Diels-Alder “Clickable” Polymer Brushes: A Versatile Catalyst-Free

Letter pubs.acs.org/macroletters

Diels-Alder “Clickable” Polymer Brushes: A Versatile Catalyst-Free Conjugation Platform Yasemin Nursel Yuksekdag,† Tugce Nihal Gevrek,† and Amitav Sanyal*,†,‡ †

Department of Chemistry and ‡Center for Life Sciences and Technologies, Bogazici University, Bebek, Istanbul 34342, Turkey S Supporting Information *

ABSTRACT: Polymeric brushes provide an attractive functional interface for a variety of applications in materials and biomedical sciences. Facile access to functionalized brushes can be realized through effective postpolymerization functionalization of reactive brushes. Over the past decade, efficient chemical transformations based on various “click” reactions have been employed for functionalization of polymeric brushes. This paper reports the first example of utilization of the Diels− Alder cycloaddition reaction based functionalization strategy that allows efficient conjugation of maleimide-containing molecules onto furan-containing polymer brushes under mild and reagent-free conditions. Polymers incorporating furan groups as side chains are “grafted from” silicon oxide surfaces and investigated toward their functionalization. Brushes are fabricated using atom transfer radical polymerization with varying amounts of furfuryl methacrylate to enable control over extent of functionalization, along with a poly(ethylene glycol) chain containing methacrylate as a comonomer to impart hydrophilic and antibiofouling characteristics. Functionalization of these reactive brushes were investigated through the immobilization of a model compound N-ethylmaleimide, a fluorescent dye BODIPY-maleimide, and a maleimide-containing biotin based ligand to direct the immobilization of streptavidin-coated quantum dots.

P

Polymer brush-coated surfaces offer an attractive platform for biomolecular immobilization for applications such as biosensing.35−39 Three dimensional dispositions of adequate functional groups on these soft nanomaterials provide high sensitivity. In particular, fabrication of functional polymer brushes wherein the matrix possesses antibiofouling properties would minimize nonspecific deposition or adsorption of biomolecules present in the environment, hence, would enable detection with high sensitivity.40 Functional polymeric brushes are generally obtained through postpolymerization functionalization of polymer brushes with appropriate molecules or biomolecules. This approach has gained impetus since the advent of “click” reactions. While initial reports employed the Huisgen-type copper-catalyzed (3 + 2) azide−alkyne cycloaddition,41−43 recent focus has shifted toward using metal-free transformations such as thiol−ene,44 thiol−yne,45,46 and the thiol-maleimide47−49 conjugations. A brief survey of the toolbox of “click” reactions reveals that the Diels−Alder cycloaddition reaction provides an attractive conjugation strategy for polymer brushes. The cycloaddition between an electron-rich diene, such as furan, and an electrondeficient dienophile, like maleimide, has been widely utilized in the fabrication of a variety of polymeric materials,50 including

ast decades have witnessed an evolution of research in polymeric surfaces from the domain of engineering and materials sciences to biological sciences.1,2 Contemporary techniques in synthesis of well-defined polymers for the design of various functional soft interfaces have promoted the usage of polymer chemistry in this area.3,4 Advances in synthesis of polymer brushes since the 1990s have enabled their fabrication with precise chemical and dimensional control to target specific applications.5,6 This evolution promoted the spread of fabrication and functionalization of polymer brushes to the field of biological sciences.7−9 Polymer brushes denote a class of polymeric coating where polymer chains are attached onto the substrate by one of their extremities through a covalent bond.10 As a result, these polymeric interfaces possess appreciable robustness when compared with coatings prepared using techniques such as spray, dip and spin coating.11 Commonly, one of the two available methods are utilized to obtain these polymeric coatings: “grafting to” and “grafting from” methods. The latter provides surface-tethered polymers in “brush” conformation with high grafting density.12,13 To date, various well-defined polymer brushes14−16 have been synthesized using contemporary polymerization techniques such as nitroxide-mediated polymerization (NMP),17−21 living anionic or cationic polymerization,22−24 atom transfer radical polymerization (ATRP),25−29 and reversible addition−fragmentation chain transfer (RAFT)30−34 polymerization to obtain precise control over polymer brush thickness, composition, and architecture. © XXXX American Chemical Society

Received: January 21, 2017 Accepted: March 21, 2017

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DOI: 10.1021/acsmacrolett.7b00041 ACS Macro Lett. 2017, 6, 415−420

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ACS Macro Letters self-healing ones,51,52 as well as for functionalization of polymeric interfaces such as hydrogels,53 nanofibers,54 and coatings.55 Generally, the reaction proceeds with high efficiency under mild conditions without the need of any catalysts. Among the various advantages of the Diels−Alder reaction, its thermoreversibility is quite relevant; the conjugation/deconjugation process can be reversible or irreversible within certain temperature ranges depending on the diene−dienophile combination.56−61 We envisioned that the utilization of a readily available diene containing reactive monomer, namely, furfuryl methacrylate (FuMA), would allow facile and rapid fabrication of reactive polymer brushes that can be easily modified with various functional molecules. Herein, we outline the fabrication and functionalization of polymer brushes that contains furan moieties as reactive side chains (Scheme 1). These polymer brushes were designed to

chain would enable facile conjugation of maleimide bearing compounds through the Diels−Alder reaction. First, the cycloaddition process is investigated using N-ethylmaleimide as a model compound; thereafter, the versatility of the platform is demonstrated by conjugation of a maleimide containing fluorescent dye, as well as a ligand enabling protein-directed immobilization of fluorescent quantum dots. Although our main focus was to evaluate these polymeric structures as platforms for facile conjugation, the reversible nature of the furan-maleimide dyad offers the potential of regenerating the parent brushes through the retro Diels−Alder reaction and their refunctionalization. This unique aspect of the dienecontaining polymeric brushes was also briefly explored. Polymerizations were initiated from halide containing initiator-coated surfaces in the presence of a Cu(I)Cl/2,2′bipyridine complex catalyst system. A mixture of methanol and water was used as reaction medium to obtain rapid polymerization and ensure solubility of both of the monomers. In order to probe the tunability of incorporation of the furan-based functional group, brushes containing three different molar ratios of PEGMEMA/FuMA in the feed (P1:90/10, P2:75/25, and P3:60/40) were synthesized, apart from PEGMEMA brushes P0. As expected, brushes with varying thicknesses could be obtained by controlling the duration of polymerization. An increase of brush thickness with polymerization time is observed. Notably, a decrease in thickness was seen upon increasing the amount of the reactive FuMA monomer in the polymer brushes (Table S1). The variation in incorporated monomer compositions for polymer brushes obtained with varying ratio of the monomers in the polymerization feed was deduced from high resolution XPS elemental scans of the O 1s peak (Figure 1a). The O 1s high resolution spectrum of the polymer brush P1 was deconvoluted with four Gaussian/Lorentzian curves that represent C−O−C (532.8 eV), O−CO (533.7 eV), and OC−O (532.3 eV) oxygen atoms and oxygen atoms belonging to the furan groups (534.9). The deconvolution of O 1s of P2 showed C−O−C at 532.6 eV, O−CO at 533.7 eV, and OC−O at 531.8 eV, and an oxygen atom belonging to the furan moiety C−O−C appeared at 534.2 eV. The O 1s signal of P3 was fitted also with four residual showing oxygen atoms from C−O−C (532.8 eV), O−CO (533.9 eV), O C−O (532.4 eV), and furan units (534.3 eV). Increasing the

Scheme 1. Schematic Illustration of Functionalization of Furan-Containing Polymer Brush through the Diels-Alder Reaction Based Conjugation

possess hydrophilicity as well as antibiofouling characteristics through utilization of poly(ethylene glycol) methyl ether methacrylate (PEGMEMA), a monomer benchmarked for this purpose.62 Utilization of a combination of these monomers is expected to allow specific conjugation of molecules of interest while reducing unwanted nonspecific adhesion, thus, providing a high signal−noise ratio during detection. Surface-initiated atom transfer radical polymerization (SI-ATRP) was employed to obtain brushes with control over chemical composition and heights. The furan functional groups present in the polymer

Figure 1. (a) High resolution XPS elemental scans of O 1s peak and (b) water contact angle of polymer brush surfaces P1, P2, and P3. 416

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ACS Macro Letters amount of the furan-containing monomer in the polymer brush resulted in an increasing of the C-O-C peak. Incorporation of the reactive monomer was calculated as 10.59, 26.30, and 43.67%, which was consistent with feed ratios of 10, 25, and 40% for P1, P2, and P3, respectively. As expected, variation in monomer composition of these polymer brushes affects their overall hydrophilicity since the furan group containing monomer is relatively hydrophobic compared to the PEG-based monomer. The water contact angles were determined to be 68°, 72°, and 79° for the polymer brushes P1, P2, and P3, respectively (Figure 1b). Thus, an increase in the amount of the furan-containing monomer in the brush results in corresponding increase in hydrophobicity of the surface. Reactive polymeric brushes containing electron-rich furan moieties were functionalized with N-ethylmaleimide, an electron-deficient model compound chosen to analyze the efficiency of functionalization via the Diels−Alder reaction. Evaluation of efficiency of functionalization of the polymer brushes containing different amounts of FuMA was performed using X-ray photoelectron spectroscopy (XPS) and infrared spectroscopy (IR). Importantly, N-ethylmaleimide contains a nitrogen atom that is not present in the original polymer backbone; hence, it provides a useful marker for the analysis of the functionalization using XPS. Polymer brush functionalization with N-ethylmaleimide was performed at two different temperatures, 40 and 60 °C. The N 1s signal in the XPS analysis revealed that higher amount of maleimide was incorporated onto the brushes at higher temperature. A further increase in reaction temperature to 80 °C did not result in an appreciable change in conjugation. The cycloaddition of furan with maleimide can produce the cycloadduct as two different isomers, namely, the endo and exo isomers. The endo isomer has lower stability and can slowly revert to starting components around 40 °C. Hence, to test the stability of the conjugates obtained at the two different temperatures, surfaces modified with N-ethyl maleimide at 40 and 60 °C were heated to 60 °C in toluene. It was observed that while the atomic percentage of N 1s for surfaces that were functionalized at 60 °C remained unchanged (1.2% vs 1.15%), surfaces functionalized at 40 °C showed a substantial loss of N signal (0.85% vs 0.38%; Figure S5). This suggests that cycloaddition at 60 °C yielded the stable exo product, while derivatization at 40 °C gave a mixture of endo and exo cycloadduct. Therefore, to obtain homogeneous linkage chemistry on the surface, all polymer brush functionalization studies were performed at 60 °C. To probe the ability to control the extent of functionalization by tailoring the amount of reactive furan group within the brushes, surfaces containing varying amounts of the reactive furan moiety were reacted with N-ethylmaleimide. After functionalization with Nethylmaleimide, the resulting polymer films were analyzed with ATR-FTIR (Figure 2a). Attachment of N-ethylmaleimide onto the polymer brushes was clearly evident from the evolution of a newly formed carbonyl (CO) stretch around 1701 cm−1, adjacent to the peak around 1730 cm−1 predominantly belonging to the ester carbonyls on the poly(PEGMEMA) side chains. This additional peak belongs to the carbonyl group of the maleimide group on the furan-maleimide cycloadduct. As expected, an increase in the amount of furan groups in the polymer brushes is accompanied by an increase in the stretching intensity of the imide carbonyl bond (Figure 2b). As expected, XPS analysis of these modified films revealed an increased amount of nitrogen in polymer brushes with higher

Figure 2. (a) Diels−Alder reaction of N-ethylmaleimide on poly(PEGMEMA-ran-FuMA) brushes; (b) ATR-FTIR spectra of carbonyl regions for P0, P1, P2, and P3; (c) Experimental and theoretical nitrogen atom percentages in N-ethylmaleimide functionalized P1, P2, and P3 obtained by XPS; (d) High-resolution XPS elemental scan of N 1s peak of P1, P2, and P3.

furan content (Figure 2d). The postpolymerization modification reactions proceeded with reasonable conversion (57.7% for P1, 53.7% for P2, and 57.4% for P3) as deduced from [N 1s]/ [C 1s] ratio. Comparison of theoretically expected and experimentally observed nitrogen atom content after cycloaddition suggests that only a moderate amount of the furan groups participate in cycloaddition (Figure 2c). Such differences are expected since the furan groups buried within the polymer brushes face considerable steric hindrance toward conjugation. Nonetheless, it is clear that the extent of functional group incorporation on these reactive polymer brushes can be easily modulated through proper choice of monomer composition (Figure 2c,d). The Diels−Alder reaction is known to be thermoreversible, that is, the cycloadduct can revert back to starting components upon heating to higher temperatures. While the main focus of this work was to demonstrate the forward reaction, that is, the Diels−Alder cycloaddition as a facile and effective method functionalization of polymeric brush interfaces, we briefly investigated the deconjugation process. Polymer brush surfaces functionalized with N-ethylmaleimide at 60 °C were exposed to higher temperatures (110 °C). The retro-Diels−Alder reaction takes place, and the polymer brush surfaces revert back to their original reactive form. The renewed polymeric surfaces can be functionalized again with a dienophile, N-ethylmaleimide, through the Diels−Alder cycloaddition reaction. Extent of deconjugation and subsequent reconjugation was followed by XPS. XPS elemental scan of N 1s peak that was clearly visible after attachment of N-ethylmaleimide on polymer brushes (Figure S6a) disappears upon subjection of these surfaces to the retro-Diels−Alder reaction (Figure S6b). Reappearance of the peak after treatment of these regenerated surfaces with Nethylmaleimide suggests efficient recyclability of these surfaces (Figure S6c). The conjugation/deconjugation processes can be easily followed using ATR-FTIR analysis of these polymer 417

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ACS Macro Letters brushes. After the first conjugation, the two carbonyl (CO) group stretches at 1701 and 1730 cm−1 belonging to ester carbonyls of side chains and imides carbonyls of maleimide, respectively, are clearly visible (Figure S6a). After deconjugation through the retro Diels−Alder reaction, the carbonyl stretch at 1701 cm−1 corresponding to maleimide group disappears (Figure S6b) which reappears upon subsequent functionalization step (Figure S6c). As a proof of concept, modification of these polymeric brushes with a maleimide-containing fluorescent dye, BODIPYmaleimide, was explored (Figure 3). Dye conjugation was

Figure 4. Modification of polymer brush with a maleimide-containing biotin based ligand and subsequent immobilization of streptavidin. (a) Fluorescence microscopy images of streptavidin conjugated Q-dots attached micropatterns and (b) control experiment with nonbiotinylated brushes.

In conclusion, novel reactive polymer brushes containing electron-rich furan-based diene moieties as side chains were synthesized using surface-initiated ATRP. Facile functionalization of these polymer brushes can be accomplished with maleimide-containing molecules of interest using the Diels− Alder reaction that proceeds under catalyst-free conditions. On demand, these polymeric surfaces can be regenerated via the retro Diels−Alder reaction to allow subsequent functionalization. Conjugation of N-ethyl maleimide as a model compound to probe the conjugation-deconjugation process; BODIPYmaleimide, a fluorescent dye; and a protein binding ligand, biotin-maleimide, was accomplished. Streptavidin-coated Qdots could be assembled onto biotinylated surfaces through specific ligand directed immobilization. It can be envisioned that facile fabrication and ability to functionalize these polymeric interfaces in a tailored fashion under relatively mild conditions will expand their utility toward fabrication of functional interfaces.

Figure 3. Fluorescence images of polymer brushes functionalized with BODIPY-maleimide (a) after Diels−Alder; (b) after retro-Diels− Alder; (c) after a second Diels−Alder reaction.

accomplished by incubation of polymer brush coated surface in a solution of furan containing fluorescent dye in toluene at 60 °C. The bright green fluorescence of thus modified surfaces observed using fluorescence microscopy reveals successful covalent conjugation of the dye to the surface (Figure 3a). Thereafter, deconjugation of dye molecules was achieved by heating these surfaces in a solution of toluene at 110 °C. After retro Diels−Alder reaction, fluorescence was substantially diminished. To prove polymer brush patterns can be reusable for subsequent functionalization, the same surface was treated with the maleimide-containing dye solution. As expected, reconjugation of the dye was evident from reappearance of fluorescence. Finally, these reactive polymeric brushes were also investigated for ligand-mediated biomolecular immobilizations (Figure 4). Widely utilized bioconjugation linkage, the biotin− streptavidin couple was chosen as a model system. Biotinylation of these polymer brushes was accomplished through treatment with a maleimide-containing biotin ligand in DMSO at 60 °C. After washing with copious amounts of solvents and water, surfaces were incubated with PBS solution of streptavidinconjugated quantum dots (Q-dots). Successful immobilization was deduced from the presence of the bright red fluorescence of the Q-dots upon fluorescence microscopy analysis (Figure 4a). Importantly, as a control, upon incubation of nonbiotinylated polymer brushes with a solution of streptavidin conjugated Q-dots, minimal fluorescence was observed (Figure 4b). This control experiment not only suggests that the immobilization occurs with specificity due to the appended biotin ligand but also illustrates that these polymer brushes are antibiofouling toward proteins, as expected due to presence of a PEG based matrix.

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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/acsmacrolett.7b00041. Synthetic protocols of BODIPY-furan dye, XPS scans, and height measurement plots (PDF).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Amitav Sanyal: 0000-0001-5122-8329 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank the Bogazici University Research Grant (Project No. 7324, 13B05P6) for financial assistance.

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DOI: 10.1021/acsmacrolett.7b00041 ACS Macro Lett. 2017, 6, 415−420

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DOI: 10.1021/acsmacrolett.7b00041 ACS Macro Lett. 2017, 6, 415−420