular weight (L) conjugates. B shows the PAGE for the higher molecular weight conjugates (H) and the L-AGA sample. Adapted with permission from Ref. (42). Copyright 2016 American Chemical Society.
In general against, the anionic Micrococcus substrate the higher molecular weight non-ionic polymers lead to a similar impacts on activity. However, with the anionic poly(acrylic acid) poly(AA) and the cationic poly((N,N-dimethyl amino)ethyl methacrylate) (DMAEMA) copolymers with acrylamide (Am) there was a noticeable reduction in activity for the AA containing polymers and a significant enhancement for the DMAEMA containing polymers. This is presumably due to the electrostatic effects induced by the attached polymers, since the DMAEMA polymer should be cationic under the studied conditions, leading to an enhancement in activity through electrostatic attractions of the cationic polymer and the anionic Micrococcus. Conversely, for the AA containing polymers, there is likely electrostatic repulsion between the anionic enzyme conjugate and the anionic Micrococcus substrate, leading to a reduction of activity. As expected, for the longer polymers steric effects reduce the activity notably. In the case of the N-acrylamidoglucosamine (NAG) based polymers, the activity was reduced, particularly at high molecular weight of the attached polymer against both substrates, and this is presumably due to the monomer being a close analogue of the substrate and it binding in the active site. 225 Matyjaszewski et al.; Reversible Deactivation Radical Polymerization: Materials and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Figure 4. Activity of bioconjugates with RAFT polymers attached. Panel A shows the activity of HEWL polymer conjugates, panel B shows the activity of low molecular weight (L) conjugates with different functional groups and panel C shows the activity of high molecular weight (H) conjugates. Adapted with permission from Ref. (42). Copyright 2016 American Chemical Society.
226 Matyjaszewski et al.; Reversible Deactivation Radical Polymerization: Materials and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Figure 5. Gel and Activity of lysozyme conjugates synthesized by HRP catalysis. (A) PAGE of standards (lane 1), native HRP (lane 2) native lysozyme (lane 3), lysozyme-oligoAmCTA conjugate (lane 4) and lysozyme-polyNiPAm-b-Am conjugate (lane 5). B) Activity data for the native lysozyme, the oligoAmCTA-lysozyme conjugate and the polyAm-b-NiPAm-lysozyme conjugate. Conditions for polyAm-b-NiPAm-lysozyme conjugate synthesis: [NiPAm]:[CTA]:[acac]:[H2O2]= 729:1:69:12, [M]=800 mM, [HRP]= 3.3 mg/mL in 0.9 mL of pH=6, 20 mM phosphate buffer at 25 °C. Reprinted with permission from Ref. (40). Copyright 2016 John Wiley and Sons.
The grafting-to followed by grafting-from strategy can be applied to the HRP catalytically initiated RAFT polymerization. In this particular case a long chain of N-isopropylacrylamide (NiPAm) was grown from the surface of the lysozymeoligo(Am) conjugate. As seen in Figure 5, the lysozyme-poly(NiPAm-b-Am) conjugate was efficiently formed by the HRP catalyzed process as evidenced by a significant shift of the conjugate to higher molecular weight on the PAGE gel. Further, the lysozyme-poly(NiPAm) conjugate grown using the HRP initiation method showed approximately 30% residual catalytic activity with respect to the small fluorescent molecule (NAG)3-MuF substrate.
Polymeric Surfactants Styrene-maleic acid copolymers have received significant interest in the lipid and membrane protein communities due to their ability to form well-defined lipid nanoparticles. These styrene-maleic acid lipid particles (SMALPs) are easily prepared and create well defined lipid nanoparticles (38, 43, 44). SMALPs find applications ranging from fundamental biophysical studies needed to understand the properties of lipid bilayers, to membrane protein structural biology studies 227 Matyjaszewski et al.; Reversible Deactivation Radical Polymerization: Materials and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
(38). This makes creating well-defined and tunable SMALPs an important target that bridges material science and biochemistry. Typical polymers used to generate SMALPs are generally synthesized by conventional free radical polymerization giving polymers with poorly defined structures and molecular weight distributions.
Scheme 4. Interaction of lipids with styrene maleic acid copolymer to generate SMALPs. Reproduced with permission from ref (45). Copyright 2016 Elsevier.
Figure 6. SEC derived molecular weight distributions of StMAn polymers of medium molecular weight (M) with varying ratios of styrene to maleic anhydride (StMAn=2,M, StMAn=3,M, StMAn=4,M). The average polymer in each trace contains 25 units of MAn. Reproduced with permission from ref (45). Copyright 2016 Elsevier. 228 Matyjaszewski et al.; Reversible Deactivation Radical Polymerization: Materials and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 2018.
Despite these issues, the SMALPs generated are typically well defined with diameters between 10 and 30 nm and relatively narrow size distributions. However, RAFT enables precision control over polymer molecular weight and microstructure, which could be an additional tool to tune the size and properties of SMALPs. A schematic description of how the RAFT synthesized styrene-maleic acid copolymer can generate SMALPs by interacting with lipid materials is given in Scheme 4. Thermally initiated RAFT polymerization was used to create well defined copolymers of styrene and maleic anhydride with flexibility in the composition and chain length. As seen in Figure 6, the polymers are well defined with narrow distributions (Mw/Mn
