Combinatorial methods for preparing polymeric biomaterials enable the rapid identification of materials useful for many applications in science, medicine, and engineering. In the work described here, we demonstrate that side-chain reactive polymers can be used as templates for the rapid preparation of a small library of diversely functionalized protein-polymer conjugates. The activated ester polymer poly(pentafluorophenyl acrylate) (PPFPA) was modified postpolymerization with substoichiometric equivalents of three hydrophilic primary amines to yield a library of amphiphilic, side-chain reactive copolymers. These copolymers were then conjugated to two receptor-targeting proteins, holotransferrin (hTF) and an engineered fibronectin-based protein (Fn3), through amine-activated ester coupling. We investigated the influence of polymer:protein ratio, side-chain chemistry (i.e., hydrophilic group identity and number of protein-reactive groups), and protein identity on conjugation efficiencies. Our results demonstrate that, for polymers of similar solubility in aqueous media, a larger polymer:protein ratio yields higher conjugation efficiencies. In addition, polymers with a greater number of reactive groups or shorter hydrophilic side chains improve protein conjugation efficiency. Finally, smaller proteins couple to the polymers more efficiently than do larger proteins. Collectively, the results described here demonstrate a modular approach for efficiently preparing bioconjugates with diverse chemistries that may be of interest in a broad range of applications.

Polymer coatings are frequently used to modify surface properties of inorganic substrates. However, the disparity in physical properties between polymer film and substrate often leads to residual stress development, which can be deleterious to the overall performance of coated materials. This work reports the molecular design of polymer films that dissipate stress upon irradiation with ultraviolet (UV) light. These polymers are synthesized by post-polymerization modification of the reactive polymer, poly(2-vinyl-4,4-dimethyl azlactone), to introduce dynamic crosslinks capable of light-initiated addition transfer fragmentation chemistry. Using a custom-built optical cantilever, contrasting film stress responses are observed between films containing dynamic bonds and analogous control films after UV light irradiation, which indicate successful stress relaxation. Further experiments demonstrate the complete relaxation of residual stress in dynamic films after an extended exposure, thereby generating a "stress-free" film. Films fabricated using this approach can be easily tailored to incorporate additional moieties to introduce desired surface properties for future application in a wide array of coatings.

We report an approach for the photomediated post-fabrication modification of reactive, azlactone-containing gels using light-initiated deprotection of amines caged with 2-(nitrophenyl)propyloxycarbonyl (NPPOC). Photomediated modification of these gels can be used to generate a gradient in chemical functionality. When functionalized with tertiary amine groups, these gradient gels exhibit rapid and reversible shape deformations in response to changes in pH.

Conjugation of proteins to drug-loaded polymeric structures is an attractive strategy for facilitating target-specific drug delivery for a variety of clinical needs. Polymers currently available for conjugation to proteins generally have limited chemical versatility for subsequent drug loading. Many polymers that do have chemical functionality useful for drug loading are often insoluble in water, making it difficult to synthesize functional protein-polymer conjugates for targeted drug delivery. In this work, we demonstrate that reactive, azlactone-functionalized polymers can be grafted to proteins, conjugated to a small-molecule fluorophore, and subsequently internalized into cells in a receptor-specific manner. Poly(2-vinyl-4,4-dimethylazlactone), synthesized using reversible addition-fragmentation chain transfer polymerization, was modified post-polymerization with substoichiometric equivalents of triethylene glycol monomethyl ether to yield reactive water-soluble, azlactone-functionalized copolymers. These reactive polymers were then conjugated to proteins holo-transferrin and ovotransferrin. Protein gel analysis verified successful conjugation of proteins to polymer, and protein-polymer conjugates were subsequently purified from unreacted proteins and polymers using size exclusion chromatography. Internalization experiments using a breast cancer cell line that overexpresses the transferrin receptor on its surface showed that the holo-transferrin-polymer conjugate was successfully internalized by cells in a mechanism consistent with receptor-mediated endocytosis. Internalization of protein-polymer conjugate demonstrated that the protein ligand maintained its overall structure and function following conjugation to polymer. Our approach to protein-polymer conjugate synthesis offers a simple, tailorable strategy for preparing bioconjugates of interest for a broad range of biomedical applications.

ABSTRACT The work reported here demonstrates an approach to the fabrication of chemically reactive and topographically patterned hydrogels using the azlactone‐functionalized polymer poly(2‐vinyl‐4,4'‐dimethylazlactone) (PVDMA) and the hydrophilic diamine Jeffamine®. Gels were initially assembled in DMSO but can be subsequently transferred into aqueous media to form hydrogels. Spectroscopic characterization of assembled gels demonstrated that variation in the stoichiometric ratio of azlactones to amines during gel synthesis permits control over the extent of crosslinking in the gels. Residual azlactones not consumed during crosslinking can be exploited to further functionalize these gels with hydrophobic, hydrophilic, and macromolecular amines that influence the physicochemical properties of these materials in aqueous solvents. The surface and bulk of these gels can be differentially functionalized (i.e., different functional groups on the gel surface relative to the bulk) by taking advantage of different rates of diffusion of macromolecular amines versus small molecule amines into assembled gels. Finally, these azlactone‐functionalized gels can be topographically patterned with microwell arrays using a replica molding technique and chemically modified postfabrication with amine nucleophiles. This reactive approach to the fabrication of topographically patterned and chemically functionalized hydrogels offers a straightforward method for the rapid synthesis of micropatterned scaffolds of interest in a broad range of applications. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017 , 55 , 3185–3194

Coiled-coil domains can direct the assembly of protein block copolymers into physically cross-linked, viscoelastic hydrogels. Here, we describe the use of fluorescence recovery after photobleaching (FRAP) to probe chain mobility in reversible hydrogels assembled from engineered proteins bearing terminal coiled-coil domains. We show that chain mobility can be related to the underlying dynamics of the coiled-coil domains by application of a three-state "hopping" model of chain migration. We further show that genetic programming allows the effective mobility of network chains to be varied 500-fold through modest changes in protein sequence. Destabilization of the coiled-coil domains by site-directed mutagenesis increases the effective diffusivity of probe chains. Conversely, probe mobility is reduced by expanding the hydrophobic surface area of the coiled-coil domains through introduction of the bulky leucine surrogate homoisoleucine. Predictions from the three-state model imply asymmetric sequential binding of the terminal domains. Brownian Dynamics simulations suggest that binding asymmetry is a general feature of reversible gels, arising from a loss in entropy as chains transition to a conformationally restricted bridged state.

Literature-based problems expose students to current, real world applications of chemistry. These types of problems are often confined to graduate and advanced undergraduate courses. We describe incorporation of literature-based problems in Organic I and II courses on quizzes and exams. We give students at least 1 week to study and discuss portions of a paper, then ask students to answer quiz and exam questions based on the paper. Students show high levels of engagement with and interest in the primary chemical literature when faced with these types of assessments.

A set of recombinant artificial proteins that can be cross-linked, by either covalent bonds or association of helical domains or both, is described. The designed proteins can be used to construct molecular networks in which the mechanism of crosslinking determines the time-dependent responses to mechanical deformation.

Virulence in Staphylococcus aureus is strongly and positively correlated with local cell density. Here we present an effective approach to modulate this group behaviour using multivalent peptide-polymer conjugates. Our results show that by attaching multiple AIP-4' units to macromolecular scaffolds, the agr QS response in S. aureus was affected strongly, while displaying a clear multivalency effect.

We report an approach to the in situ synthesis of oligonucleotide arrays on surfaces coated with crosslinked polymer multilayers. Our approach makes use of methods for the 'reactive' layer-by-layer assembly of thin, amine-reactive multilayers using branched polyethyleneimine (PEI) and the azlactone-functionalized polymer poly(2-vinyl-4,4'-dimethylazlactone) (PVDMA). Post-fabrication treatment of film-coated glass substrates with d-glucamine or 4-amino-1-butanol yielded hydroxyl-functionalized films suitable for the Maskless Array Synthesis (MAS) of oligonucleotide arrays. Glucamine-functionalized films yielded arrays of oligonucleotides with fluorescence intensities and signal-to-noise ratios (after hybridization with fluorescently labeled complementary strands) comparable to those of arrays fabricated on conventional silanized glass substrates. These arrays could be exposed to multiple hybridization-dehybridization cycles with only moderate loss of hybridization density. The versatility of the layer-by-layer approach also permitted synthesis directly on thin sheets of film-coated poly(ethylene terephthalate) (PET) to yield flexible oligonucleotide arrays that could be readily manipulated (e.g., bent) and cut into smaller arrays. To our knowledge, this work presents the first use of polymer multilayers as a substrate for the multi-step synthesis of complex molecules. Our results demonstrate that these films are robust and able to withstand the ~450 individual chemical processing steps associated with MAS (as well as manipulations required to hybridize, image, and dehybridize the arrays) without large-scale cracking, peeling, or delamination of the thin films. The combination of layer-by-layer assembly and MAS provides a means of fabricating functional oligonucleotide arrays on a range of different materials and substrates. This approach may also prove useful for the fabrication of supports for the solid-phase synthesis and screening of other macromolecular or small-molecule agents.

We report an approach to the fabrication and selective functionalization of amine-reactive polymer multilayers on the surfaces of 3-D polyurethane-based microwell cell culture arrays. "Reactive" layer-by-layer assembly of multilayers using branched polyethyleneimine (BPEI) and the azlactone-functionalized polymer poly(2-vinyl-4,4'-dimethylazlactone) (PVDMA) yielded film-coated microwell arrays that could be chemically functionalized postfabrication by treatment with different amine-functionalized macromolecules or small molecule primary amines. Treatment of film-coated arrays with the small molecule amine d-glucamine resulted in microwell surfaces that resisted the adhesion and proliferation of mammalian fibroblast cells in vitro. These and other experiments demonstrated that it was possible to functionalize different structural features of these arrays in a spatially resolved manner to create dual-functionalized substrates (e.g., to create arrays having either (i) azlactone-functionalized wells, with regions between the wells functionalized with glucamine or (ii) substrates with spatially resolved regions of two different cationic polymers). In particular, spatial control over glucamine functionalization yielded 3-D substrates that could be used to confine cell attachment and growth to microwells for periods of up to 28 days and support the 3-D culture of arrays of cuboidal cell clusters. These approaches to dual functionalization could prove useful for the long-term culture and maintenance of cell types for which the presentation of specific and chemically well-defined 3-D culture environments is required for control over cell growth, differentiation, and other important behaviors. More generally, our approach provides methods for the straightforward chemical functionalization of otherwise unreactive topographically patterned substrates that could prove to be useful in a range of other fundamental and applied contexts.

Polymers functionalized with azlactone (or oxazolone) functionality have become increasingly useful for the rapid and modular design of functional materials. Because azlactones can react via ring-opening reactions with a variety of different nucleophilic species (e.g., primary amines, hydroxyl groups, and thiol functionality), azlactone-functionalized materials can serve as convenient 'reactive' platforms for the post-synthesis or post-fabrication introduction of a broad range of chemical functionality to soluble polymers, insoluble supports, and surfaces/interfaces. The last decade has seen an increase in both the number and the variety of reports that exploit the properties and the reactivities of azlactone-functionalized polymers. Here, we highlight recent work from several different laboratories, including our own, toward the design and characterization of azlactone-functionalized polymers, with a particular emphasis on: (i) new synthetic approaches for the preparation of well-defined azlactone-functionalized polymers using living/controlled methods of polymerization, (ii) the design and modular synthesis of side-chain functionalized polymers and block copolymers via post-polymerization modification of azlactone-functionalized polymers, (iii) the development of reactive polymeric supports useful in the contexts of separations and catalysis, and (iv) methods for the fabrication of reactive thin films and other approaches to the immobilization of azlactone functionality on surfaces and interfaces. Examples discussed herein reveal a growing awareness of azlactone functionality as a useful tool for polymer chemists, and highlight several ways that the unique reactivity of these materials can both complement and provide useful alternatives to other reactive polymers currently used to design functional materials.

We report a method for modulating the physicochemical properties of surfaces that is based on the reactive layer-by-layer fabrication of covalently crosslinked thin films using azlactone-functionalized copolymers. We demonstrate that copolymers containing different molar ratios of methyl methacrylate (MMA) and 2-vinyl-4,4-dimethylazlactone (VDMA) can be alternately deposited with poly(ethyleneimine) to assemble covalently crosslinked thin films. Characterization using ellipsometry demonstrates that, in general, film growth and thickness decrease as the content of reactive, azlactone functionality in the copolymer used to assemble the film decreases. Reflective infrared spectroscopy experiments demonstrate that films fabricated from MMA: VDMA copolymers contain residual azlactone functionality and that these reactive groups can be exploited to modify film-coated surfaces. Fabricating films from MMA: VDMA copolymers containing different compositions permitted modulation of the density of reactive groups within the films and, thus, the extent to which the films are functionalized by exposure to small molecule amines. For example, functionalization of MMA: VDMA copolymer films with the small molecule D-glucamine resulted in films with water contact angles that varied with the composition of the copolymer used to fabricate the film (e.g., as the azlactone content in the film increased, glucamine-modified films became more hydrophilic). We demonstrate further that treatment of copolymer-containing films with glucamine resulted in changes in the numbers of mammalian cells that grow on the surfaces of the films. Our results suggest the basis of methods that could be used to modulate or tune the density of chemical and biological functionality presented on surfaces of interest in a variety of fundamental and applied contexts.

We report on the fabrication of covalently crosslinked and amine-reactive hollow microcapsules using 'reactive' layer-by-layer assembly to deposit thin polymer films on sacrificial microparticle templates. Our approach is based on the alternating deposition of layers of a synthetic polyamine and a polymer containing reactive azlactone functionality. Multilayered films composed of branched poly(ethylene imine) (BPEI) and poly(2-vinyl-4,4-dimethylazlactone) (PVDMA) were fabricated layer-by-layer on the surfaces of calcium carbonate and glass microparticle templates. After fabrication, these films contained residual azlactone functionality that was accessible for reaction with amine-containing molecules. Dissolution of the calcium carbonate or glass cores using aqueous ethylenediaminetetraacetic acid (EDTA) or hydrofluoric acid (HF), respectively, led to the formation of hollow polymer microcapsules. These microcapsules were robust enough to encapsulate and retain a model macromolecule (FITC-dextran) and were stable for at least 22 hours in high ionic strength environments, in low and high pH solutions, and in several common organic solvents. Significant differences in the behaviors of capsules fabricated on CaCO3 and glass cores were observed and characterized using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). Whereas capsules fabricated on CaCO3 templates collapsed upon drying, capsules fabricated on glass templates remained rigid and spherical. Characterization using EDS suggested that this latter behavior results, at least in part, from the presence of insoluble metal fluoride salts that are trapped or precipitated on or within the walls of capsules after etching the glass cores using HF. Our results demonstrate that the assembly of BPEI/PVDMA films on sacrificial templates can be used to fabricate reactive microcapsules of potential use in a wide range of fields, including catalysis, drug and gene delivery, imaging, and biomedical research.

We report an approach to the fabrication of free-standing and amine-reactive thin films that is based on the reactive layer-by-layer assembly and subsequent lift-off of azlactone-containing polymer multilayers. We demonstrate that covalently cross-linked multilayers fabricated using the azlactone-functionalized polymer poly(2-vinyl-4,4-dimethylazlactone) (PVDMA) and a primary amine-containing polymer [poly(ethyleneimine) (PEI)] can be delaminated from planar glass and silicon surfaces by immersion in mildly acidic aqueous environments to yield flexible freestanding membranes. These free-standing membranes are robust and can withstand exposure to strong acid, strong base, or incubation in high ionic strength solutions that typically lead to the disruption and erosion of polymer multilayers assembled by reversible weak interactions (e.g., "polyelectrolyte multilayers" assembled by electrostatic interactions or hydrogen bonding). We demonstrate further that these PEI/PVDMA assemblies contain residual reactive azlactone functionality that can be exploited to chemically modify the films (either directly after fabrication or after they have been lifted off of the substrates on which they were fabricated) using a variety of amine-functionalized small molecules. These free-standing membranes can also be transferred readily onto other objects (for example, onto the surfaces of planar substrates containing holes or pores) to fabricate suspended polymer membranes and other film-functionalized interfaces. In addition to planar, two-dimensional free-standing films, this approach can be used to fabricate and isolate three-dimensional free-standing membranes (e.g., curved films or tubes) by layer-by-layer assembly on, and subsequent lift-off from, the surfaces of topologically complex substrates (e.g., the curved ends of glass tubing, etc.). The results of this investigation, when combined, suggest the basis of methods for the fabrication of stable, chemically reactive, and flexible polymer thin films and membranes of potential utility in a variety of fundamental and applied contexts.