Synthesis of Modular Brush PolymerâProtein Hybrids Using

Article pubs.acs.org/bc

Cite This: Bioconjugate Chem. 2018, 29, 2594−2605

Synthesis of Modular Brush Polymer−Protein Hybrids Using Diazotransfer and Copper Click Chemistry Luis A. Navarro,* Daniel L. French, and Stefan Zauscher* Department of Mechanical Engineering and Materials Science, Duke University, 101 Science Drive, Durham, North Carolina 27708, United States

Bioconjugate Chem. 2018.29:2594-2605. Downloaded from pubs.acs.org by KAROLINSKA INST on 08/18/18. For personal use only.

S Supporting Information *

ABSTRACT: Proteoglycans are important brush-like biomacromolecules, which serve a variety of functions in the human body. While protein− bottlebrush hybrids are promising proteoglycan mimics, many challenges still exist to robustly produce such polymers. In this paper, we report the modular synthesis of protein−brush hybrids containing elastin-like polypeptides (ELP) as model proteins by copper-catalyzed azide−alkyne cycloaddition. We exploit the recently discovered imidazole-1-sulfonyl azide (ISA) in a diazotransfer reaction to introduce an N-terminal azide onto an ELP. Next, we use a click reaction to couple the azido-ELP to an alkyne-terminated amine-rich polymer followed by a second diazotransfer step to produce an azide-rich backbone that serves as a scaffold. Finally, we used a second click reaction to graft alkyne-terminated poly(oligoethylene glycol methacrylate) (POEGMA) bristles to the azide-rich backbone to produce the final protein−bottlebrush hybrid. We demonstrate the effectiveness of this synthetic path at each step through careful characterization with 1H NMR, FTIR, GPC, and diagnostic test reactions on SDS-PAGE. Final reaction products could be consistently obtained for a variety of different molecular weight backbones with final total grafting efficiencies around 70%. The high-yielding reactions employed in this highly modular approach allow for the synthesis of protein−bottlebrush hybrids with different proteins and brush polymers. Additionally, the mild reaction conditions used have the potential to avoid damage to proteins during synthesis.

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INTRODUCTION

Although bottlebrush polymers have attracted a large amount of attention due to their ability to mimic proteoglycans, they additionally offer some unique advantages over linear polymers. Where linear polymers have a persistence length which is fixed by the choice of monomer, bottlebrushes of the same chemical composition can exhibit different persistence lengths by varying the lengths or grafting density of the bristles.18−22 Furthermore, bottlebrushes can be used to make extremely soft structural materials.23 In conjunction with existing proteoglycan work, there is thus great interest in making hybrids with structural proteins and synthetic brush polymers. Recently, click chemistry has revolutionized the field of bioconjugation.24 Specifically, copper-catalyzed azide−alkyne cycloaddition has gained popularity in the bioconjugate space due to its high conjugation efficiency, fast reaction times, compatibility with aqueous media, and high functional group tolerance.25 These click reactions are especially effective for the preparation of bottlebrushes, where they enable high grafting densities.26−28 Issues in these reactions generally arise from the incorporation of functional azide or alkyne groups into

Proteoglycans are a class of biomacromolecules with important functional roles in a variety of human tissues. They serve key roles in maintaining osmotic pressure in cartilage,1 protecting cartilage from cell overgrowth and degradation,2−6 protecting the respiratory tract,7 lubricating eyes,8 and protecting the epithelium.9 The functions of these brush-like macromolecules critically depend on the oligosaccharide chains that are densely grafted to a polypeptide backbone. For example, stripping a single saccharide unit from proteoglycan 4’s side chains dramatically reduces its boundary lubrication ability.10 Despite the interest in designing new proteoglycans, their synthesis typically requires mammalian cells to achieve a large degree of post-translational modification, making it challenging to generate authentic proteoglycans in large quantities.11−13 In spite of this, researchers have been able to take major structural inspiration from various proteoglycans to produce functional biomimetic macromolecules. For example, Banquy et al. made a purely synthetic proteoglycan mimic that served as a boundary lubricant on mica,14 while Lawrence et al. showed that a purely biological mimic reduces friction between mated cartilage surfaces.15 This growing body of work is indicative of a rising demand for ways to produce heavily hydrated brushlike structures that mimic proteoglycans.6,16,17 © 2018 American Chemical Society

Received: May 3, 2018 Revised: July 11, 2018 Published: July 12, 2018 2594

DOI: 10.1021/acs.bioconjchem.8b00309 Bioconjugate Chem. 2018, 29, 2594−2605

Article

Bioconjugate Chemistry

the use of click reactions allows for the modular assembly of the brush with high conversion. To demonstrate the modularity and reproducibility of this approach, we carried out the synthesis with three different polymer backbone lengths (see Table 1). To discern the effect of the protein

synthetic polymer backbones. Most notably, alkynes are vulnerable to chain transfer,29 polymerization through the triple bond,30 and Glaser coupling.31 Azides suffer from side reactions especially at elevated temperatures that allow either cyclization of azide with monomer alkenes32 or decomposition with evolution of nitrogen.30 One popular approach for alkyne incorporation is to either use protected alkynes33−35 or introduce alkyne functional groups into polymers after polymerization. For example, Gao used carbodiimide chemistry to couple alkynes to poly(2-hydroxyethyl methacrylate) (PHEMA).36 Azides are typically included in monomers32,37,38 or incorporated by nucleophilic substitution using sodium azide.31,35 Azides can also be incorporated by diazotransfer, which converts primary amines to azides. However, to date, diazotransfer has been a far less popular choice due to the extremely hazardous and harsh in situ preparation of the typically employed tosyl azide in dichloromethane.39,40 Recently, diazotransfer has become much more popular due to the discovery of imidazole-1-sulfonyl azide salts (ISA), which are the first shelf-stable diazotransfer reagents usable in purely aqueous media.40,41 ISA can be used to functionalize amines in polypeptide N-termini and lysine side chains.42,43 This development opens new opportunities for the synthesis of protein−brush hybrids. Here, we demonstrate the synthesis of modular protein− bottlebrush hybrids using copper-assisted click chemistry. The critical advance in this paper is the way in which we exploit recent advances in diazotransfer to generate these hybrids. Specifically, we use diazotransfer to prepare an azidecontaining protein and use that azide group to conjugate it to an alkyne-terminated, amine-rich polymer backbone (Scheme 1). We then use diazotransfer to convert the amines on the backbone to azides and then graft alkyne-terminated polymer bristles. Notably, the use of ISA allows these transformations to be carried out in aqueous media, which is essential for handling a wide variety of proteins. Furthermore,

Table 1. Molecular Weight Statistics of Polymers Employed in This Study light scatteringa sample alkyne-POEGMA P(azido-AMA-coHEMA) 30 P(azido-AMA-coHEMA) 92 P(azido-AMA-coHEMA)101

Mw (kDa)

Rg (nm)

40.06c 38.46 83.48 127.7

GPCb Rh (nm)

Mn (kDa)

PDI

28.9c 12.6

7.5

19.43d 30.70

1.555d 1.264

19.3

12.3

92.57

1.588

20.0

11.5

101.01

1.389

NMRe brush samplef TIPS-brush TIPS-brush TIPS-brush ELP -brush ELP -brush ELP -brush

(30 kDa) (92 kDa) (101 kDa) (30 kDa) (92 kDa) (101 kDa)

GPCd

bristle Xn

ρ(%)

Mn (kDa)

Mn (kDa)

PDI

42.8 21.8 26.2 19.6 26.7 26.7

68.3 70.1 63.6 96.5 75.4 71.7

543.9 596.5 924.1 381.9 778.1 1063

237.2 690.2 803.9 1013 608.6 709.1

1.19 1.52 1.31 1.24 1.35 1.31

a

Light scattering in 1 M acetic acid, 0.3 M NaH2PO4 (pH 3.3) at 25 °C prior to diazotransfer. bGPC (MALS) with DMF (10 mM LiBr) mobile phase after diazotransfer. cLight scattering in PBS at 25 °C. dGPC (MALS) with THF. e1H NMR in pyridine-d5 at 500 MHz with statistics determined by integrals for triazole (8.4 ppm), PHEMA methylene (4.1 ppm), and POEGMA methoxy (3.4 ppm). For reference, bristle Xn determined by THF GPC of precursors was 64.7. f Brush samples are P((AMA-g-POEGMA)-co-HEMA) where the number in parentheses corresponds to the molar mass of the P(AMAco-HEMA) backbone polymer.

Scheme 1. Modular Synthesis of ELP−Brush Polymer Hybrid by Sequential Copper Click and Diazotransfer Reactionsa

block on the physical properties of the final products, we compare our protein−brush hybrids to bottlebrush polymers lacking the terminal protein.

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RESULTS AND DISCUSSION Conjugate Design. We chose to use poly(2-aminoethyl methacrylate-co-2-hydroxyethyl methacrylate) (P(AMA-coHEMA)) as a polymer backbone. The amine-containing monomers serve as handles to introduce azide groups for later grafting of alkyne-terminated polymer bristles. PHEMA was copolymerized into the backbone to serve as a spacer between grafting points and to determine grafting efficiency in the final product. Poly(oligoethylene glycol methacrylate) (POEGMA) was chosen as a bristle polymer due to its hydrophilicity, relevance as a biocompatibilizer in biotechnology, compatibility with radical polymerization, and ability to be handled and characterized in both organic and aqueous media. We used triisopropylsilyl (TIPS) protecting groups on the backbone alkyne to prevent potential side reactions during polymerization. We chose an elastin-like polypeptide (ELP) to serve as a model protein for conjugation due to its lack of secondary structure, lack of extraneous amines, ease of purification, and repetitive nature, which allowed for simpler characterization. Without a loss of generality, the ELP could be replaced with another protein.

HEMA and fluorescein-O-methacrylate comonomers present in the amine-rich polymer have been omitted for clarity. a

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Bioconjugate Chemistry ELP Synthesis. Here, we chose to use ELP (sequence (GVGVP) 30 with a C-terminal collagen affinity tag (WYRGRL),15 for further detail, see Table S1) because it is an intrinsically disordered model protein for its ease of purification, lack of susceptibility to denaturation in organic solvents, lack of lysine residues, and repetitive nature producing relatively simple NMR spectra. We introduced azide functionality into the ELP by diazotransfer with ISA. Because the entire protein lacks lysines, the only amine available for diazotransfer is that on the N-terminus. This allows us to controllably add no more than one brush polymer to each protein. After diazotransfer, a combination of 1H NMR, IR, MALDI-TOF, ESI-TOF, and diagnostic SDS-PAGE was used to confirm the purity of the protein and introduction of an azide. Diagnostic gels show reaction with Cy3-alkyne after diazotransfer (Figure S31), thus confirming the introduction of at least one azide group into the protein. Cyanine labeling further reveals the presence of an ELP-V30 impurity (i.e., ELP lacking the collagen binding tag) in the ELP sample. ELP-V30 impurities are nearly invisible through routine Coomassie staining because the only available positive charge is on the N-terminus, while the collagen binding tag contains several positively charged groups. Cyanine labeling also allowed for accurate quantification of ELP-V30 impurities. Bristle Synthesis. We used AGET ATRP to synthesize alkyne-terminated POEGMA bristles (see Supporting Information). Their chemical composition and purity were verified by NMR and IR. GPC traces show a bimodal molecular weight distribution with a second peak containing less polymer. For the bristle synthesis, we used unprotected alkyne initiators to obtain enough material to produce brush polymers with a large excess of bristles. However, the use of unprotected alkynes also led to undesirable side reactions, most notably Glaser coupling, which negates the ability of terminal alkynes to react. We know this because the same reaction conditions performed on a small scale with TIPSprotected alkyne initiators showed unimodal distributions (data not shown). A diagnostic gel reveals a successful reaction between Cy3-N3 and alkyne-POEGMA bristles, demonstrating the presence of active alkyne groups after polymerization (Figure 1). Backbone Design. AGET ATRP proved to be effective for producing the high molecular weight backbone polymers (see Supporting Information). We note that switching from PMDETA to the stronger, pyridyl-based ligand, TPMA,

allowed the polymerization to reach high conversion while following first order rate kinetics. Backbone polymer chemical composition and purity were determined by 1H NMR and IR, with 1H NMR additionally revealing comonomer incorporation ratios that closely matched the monomer feed ratios in every batch, independent of conversion or molecular weight. This observation is further supported by research from Quiñ onez-Angulo, who showed that the kinetics of P(DEAEAM-co-OEGMA) random copolymers proceed independent of the ratio of OEGMA to amine-based monomer.44 Therefore, we suspect that, despite the charged monomers, the polymers produced with this method are truly random copolymers. This is important for the production of brush polymers with roughly uniform bristle spacing. In some cases, alkyne headgroup activity in smaller polymers can be determined directly by 1H NMR.29,33 However, such direct approaches fail for larger polymers where the mass of the polymer dwarfs signals from headgroups. Verification of the activity of single alkynes or azides is thus typically performed directly by mass spectrometry42,43,45,46 and/or indirectly by reaction with an azide followed by mass spectrometry,33,45 GPC,47 or fluorescent detection.48 However, polydisperse polyelectrolytes yield exceedingly complex mass spectra and typically have major issues in chromatographic separation due to complications arising from interactions between the stationary phase, polymers, and their counterions. Furthermore, neutralization of PAMA-based polymers causes degradation through intramolecular and intermolecular reactions with the amine,49 potentially complicating analysis dramatically. We overcame these obstacles by using a diagnostic click reaction in which we labeled the reactive headgroups with cyanine dyes (Figure 2). Fortunately, the polycations employed in this work migrate well in SDS-PAGE, enabling us to use this technique to separate the reaction products. The inclusion of a different fluorophore (0.5 mol % fluorescein Omethacrylate) in the backbone copolymers enabled the fluorescent detection of polymer chains independent of their reaction with cyanine dye, allowing us to verify that both signals are produced by the same species. Additionally, we discovered that these polycations could be intensely stained with Coomassie brilliant blue, a dye that is routinely used to stain proteins through interactions with positively charged groups. Coomassie staining effectively adds an orthogonal, redundant check for visualizing these migrating polymers. These diagnostic click reactions on SDS-PAGE were paired with positive controls (PEG-azide or PEG-alkyne) to verify that the test reactions were working. Similarly, negative controls (polymer without dye) were included to ensure cyanine signals came only from reactive dyes and not partial fluorescein excitation. This approach allowed us to simultaneously screen for functional azides and/or alkyne groups in the desired polymers as well as unreacted starting material impurities. Diagnostic gels of TIPS-protected P(AMA-co-HEMA) with Cy3-N3 reveal that a small amount of the TIPS protecting group was removed during polymerization, which is known to happen to some degree in the presence of bases.33 However, after deprotection with AgF, backbone polymers show much more reaction with cyanine dyes (Figure 2A). Despite challenges in quantitatively determining the degree of deprotection, this result indicates that AgF successfully deprotects these alkynes. The position of the cyanine signal matches the position of fluorescein and Coomassie signals in

Figure 1. Cyanine3 (Cy3) scan of a diagnostic SDS-PAGE of alkynePOEGMA bristles. The gel shows a protein ladder (lane 1), positive control alkyne-PEG (20 kDa) reacted with Cy3-azide (lane 2), unreacted Cy3-azide dye (lane 3), alkyne-POEGMA (19 kDa) without dye (lane 4), and alkyne-POEGMA reacted with Cy3-azide (lane 5). 2596


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Figure 2. Diagnostic SDS-PAGE to test alkyne activity in different molecular weight P(AMA-co-HEMA) by reaction with Cy3 dyes and visualization by Cy3 (top), fluorescein (middle), and Coomassie (bottom). For reference, gels show protein ladder (lane 1), positive control alkyne-PEG (20 kDa, lane 2), azido-PEG (10 kDa, gel B, lane 3), and azido-ELP (gel B, lane 4). (A) Gel A shows polymers before (AgF −) and after (AgF +) AgF deprotection by reaction with (Cy3 +) or without (Cy3 −) Cy3-azide (color overlay available in Figure S65). (B) Gel B shows deprotected P(AMA-co-HEMA) before (lane 5) and after (lanes 6−11) conjugation with azido-ELP and reacted with Cy3-azide (N3) or Cy3alkyne (≡).

Figure 3. THF GPC chromatograms of P((AMA-g-POEGMA)-co-HEMA) brush polymers with 30 kDa (black), 92 kDa (red), and 101 kDa (blue) backbone lengths conjugated to ELP (dash) or not (solid). Unreacted POEGMA bristle (black dot) is shown for reference. Chromatograms are shown for detection by (A) light scattering and (B) refractive index.

the conjugate contains a mixture of Cy3-active, lower mobility species and a Cy3-inactive, higher mobility species. We therefore hypothesize that the proteins employed potentially contained multiple azides, allowing some proteins to react with both polymer and dye. Furthermore, the presence of the Cy3inactive, high mobility fluorescein signal indicates the potential degradation of the polymers’ alkyne head groups. Backbone Diazotransfer. The presence of a strong peak at 2100 cm−1 in polymer IR spectra confirms the successful incorporation of azide groups into the amine-rich polymers. No unreacted amines are visible in 1H NMR (DMSO-d6), although trace acetate ion and/or acetic acid is visible (>87% conversion). Unfortunately, low water solubility made SDSPAGE diagnostic analysis unreliable. Brush Polymer Synthesis. We chose to use copperassisted click reactions for polymer brush assembly to achieve high grafting efficiency26,36 and to avoid reactive intermediates present in many nucleophilic substitution-based approaches.27 Furthermore, it is known that the triazole products of brush

the same gel and properly correlates to less migration for higher molecular weight polymers. Alkyne-terminated backbones were then conjugated to azido-ELP with a large excess of protein to ensure minimal residual unfunctionalized polymer. SDS-PAGE revealed that no high molecular weight polycations reacted with Cy3-N3 following conjugation (Figure 2B), implying that nearly all available alkyne head groups were consumed. The gel also revealed that a significant amount of unreacted protein impurity is present in the final products. At this step, the majority of ELP was removed by precipitation, while the remaining protein was later removed after the second diazotransfer reaction. We also noted that a small amount of conjugated polycation unexpectedly reacted with Cy3-alkyne in lanes 7, 9, and 11 (Figure 2B). Upon closer inspection, the ratio of fluorescein to cyanine signal intensity of these bands increases with migration, whereas the alkyne-P(AMA-coHEMA) precursor has a constant ratio of fluorescein to cyanine intensity (Figure 2A). Specifically, it seems as though 2597


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Figure 4. Representative tapping mode AFM images in air of (left) TIPS-P((AMA-g-POEGMA)-co-HEMA) and (right) ELP-P((AMA-gPOEGMA)-co-HEMA) spin coated onto freshly cleaved mica. Apparent bottlebrush polymer contour lengths were 87 ± 30 nm (40 brushes) with protein tag and 102 ± 33 nm (17 brushes) without protein tag (t test p = 0.123) when averaged across several AFM images. The brush diameters for both bottlebrush polymers were approximately 17 nm.

polymers in “grafting to” approaches serve as copper ligands which autocatalyze the coupling reaction.50 Copper-based click reactions are robust against a wide variety of functional groups which may be found in proteins or synthetic polymers, making it suitable for this modular synthesis. To detect the presence of remaining bristles after purification, the brush polymers were passed through GPC with a THF mobile phase (Figure 3). Chromatograms clearly show longer retention times for brush polymers with longer backbone lengths. This seemingly contradictory trend has been previously reported for other brush polymers.51 Furthermore, brush polymer samples with short peptide units showed consistently shorter retention times than their peptide-free counterparts. The observed 1 min reduction in retention time roughly corresponds to a 3-fold difference in the molecular weight for a linear POEGMA based polymer on this column (data not shown). Therefore, we argue that this large difference in retention time can only come from a conformational change in the brush polymer to accommodate the extremely THF-insoluble protein with the hydrophilic POEGMA side chains. 1 H NMR of the brush polymers in pyridine proved to be the most effective way to determine brush polymer molecular weight, grafting density, and bristle length (see Table 1). Specifically, pyridine substantially shifts protons near alcohol groups downfield relative to their positions in more common NMR solvents, which allowed us to effectively separate peaks for our PHEMA-rich backbone from those of the POEGMA bristles. In this medium, separate peaks were observed for the POEGMA terminal methoxy group (3.3 ppm), triazole headgroup (8.4 ppm), and backbone PHEMA CH 2 (4.1 ppm), which allowed for determination of the effective grafted bristle lengths and grafting efficiency. NMR spectra clearly showed incorporated bristle lengths that are consistently shorter than those determined by GPC of the precursors. Therefore, it is plausible that this grafting process proceeds with a strong preference for the shortest available chains. Furthermore, the apparent grafting efficiencies consistently reached 70% despite the bulky nature of the POEGMA bristles. This figure includes the reaction efficiency of the diazotransfer and click reactions. However, overlap

between then PHEMA CH2 and POEGMA CH2 peaks in NMR causes this figure to be a slight underestimate. We note that light scattering proved to be quite limited for molecular weight determination of the brush polymers, consistent with previous reports on other bottlebrushes (see also Supporting Information).51 To confirm the final bottlebrush architecture, we imaged the brush polymer on mica surfaces by tapping mode atomic force microscopy (AFM) in the air (Figure 4). AFM images show bottlebrush-like objects of comparable lengths (87.2 ± 30.4 nm with ELP, and 102.2 ± 33.4 nm without ELP) and diameters (∼17 nm for both). The apparent dimensions are consistent with the estimated contour lengths of the backbone (179 nm) and bristles (16 nm), which set upper bounds for the dimensions of the polymers at maximum extension. Synthesis of a Biohybrid with a Folded Protein. Certain applications require biohybrid constructs in which the protein secondary/tertiary structure must be preserved. The biggest obstacle inherent to extending our approach to folded proteins is its potential denaturation due to interactions with the polycationic AMA-rich backbone, as the use of organic solvents can be avoided with different choices of filler comonomers and bristle polymers (for an extended discussion, see the Supporting Information). To demonstrate that AMAbased copolymers are not intrinsically destructive to folded protein structures, we show the synthesis of a functional streptavidin-P(AMA-co-HEMA) conjugate by diazotransfer and Huisgen cycloaddition (Scheme 1). The gel in Figure 5 shows the introduction of azides into streptavidin following diazotransfer (lane 7) and the consumption of some of those azides following conjugation to alkyne-P(AMA-co-HEMA) (lane 10). Importantly, streptavidin-P(AMA-co-HEMA) retains its biotin-binding ability, as visualized by reaction with Cy3-labeled biotin (lanes 3, 6, and 9). This suggests that our AMA-based copolymers can be compatible with functional proteins.

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CONCLUSIONS We have demonstrated that modern diazotransfer reagents (specifically, ISA) can be exploited in new ways to produce complex, branched protein−polymer hybrids, such as the 2598


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Aldrich), ammonium sulfate (99%, MP Biomedicals), Lascorbic acid (99%, Sigma-Aldrich), azide-PEG3-biotin (444.6 Da, 89.5%, Sigma-Aldrich), biotin (99%, SigmaAldrich), biotin-labeled bovine serum albumin (biotin-BSA, 80% protein, 8−16 biotin per protein, Sigma-Aldrich), αbromoisobutyryl bromide (98%, Sigma-Aldrich), 3-bromo-1propanol (97%, Sigma-Aldrich), chloroform-d (CDCl3, 99.8% D, Sigma-Aldrich), copper(II) bromide (CuBr2, anhydrous, 99%, Acros), copper(II) chloride (CuCl2, 99.999%, SigmaAldrich), copper(II) sulfate pentahydrate (CuSO4·5H2O, 98.9%, Fisher Chemicals), cyanaine3-alkyne (Cy3-alkyne, Lumiprobe), cyanine3-azide (Cy3-azide, 10 mM in DMSO, Lumiprobe), deuterium oxide (D2O, 99.9% D, Sigma-Aldrich), dichloromethane (DCM, 99.5%, Alfa Aesar), diethyl ether (anhydrous, 99.7%, Sigma-Aldrich), N,N-dimethylformamide (DMF, HPLC grade, 99.9%, Honeywell Research Chemicals), dimethyl sulfoxide (DMSO, 99.99%, Mallinckrodt/Covidien), dimethyl sulfoxide-d6 (DMSO-d6, 99.9% D, Cambridge Isotope Laboratories), ethanol (EtOH, absolute, KOPTEC), ethyl acetate (99%, Fisher Chemicals), ethyl magnesium bromide (3.0 M in ether, Sigma-Aldrich), ethylenediaminetetraacetic acid disodium salt (Na2EDTA, 99%, Sigma-Aldrich), fluorescein O-methacrylate (97%, Sigma-Aldrich), glycerol (99.5%, EM Science), heptane (99% Aldrich), hexane (98.5%, BDH, VWR Analytical), hydrochloric acid (37% solution, Mallinckrodt), hydroquinone (99.5%, Sigma-Aldrich), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 99.5%, Sigma Life Science), 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid sodium salt (HEPES, 99.5%, Sigma), imidazole (Mallinckrodt), isopropyl alcohol (99.5%, BDH, VWR Analytical), Laemli buffer (2×, Bio-Rad), lithium bromide (LiBr, anhydrous, 99.995%, Beantown Chemical), magnesium sulfate (MgSO4, anhydrous, 99%, Mallinckrodt), methacryloyl chloride (97%, Sigma-Aldrich), methanol (MeOH, 99.8%, EMD Millipore Corporation), N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA, 99% Sigma-Aldrich), polyethylene glycol methyl ether alkyne (PEG-alkyne, Laysan Bio, Inc., MW 20,000, Lot #134-165), polyethylene glycol methyl ether azide (PEG-azide, Laysan Bio, Inc., MW 10 000, Lot #134-77), potassium chloride (99.995%, Alfa Aesar), potassium permanganate (99%, SigmaAldrich), potassium phosphate monobasic (99.8%, J.T. Baker), propargyl alcohol (99%, Sigma-Aldrich), pyridine-d5 (99.5% D, Cambridge Isotope Laboratories), silica gel (100−200 mesh, Fisher Chemicals), silver(I) fluoride (AgF, 99.9%, SigmaAldrich), sodium azide (99%, EMD Millipore), sodium bicarbonate (99.5%, Sigma-Aldrich), sodium chloride (99%, Macron Chemicals), sodium hydroxide (98.2%, Fisher Chemicals), sodium L-ascorbate (99%, Sigma-Aldrich), sodium nitrite (NaNO2, 97%, Sigma-Aldrich), sodium phosphate dibasic (99%, Sigma-Aldrich), streptavidin (from Streptomyces avidinii, 13 U/mg, 65−100% protein, Sigma-Aldrich), sulfuric acid (95%, EM Science), sulfuryl chloride (SO2Cl2, 98.5%, Fischer Scientific), tert-butyl alcohol (tBuOH, 99%, Alfa Aesar), tetrahydrofuran (THF, anhydrous, 99.9%, SigmaAldrich), triethylamine (NEt3, 99%, Sigma-Aldrich), trifluoroacetic acid (TFA, 99%, Sigma-Aldrich), triisopropylsilyl chloride (TIPS-Cl, 97%, Sigma-Aldrich), tripropargylamine (98%, Sigma-Aldrich), tris(2-pyridylmethyl)amine (TPMA, 98%, Sigma-Aldrich), and tris(3hydroxypropyltriazolylmethyl)amine (THPTA, 95%, SigmaAldrich) were purchased and used as received. Potassium carbonate (anhydrous, 99%, Fluka) was heated in an oven for

Figure 5. Diagnostic SDS-PAGE to test azide activity in different streptavidin derivatives. The gel shows native streptavidin (lanes 2− 4), azido-streptavidin (lanes 5−7), and streptavidin-P(AMA-coHEMA) hybrid (lanes 8−10) reacted with Cy3-alkyne (lanes 4, 7, and 10), biotin-Cy3 (lanes 3, 6, and 9), or with no additive (lanes 2, 5, and 8). A ladder is included for reference (lane 1). Scans for Cy3 and Coomassie are shown. For color overlay and fluorescein scan, see Figure S76.

protein−bottlebrushes shown here. We employ mild reaction conditions that are favorable for the preparation of hybrids because the protein only needs to be present during two diazotransfer reactions and two click reactions, both of which can be performed at room temperature in buffered aqueous media. Furthermore, our “grafting to” approach allows synthetic polymers to be separately produced in bulk, handled with organic solvents, and characterized for their molecular weight distributions. This is valuable in situations where the protein of interest is precious, as the molecular weight distribution of the final products can be estimated a priori. We showed that our amine-rich, synthetic polymer backbones are amenable to characterization by SDS-PAGE using fluorescent detection and staining procedures that are routine in protein analysis. Diazotransfer to these polymers reliably yielded azide-rich backbones suitable for the preparation of brush polymers. Furthermore, our click-based grafting of POEGMA to backbone azides consistently produced high grafting densities despite our use of sterically encumbering bristles. Finally, our synthetic strategy can be compatible with folded proteins, as shown by the synthesis of a functional streptavidin-P(AMA-co-HEMA) hybrid. The synthetic approach taken in this paper paves the way for the design of a general class of highly branched biohybrids, such as proteoglycan mimics. While we showed successful coupling to an ELP N-terminus, our approach extends to any protein N-terminal amine, protein lysine residue, DNA nucleobase excepting thymidine,52 amine-bearing polysaccharide, or existing azide modification. Furthermore, our procedures can be easily adapted to prepare hybrids with different bristle chemistries, including hydrophilic synthetic polymers, proteins, and glycopolymers. Last, by using alkyneP(AMA-co-HEMA) in place of the bristles, more highly branched architectures should be accessible.

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EXPERIMENTAL PROCEDURE Materials. Acetic acid (glacial, 99.5%, Mallinckrodt), acetone (99.5%, Sigma-Aldrich), acetonitrile (99.99%, EM Science), N-acetyl-L-cysteine (99%, Sigma-Aldrich), alumina (basic, 150 mesh, Sigma-Aldrich), 2-aminoethyl hydrochloride (99%, Sigma-Aldrich), ammonium chloride (99.5%, Sigma2599


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ATR; 500−4000 cm−1) spectra were obtained using an ABB FTLA2000 spectrometer and processed using GRAMS/AI (Thermo Galactic, v7.00). Direct injection mass spectra were obtained using an Agilent Technologies 1100 series LC/MSD Trap with electrospray ionization. Mass spectra were analyzed using DataAnalysis for LC/MSD Trap (v3.3). Mass spectroscopy samples were loaded by direct injection using a Harvard Apparatus 20 syringe pump. LCMS was obtained with an Agilent 1100 series HPLC with capillary pump (G1376A), degasser (G1379A), microALS (G1389A), ALS Therm (G1330A), and column compartment (G1316A). The HPLC fed into a Luna C18(2) column (Phenomenex, 3 μm, 100 Å, 50 × 1 mm) with detection of UV absorbance by a diode array detector (DAD, G1315B). Mass spectrometry data for LCMS was recorded and processed with the same hardware and software as directly injected samples. Melting points were obtained manually using a heated oil bath. Liquid densities were obtained gravimetrically using a 1 mL volumetric flask. Biological Characterization. Matrix-assisted laser desorption/ionization with time-of-flight detection (MALDI-TOF) was performed using a Bruker AutoflexSpeed MALDI Mass Spectrometer (Billerica, MA) calibrated with an aldolase standard. MALDI samples were prepared by mixing 5 μL of analyte (10 μM in H2O) with 5 μL of sinapinic acid (saturated solution in 69.9% water, 30% acetonitrile, 0.1% trifluoroacetic acid) and spotting the mixture onto MALDI sample plates. ESI-TOF was taken using an Agilent 6224 TOF LC/MS system with a dual ESI source and a 1200 HPLC, using Agilent MassHunter software. When applicable, C18 ZipTip pipet tips (Millipore-Sigma) were used to desalt samples. DNA sequencing was performed by submitting plasmid samples to Eton Bioscience (Durham, NC branch), and sequencing data were analyzed using SeqBuilder (v14.1, DNASTAR). DNA sequencing chromatograms were analyzed by FinchTV (v1.4) to ensure data quality. Ion Exchange Chromatography. Protein purification was carried out with an AKTApurifier system (GE Healthcare) equipped with modules P-960, PV-908, PV-907, NV-907, and M-925. Samples were separated by cation exchange columns (three HI-Trap SPFF columns in series) with 300−600 μL injections of roughly 40 mg/mL protein. Separation was monitored using an inline UV-detector (UPC-900) and analyzed by Unicorn (v5.1). Gel Permeation Chromatography. Gel permeation chromatography (GPC) with THF eluent (HPLC grade, 100 ppm BHT) was performed on two Agilent PLgel 105 Å, 7.5 × 300 mm, 5 μm columns (part number PL1110−6550) at room temperature at a flow rate of 1.0 mL/min. The flow rate was set using an Agilent 1260 Infinity Isocratic pump. THF GPC samples were filtered through a PTFE membrane with 0.2 μm pores prior to injection. UV absorbance was measured with an in line Agilent 1260 Infinity UV detector (Agilent Technologies) in the range of 190−800 nm with a 2.0 nm step and 4.0 nm slit width. Molecular weights were calculated using an inline Wyatt Optilab T-rEX refractive index detector (RI, Wyatt Technologies Corp.) and Wyatt miniDAWN TREOS multiangle light scattering detector (MALS, Wyatt Technologies Corp.). Refractive index increment (dn/dc) values for POEGMA polymers were determined inline, assuming 100% mass recovery based on known injection mass. These dn/dc values were corroborated with data from an Abbemat 500 refractometer (Anton Paar). dn/dc values for brush polymers were assumed to match bristle dn/dc’s for

several hours and allowed to cool in a desiccator before use to ensure accurate stoichiometry. 2-Hydroxyethyl methacrylate (HEMA, 99%, Sigma-Aldrich) and poly(ethylene glycol) methyl ether methacrylate (OEGMA, Mn = 300, Sigma-Aldrich) were passed through basic alumina to remove inhibitor and used within 3 h prior to any polymerization. While technical grade 2-aminoethyl methacrylate hydrochloride (AMA-HCl) is commercially available, its low 90% purity prohibits its use in this synthesis. AMA-HCl was thus prepared according to literature procedures.49 Diagnostic click reactions for gels in Figures 1 and 2A were conducted using THPTA purchased from SigmaAldrich. THPTA used for the gel shown in Figure 2B, conjugation reactions, and grafting reactions was prepared on a large scale synthetically. Initiators, polymers, proteins, ISA, buffers, and staining solutions were prepared with full synthesis procedures available in Supporting Information. For biosynthesis, AcuI (New England Biolabs, Ipswich, MA), agar (Ultrapure, Affymetrix), agarose (molecular biology grade, Bio-Rad), aldolase (Sigma-Aldrich), BamHI (as BamHI-HF, New England Biolabs), BglI (New England Biolabs), BL21 (DE3) competent E. coli (New England Biolabs), BseRI (New England Biolabs), calf intestinal phosphatase (CIP, New England Biolabs), Cutsmart Buffer (New England Biolabs), isopropyl β-D-1-thiogalactopyranoside (IPTG, 99%, Gold Biotechnology), kanamycin (USP grade, Gold Biotechnology), NEB10β competent E. coli (New England Biolabs), polyethylenimine (PEI, 50% aqueous solution, 50−100 kDa, MP Biomedicals), QIAprep Spin Miniprep Kit (Qiagen, Valencia, CA), QIAquick Gel Extraction Kit (Qiagen), Quick Ligase (New England Biolabs), Quick Ligase Buffer (New England Biolabs), SDS-Tris-Glycine Buffer (Bio-Rad), SybrSafe (Invitrogen), Terrific Broth II (MO Bio), tris-acetate-EDTA buffer (TAE, Bio-Rad), trypsin (Trypsin-ultra, mass spectrometry grade, New England Biolabs), Trypsin-ultra reaction buffer (2× concentrated, 50 mM tris HCl, 20 mM CaCl2, pH 8, New England Biolabs), and XbaI (New England Biolabs) were purchased and used as received. Forward and reverse oligonucleotides (Ultramer) encoding for a collagen-binding domain (WYRGRL) and flexible linker (G4S)3 were ordered from Integrated DNA Technologies. The plasmid, JMD2, was donated from the Chilkoti Laboratory (Duke University, Durham, NC). JMD2 was adapted from pET-24 via previously described procedures.53 A JMD2 plasmid containing a gene for (VPGVG)30 (abbreviated ELPV30) was supplied by Prof. Ashutosh Chilkoti’s laboratory (Duke University) and was produced through the same procedures.53 Full protein and DNA sequence information is available in Table S1. All water used was Milli-Q grade and obtained using a NANOpure Diamond water purification system (Barnstead, 18.2 MΩ·cm) and filtered through a 0.22 μm Millipak 40 Gamma Gold (Millipore) filter. Regenerated cellulose dialysis membranes (Spectra/Por, molecular weight cutoff 8−10 kDa and 25 kDa) were purchased from VWR and used as received. Regenerated cellulose dialysis membranes (Snakeskin, molecular weight cutoff 3.5 kDa) were purchased from Thermo Scientific and used as received. Instrumentation. Chemical Characterization. NMR spectra were obtained using Bruker 500 MHz and Bruker 400 MHz spectrometers at 25 °C with VnmrJ version 3.2. NMR data were analyzed using MestReNova (v8.0.1−10878). Fourier transform infrared-attenuated total reflection (FTIR2600


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(filtered through a 0.2 μm regenerated cellulose filter, 20 μL of solution onto a 1.5 cm diameter disc, spun at 3000 rpm for 60 s). Samples were imaged using TESPW AFM probes (Veeco, 20−80 N/m, 230−410 kHz) for tapping mode in the air using a Bruker MultiMode AFM with Nanoscope V controller and Research Nanoscope (Bruker, v9.0, build R1Sr4.98954) software. Images were processed with Nanoscope Analysis (Bruker, v1.50, build R2.103555) to plane fit, flatten, and export images, then analyzed with ImageJ (v2.0.0rc-59) with Fiji to manually estimate brush polymer lengths and diameters. Highlighted Synthetic Procedures. α-(Triisopropylsilylacetylene)-ω-bromo-poly(2-aminoethyl methacrylate-co-2hydroxyethyl methacrylate-co-fluorescein O-methacrylate) (TIPS-P(AMA-co-HEMA)). A mixture of 90% methanol with water was purged with nitrogen gas for 1 h. Excess 2hydroxyethyl methacrylate (HEMA) was passed through basic alumina to remove the inhibitor, while the 2-aminoethyl methacrylate (AMA-HCl) was purified from its inhibitor during its synthesis. To a 25 mL Schlenk tube was added CuBr2 (22.5 mg, 0.100 mmol, 5 Eq), 100 μL of 90% MeOH, TPMA (35.1 mg, 0.121 mmol, 6 Eq), and HEMA (294.0 μL, 315.5 mg, 2.424 mmol, 120 Eq). The mixture was allowed to stir at room temperature for 15 min to allow the copper complex to fully form. To the lime green solution was added 90% MeOH (965 μL), 3-(triisopropylsilyl)-2-propynyl-2bromo-2-methylpropanoate initiator (7.3 mg from a 100 mg/ mL stock solution, 20. μmol, 1 Eq), fluorescein O-methacrylate (8.0 mg, 20.2 μmol, 1 Eq), and AMA-HCl (100. mg, 0.606 mmol, 30 Eq). In a 50 mL two-neck round-bottom flask was prepared excess L-ascorbic acid solution (40 mg/mL in 90% MeOH). Each flask was placed under a nitrogen atmosphere with four freeze−pump−thaw cycles. The reaction mixture was allowed to stir at 30 °C, and once the temperature had equilibrated, the reaction was started by injecting Lascorbic acid solution (178 μL, 7.12 mg, 40.4 μmol, 2 Eq). The lime green solution was covered in foil and allowed to continue stirring at 30 °C for 22 h, at which point the reaction was stopped by exposing it to the air. Reaction progress was monitored by 1H NMR and stopped at 88.3% conversion. The following day, an increase in viscosity had caused all stirring to stop. Polymer solutions were recollected with water and dialyzed in 8−10 kDa MWCO Spectra/Por RC dialysis tubing against 0.1 mM acetic acid for 1 day with frequent buffer exchanges. Polyethylenimine (acetate salt) was added to dialysis buffer overnight (600 mg/L, 50−100 kDa) to reduce swelling. The final deep highlighter-yellow dialyte was concentrated under reduced pressure and lyophilized to yield 364.8 mg of deep yellow solid. The 30 kDa, 92 kDa, and 101 kDa polymer samples were obtained by modifying the molar ratios of initiator/AMA/HEMA/fluorescein to match 1:30:120:1, 1:60:240:2, and 1:90:360:3, respectively. Batches were prepared with equal amounts of initiator, and the amount of 90% MeOH added was altered to maintain equal molar concentrations of all nonmonomer reagents across batches. A polymer dn/dc of 0.0802 mL/g in DMF (10 mM LiBr) was obtained during GPC by the detector dRI signal assuming 100% recovery of all polymer. A polymer dn/dc of 0.1620 ± 0.0174 mL/g (95% confidence) in buffer (pH 3.3, 1 M HOAc, 0.3 M NaH2PO4, 24.5 °C) was determined by making five polymer stock solutions of known concentrations in buffer, dialyzing each for 2.5 h against a common beaker of buffer to reach Donnan equilibrium, gravimetrically correcting the

purposes of molecular weight determination. GPC data were analyzed using Astra (v6.1.5.22, Wyatt Technologies Inc.). POEGMA molecular weights and polydispersities were determined using MALS and RI data fit to a Zimm model. GPC with dimethylformamide (DMF, HPLC grade, 10 mM LiBr) eluent was performed with Styragel HR 4, Styragel HR 4E, and Styragel HR 3 columns (Mn ranges: 5−600, 0.05−100, and 0.5−30 kDa) in series at 25 °C with a flow rate of 0.25 mL/min. The flow rate was set using a Waters 2695 separations module. DMF GPC samples were filtered through a polypropylene membrane with a 0.2 μm pore prior to injection. Molecular weights were calculated using an inline Wyatt Optilab Rex refractive index detector (RI, Wyatt Technologies Corp.) and Wyatt miniDawn multi-angle light scattering detector (MALS, Wyatt Technologies Corp.). The light scattering was manually calibrated using six polystyrene standards (Fluka) ranging from 2 to 700 kDa. Refractive index increment (dn/dc) values for P(AMA-co-HEMA) polymers in DMF were determined inline, assuming 100% mass recovery based on known injection mass. GPC data were analyzed using Astra (v6.1.5.22, Wyatt Technologies Inc.). P(AMA-coHEMA) molecular weights and polydispersities were determined using MALS and RI data fit to a Zimm model. Light Scattering. Static light scattering (SLS) and dynamic light scattering (DLS) data were obtained with a CGS-3 compact goniometer system (ALV) and LSE-5004 correlator (ALV). SLS data were fit to a Zimm formalism using ALV Static and Dynamic FIT and PLOT software (v4.48). DLS data were processed using AfterALV (v1.0). Autocorrelation functions were fit at each acquired angle. A line was fit for hydrodynamic radius versus scattering angle, and the reported Rh corresponds to the extrapolated hydrodynamic radius at zero angle. SLS samples containing POEGMA were filtered through a cellulose acetate membrane with 0.2 μm pores prior to measurement. DLS and SLS samples containing P(AMA-coHEMA) were filtered through an Anotop 10 0.1 μm pore syringe filter followed by an Anotop 10 0.02 μm pore filter immediately prior to measurement. Gel Electrophoresis. SDS-PAGE gels were run using a MiniPROTEAN Tetra cell, 4−20% TGX Stain Free polyacrylamide gel, and PowerPac Basic power supply purchased from BioRad. The chambers were filled with 1× tris/glycine/SDS buffer diluted from a 10× concentrated solution purchased from BioRad. Precision Plus Protein Dual Color (Bio-Rad) was loaded onto gels as a protein ladder. Gels were imaged using a Typhoon 9410 for Cy3 and fluorescein signals (532 nm laser with 580 nm emission filter) and imaged using a Universal Hood II (Bio-Rad) for Coomassie stained gels. Gels to be stained with Coomassie were imaged for Cy3 prior to staining. Coomassie staining was achieved by rocking the gel in a 0.05% solution of Coomassie Brilliant Blue R-250 (Bio-Rad) in 10% acetic acid and 50% methanol for 10−15 min and then destaining in deionized water for a day. Gel data were analyzed using ImageJ (v2.0.0-rc-59) with Fiji to determine absorbed intensities relative to background. DNA gels were taken by loading samples into a 1.5% agarose gel (with 0.01% SybrSafe dye) and separated electrophoretically (130 mV, 30 min). The DNA ladder used was a 1 kbp DNA ladder (N3232S, New England Biolabs). The gel was imaged using a Universal Hood II (Bio-Rad) and assessed qualitatively for plasmid gene insertion. Atomic Force Microscopy. Samples were spin coated onto fresh-cleaved mica from a 100 μg/mL aqueous solution 2601


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inversion into a homogeneous, deep highlighter-yellow solution, followed by addition of L-ascorbic acid solution (578 μg, 3 mg/mL in H2O, 192.5 μL, 3.279 μmol, 15 Eq), leading to a total solution volume of 1093 μL. Argon was blown into the vial before sealing with parafilm and shaking briefly. The unchanged, homogeneous solution was covered in foil and allowed to mix by inversion for 23 h. To the homogeneous yellow solution was added (NH4)2SO4 solution (400 mg/mL in H2O) to a final concentration of 66.6 mg/mL (NH4)2SO4, instantly forming a large amount of white precipitate. The tube was then spun at 10 000 rcf for 10 min, and the lime supernatant was collected. The pellet was redissolved into 833 μL of H2O, precipitated with 166 μL of (NH4)2SO4 solution, and spun down, and the lime supernatant was collected twice more for a total of three precipitations. The combined lime supernatants were dialyzed against 0.1 mM acetic acid in 25 kMWCO Spectra/Por RC dialysis tubing for a day with two buffer exchanges. The dialyte was transferred with H2O and lyophilized to yield 19.5 mg of a deep yellow fluffy solid. Due to material constraints, the pellet, consisting mostly of protein, was also purified by dialysis against 0.1 mM acetic acid in 3.5 kMWCO Snakeskin RC dialysis tubing for a day with three buffer exchanges, recollected with H2O, and lyophilized to yield 28.0 mg of a pale beige fluffy solid as the final product. The pellet’s purity was verified quickly by FTIR (neat) to show it predominantly consisted of protein. The pellet was then reused for a second batch of protein−polymer conjugate while taking precautions to never potentially cross-contaminate different molecular weight backbones by keeping those sample pellets separate. FTIR (neat) of supernatant samples matched a combination of ELP and polymer precursors. FTIR (neat) of pellets matched that of ELP precursors. Functionalization was confirmed by diagnostic SDS-PAGE (Figure 2). More thorough removal of ELP impurities from the polymers was performed during the following diazotransfer step. Diazotransfer Reaction for α-(ELP)-ω-Bromo-poly(2-azidoethyl methacrylate-co-2-hydroxyethyl methacrylate) (ELP-P(azido-AMA-co-HEMA)). This procedure for 108 kDa ELP-P(AMA-co-HEMA) is representative, as stoichiometry was adjusted to maintain constant molar concentrations and equivalents of amine across samples. To a 50 mL roundbottom flask was added 7.68 mL of K2CO3 (10 mg/mL in 70% MeOH, 76.1 mg, 556 μmol, 15 Eq), 12.9 mL of 70% methanol, and imidazole-1-sulfonyl azide hydrogen sulfate (1.00 mL of 100 mg/mL in 70% MeOH, 100. mg, 370 μmol, 10 Eq). The reaction solution became finely cloudy white with the precipitation of potassium bicarbonate. After switching to incandescent lighting, 92.3 μL of CuSO4 pentahydrate (10.0 mg/mL in H2O, 923 μg, 3.70 μmol, 0.1 Eq) and ELPP(AMA-co-HEMA) (32.2 mg in 2 mL 70% MeOH, 37.0 μmol amine, 1 Eq) were added to the reaction mixture, causing it to become turbid yellow. The round-bottom flask was capped with a septum, covered with foil, and stirred at room temperature for 22 h, ending with a finely turbid green-yellow solution. The reaction was stopped by the addition of 1 M acetic acid (1.11 mL, 30 Eq), causing the yellow color to immediately fade and precipitate white solid. After stirring for 15 min at room temperature, the mixture was concentrated under reduced pressure, and transferred to 25 kDa MWCO SpectraPor RC dialysis tubing with 2 mL of DMF to dissolve the precipitate. The sample was dialyzed against pH 4 acetic acid (0.1 mM) for a day, yielding an agglomerated beige

concentration by accounting for swelling, measuring refractive index by refractometry, and fitting a line (r2 = 0.9745). When such measures were not taken, refractive index values showed extreme deviations from linearity (Figure S54). 1H NMR (500 MHz, D2O): δ 8.20−6.77 (8.20, 7.90, 7.47, 7.27, 6.84, 6.77, s, 10H, fluorescein), 4.23 (s, 2H, PAMA CH2 on ester), 4.13 (s, 8H PHEMA CH2 on ester), 3.86 (s, 8H, PHEMA CH2 on OH), 3.28 (s, 2H, PAMA CH2 on NH3), 2.01−1.69 (s, 10H, methacryl CH2), 1.92 (s, 3H, acetate CH3), 1.12−0.94 (m, 15H, methacryl CH3) ppm. 13C NMR (125 MHz, D2O): δ 179.8 (ester), 67.7, 67.0, 59.2, 53.5 (methacryl quaternary C), 45.2 and 44.7 (methacryl CH2), 23.27, 16.8, and 9.74 ppm. 1 H NMR (500 MHz, pyridine-d5): δ 5.50 (br s, 14H, H2O/ OH), 4.48 (s, 2H, PAMA CH2 on ester), 4.39 (s, 8H PHEMA CH2 on ester), 4.27 (s, 1H), 4.21 (s, 1H), 4.09 (s, 8H, PHEMA CH2 on OH), 3.06 (s, 2H, PAMA CH2 on NH3), 2.37−2.25 (s, 10H, methacryl CH2), 2.15 (s, 3H, acetate CH3), 1.85−1.53 (s, 15H, methacryl CH3) ppm. Small peaks visible from 7 to 8 ppm omitted due to potential confusion with solvent 13C satelites. FTIR-ATR (neat): 3256.1 (O−H), 2995.8, 2949.6 (C−H), 2884.3, 1717.7 (CO), 1642.2, 1563.2, 1481.4, 1451.4, 1405.2, 1391.6, 1334.4, 1271.7, 1246.9, 1152.6 (C−O), 1075.8 (C−O), 1021.2, 969.3, 947.5, 901.3, and 852.2 cm−1. For molecular weight and size statistics determined by GPC, SLS, and DLS, see Table 1. Protection of the alkyne group was verified by diagnostic SDS-PAGE (Figure 2). α-(Acetylene)-ω-bromo-poly(2-aminoethyl methacrylateco-2-hydroxyethyl methacrylate) (Alkyne-P(AMA-coHEMA)). In a 1.5 mL Eppendorf centrifuge tube was dissolved TIPS-P(AMA-co-HEMA) (150. mg, ∼1.5 μmol, 1 Eq) into 320 μL of methanol. In darkness was added 380 μL of 10 mg/ mL AgF in methanol solution (3.80 mg, 30 μmol, ∼15−45 Eq per TIPS group). With the minimal light in the room, it was possible to see orange precipitate immediately form and then quickly redissolve. Argon was blown into the tube. It was capped and sealed with parafilm, foiled, and allowed to mix at room temperature by inversion for 24 h. One molar HCl was added (45 μL, 45 μmol, 1.5 Eq per AgF), and no precipitate was observed as it was mixed at room temperature for 30 min by inversion. The sample was dialyzed in darkness with 25 kDa MWCO Spectra/por RC dialysis tubing against 0.1 mM acetic acid for a day. The samples were then lyophilized to yield 149.4−155.3 mg of fluffy intense yellow solid, corresponding to quantitative recovery. 1H NMR (500 MHz, D2O) and FTIR (neat) of these compounds were identical to their precursor polymers. Alkyne deprotection was verified by a diagnostic click reaction on an SDS-PAGE gel (Figure 2). α-(ELP)-ω-Bromo-poly(2-aminoethyl methacrylate-co-2hydroxyethyl methacrylate) (ELP-P(AMA-co-HEMA)). The stoichiometry for this reaction was altered for each polymer to maintain constant molar concentrations of all reagents by using with different alkyne mass concentrations (according to P(AMA-co-HEMA) Mn’s determined by DMF GPC prior to diazotransfer of 37.0 kDa, 73.1 kDa, and 104.9 kDa for the reported 30 kDa, 92 kDa, and 101 kDa samples respectively). This procedure for 92 kDa polymer is representative for all samples. To a 1.5 mL Eppendorf centrifuge tube was added alkyne-P(AMA-co-HEMA) (16.0 mg, 0.219 μmol, 1 Eq), azido-ELP (35.8 mg, 2.19 μmol, 10 Eq), H2O (463 μL), 2 M NEt3 HOAc buffer (109 μL, pH 7.1, 10× concentrated), and premixed CuSO4-THPTA solution (10 mM in H2O, 328 μL, 3.28 μmol, 15 Eq). The contents of the vial were mixed by 2602


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Bioconjugate Chemistry precipitate and clear solution. The final dialyte was spun down at 2000 rcf for 10 min, and the clear supernatant was decanted. The hybrid-rich pellet was dried by lyophalization for 23.2 mg of pale beige solid. The combined dry masses of the pellet and supernatant matched quantitative recovery, with the supernatant (ELP impurity) corresponding to roughly 20−30% of the input polymer. FTIR-ATR and 1H NMR were consistent with a combination of P(azido-AMA-co-HEMA) and ELP. Ratios of ELP to synthetic polymer by 1H NMR and FTIR were equal across all samples. The presence of azide is confirmed by FTIR and disappearance of the precursor amine peak (8.16 ppm, 1H NMR). For P(azido-AMA-co-HEMA) samples, diagnostic SDS-PAGE gave variable results for the same sample due to solubility issues. 1H NMR (500 MHz, DMSO-d6): δ 8.21−6.62 (8.04, 7.97, 7.76, 6.90, and 6.62 ppm, s, ELP amide N-H and fluorescein), 4.81 (s, 4H, PHEMA OH), 4.31 (s, ELP alpha CH), 4.03 (s, 2H, PAMA CH2 on ester), 3.90 (s, 8H PHEMA CH2 on ester), 3.58 (s, 10H, PHEMA CH2 on OH and PAMA CH2 on N3), 3.38 (s, illresolved, PAMA CH2 on azide), 1.83 (s, 8H, PHEMA methacryl CH2), 1.52 (s, 1H, PAMA methacryl CH2), 1.24 (s, 3H, residual acetate and/or acetic acid CH3, integral corresponds to >93% conversion), and 0.96−0.79 ppm (m, 15H, methacryl CH3). The 3.38 ppm (s, PAMA CH2 on azide) was not resolvable on most samples due to overlap with water. FTIR-ATR (neat): 3319.2 (O−H), 2958.3 (C−H), 2884.0, 2107.4 (NNN), 1722.8 (CO), 1652.8 (amide I), 1526.2 (amide II), 1449.0, 1389.5, 1245.2, 1152.5 (C−O), 1074.5 (C−O), 1022.2, 964.4, 989.3, 851.3, and 746.7 cm−1. Grafting of Alkyne-Terminated POEGMA to Poly(2azidoethyl methacrylate-co-2-hydroxyethyl methacrylate) (P((AMA-g-POEGMA)-co-HEMA)). This procedure is representative for the preparation of all brush polymers regardless of backbone length and ELP-incorporation. P(azido-AMA-coHEMA) (5.0 mg, 5.04 μmol azide, 1 Eq) was dissolved into 900 μL of DMF and 100 μL of H2O, a process which took a few minutes for ELP-lacking polymers and several hours for ELP-containing polymers. To a separate 50 mL round-bottom flask was added CuSO4 pentahydrate (10 mg/mL in H2O from ground solid, 1.89 mL, 18.9 mg, 75.6 μmol, 15 Eq) and THPTA (30 mg/mL in H2O, 1.46 mL, 43.7 mg, 101 μmol, 20 Eq), producing a medium intensity sapphire-blue colored solution. To the mixture was added 2 M NEt3 HOAc (1.74 mL, pH 7.1, 10× concentation), 9.4 mL of methanol, and 370 μL of H2O. After degassing with argon for 2 h, to the mixture was added P(azido-AMA-co-HEMA) solution and alkyne-POEGMA (491.8 mg, 25.19 μmol, 5 Eq) dissolved in 1 mL of methanol. Argon was blown into the reaction flask before finally adding sodium L-ascorbate (10 mg/mL in H2O, 1.50 mL, 15.0 mg, 75.6 μmol, 15 Eq), causing the mixture to quickly turn pale turquoise. The final reaction medium was thus 17.4 mL of 0.2 M NEt3 HOAc in 60% (v/v) methanol (40% water) with trace DMF. The flask was septum-sealed, covered in foil, and allowed to stir at room temperature for 22 h. The resulting pale yellow mixture was exposed to air, concentrated under reduced pressure, and combined with 1 mL of 0.1 M EDTA (disodium salt), causing the solution to turn medium intensity green-yellow. The solution was dialyzed in 25 kMWCO Spectra/Por RC dialysis tubing against 0.1 mM EDTA (disodium salt) for 5 h, 0.1 mM acetic acid overnight, and then Milli-Q H2O for 6 h with several buffer exchanges. The dialyte was concentrated under reduced pressure, transferred to a glass centrifuge tube with water, and

lyophilized. To the medium intensity yellow goo was added 600 μL of methanol to redissolve, then adding 12 mL of diethyl ether, causing the solution to immediately turn cloudy white. The tube was spun down. The supernatant was carefully removed by pipet, and the small amount of residual diethyl ether and methanol was removed by airstream. The process of redissolving and precipitating was repeated three more times with 1.0 mL, 1.0 mL, and 1.4 mL of methanol in each consecutive cycle. We have found the separation of bristle from the brush to very critically depend on the amount of methanol added, requiring roughly 200 μL of methanol for 4 kDa POEGMA to 1.4 mL of methanol for 20 kDa POEGMA (data not shown). Generally, the polymer is repeatedly precipitated until both a sufficient mass is obtained and GPC (THF) indicates that all bristle has been removed (Figure 3). 1 H NMR (500 MHz, D2O) was identical to that of alkynePOEGMA precursors with an additional peak at 8.21 ppm, corresponding to triazole C−H. FTIR-ATR (neat) was identical to alkyne-POEGMA. NMR molecular weight statistics were calculated based on data from 1H NMR in pyridine-d5. For consistency, all integral regions were precisely matched across polymer samples. Bristle degree of polymerization (Xn) was calculated from the ratio between the POEGMA terminal methoxy CH3 (3.4 ppm) and triazole CH (8.4 ppm). The product of grafting efficiency and bristle degree of polymerization was calculated from the ratio of PHEMA CH2 (4.12 ppm) and POEGMA terminal methoxy CH3 (3.4 ppm). Grafting efficiency was then calculated by division of the NMR-derived values. 1H NMR (500 MHz, pyridine-d5): δ 8.41 (s, triazole C−H), 6.54 (s, annihilated by D2O shake, PHEMA OH), 5.65 (s, 2H), 4.42 (s, POEGMA CH2 on ester, PAMA CH2 on ester, and PHEMA CH2 on ester), 4.12 (s, PHEMA CH2 on O−H), 3.82 (s, POEGMA bulk CH2), 3.64 (s, POEGMA last CH2), 3.40 (s, POEGMA terminal OCH3), 2.27 (s, methacryl CH2), and 1.53−1.44 ppm (s, methacryl CH3). Diagnostic Click Test Reaction. A fresh stock aqueous solution of 3 mg/mL L-ascorbic acid and 1.3 mM solution of the polymer to be tested was made the day of the test reaction. To a 0.5 mL plastic centrifuge tube was added 3 μL of test polymer solution (4 nmol of azide/alkyne), 0.8 μL of 10 mM Cy3 dye in DMSO (8 nmol, azide or alkyne terminated), 6.0 μL of 10 mM CuSO4 solution with 10 mM THPTA (60 nmol, premixed), 2.0 μL of pH 7 2.0 M NEt3 HOAc buffer, and 4.7 μL of water. The final buffer conditions were 0.2 M NEt3 HOAc at pH 7. For test reactions involving azide backbones, all samples to be tested on the same gel were reacted using 40% (v/v) ethanol in place of water. The 20 μL of magenta solution was combined by light centrifugation and mixed by pipet. The reaction pot was sealed and allowed to react at room temperature overnight. A gel loading sample was prepared by mixing 2 μL of reaction mixture, 10 μL of 2× Laemli buffer/glycerol in a 4:1 v/v mixture, and 8 μL of water. A total of 10 μL of gel loading sample was run on a 4−20% polyacrylamide gel alongside a protein ladder and positive control click reaction mixture (using 10 kDa PEG azide or 20 kDa PEG alkyne) at 120 V for an hour. The gels were fluorescently imaged for the presence of Cy3, and total Cy3 fluorescent intensity was determined using ImageJ. Afterward, gels with amine-containing polymer samples were stained using Coomassie brilliant blue for 10 min, and subsequently destained in water overnight while changing the water four times. The stained gel was then imaged to corroborate the 2603


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migration and shape of the polymer band in the Cy3 scan and verify the presence of loaded polymer.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.8b00309.

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Detailed synthetic procedures, characterization of all prepared compounds, and an extended discussion (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Stefan Zauscher: 0000-0002-2290-7178 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. L.A.N. prepared and characterized all small molecule reagents, synthetic polymers, and bioconjugates. D.L.F. prepared unfunctionalized ELPs used in this study. S.Z. aided in development. Funding

Funding was provided by the National Science Foundation (NSF) Triangle Materials Research Science and Engineering Center (MRSEC; DMR-1121107). We are also grateful for generous support from Prof. Farshid Guilak. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Yeongun Ko in Prof. Jan Genzer’s group (North Carolina State University) for performing DMF GPC of backbone polymer samples. We also thank Meredith Barbee in Prof. Steven Craig’s group (Duke University) for aid in performing THF GPC of POEGMA-based polymers. We thank Prof. Ashutosh Chilkoti’s laboratory for supplying plasmids for preparing ELP and offering use of their light scattering and biosynthesis equipment. We thank Peter Silinski for acquiring and processing ESI-TOF data.

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ABBREVIATIONS Cy3, cyanine 3 (dye); ELP, elastin-like polypeptide (with the inclusion of a collagen-binding peptide, model protein); ISA, imidazole-1-sulfonyl azide (hydrogen sulfate salt, diazotransfer reagent); PAMA, poly(2-aminoethyl methacrylate) (aminerich backbone); PHEMA, poly(2-hydroxyethyl methacrylate) (backbone spacer); POEGMA, poly(oligoethylene glycol methacrylate) (monomer Mn = 300, bristle); TIPS, triisopropylsilyl (alkyne protecting group)

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REFERENCES

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