L. Andrew Lee

Surface Modification of Tobacco Mosaic Virus with “Click” Chemistry

Cu-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) reaction, renascent of the well-known Huisgen reaction, has recently flourished with applications in organic synthesis, drug discovery, polymer and materials science, and biotechnology. The high reaction yield, simple reaction and purification conditions, wide range of solvent and pH stabilities, and functional group tolerance make the CuAAC reaction a prototypical “click chemistry”, ideal for incorporating functionalities onto desired scaffolds. Over the years it has been widely employed to construct and functionalize polymeric and polyvalent display systems, including polymers, dendrimers, nanoparticles, and surfaces, where an extremely high reaction efficiency for every unit reaction is desirable. In particular, as organic azides and alkynes are almost unreactive with biomolecules and water, CuAAC reactions have been employed in derivatizing biomacromolecules, viruses, and cells with high efficacy under mild reaction conditions. Recently tyrosine residues have been considered as a particularly attractive target for chemoselective modification of proteins because of its subabundant distribution. Francis and coworkers have reported a number of transformations, ranging from the Mannich-type reaction to a transition metal mediated allylation reaction to a diazonium-coupling reaction, which can efficiently target the phenolic group of tyrosine residues at physiological conditions. To overcome the sluggish reactivity with electron-enriched diazonium salts, a sequential reduction/oxidation/Diels–Alder reaction was developed to break the limitation of functionalities being incorporated. In this communication, we report that CuAAC reactions can be combined with a diazonium-coupling reaction to quantitatively functionalize tyrosine residues with a wide array of starting materials. Tobacco Mosaic Virus (TMV) is a classic example of rodlike plant viruses consisting of 2130 identical protein subunits arranged helically around genomic single RNA strand. The length of TMV, that is, 300 nm, is defined by the encapsulated genomic RNA that stabilizes the coat protein assembly. The polar outer and inner surfaces of TMV have been exploited as templates to grow metal or metal oxide nanowires, and conductive polymers have been coated on 1D assembled TMV to produce conductive nanowires. TMV based materials have recently shown great potential with applications in nanoelectronics and energy harvesting devices. In addition, it has been reported that tyrosine residues (Y139) of TMV are viable for chemical ligation using the electrophilic substitution reaction at the ortho position of the phenol ring with diazonium salts. This reaction is very efficient, yet has two distinct disadvantages for broader applications. First, it is difficult to synthesize desired starting materials; and second, the reaction is not compatible with acid-labile functional groups and suitable for electron-deficient anilines only. To embrace the structural diversity of various starting materials, TMV offers an ideal polyvalent display system which allows us to test the efficiency of CuAAC reaction in combining with the tyrosine ligation reaction. As shown in Scheme 1, TMV was first treated with the diazonium salt generated from 3-ethynylaniline 1 in situ adapted from the protocol reported by Francis and co-workers. MALDI-TOF MS analysis indicated that >95% of the capsid monomers were converted into alkyne derivatives 2 (Figure 1) despite the absence of a strong electron withdrawing group in the diazonium reagent. Encouraged by this result, the CuAAC reactions between 2 and azides were explored. For bioconjugation reactions using CuAAC, the Cu catalysts are either generated directly by addition of Cu salts, or in situ from soluble Cu sources and a reducing agent, such as a copper wire, phosphines, thiols, or ascorbate. Multidentate heterocyclic ligands are often required for enhancing the reaction efficiency. Upon screening a series of reaction conditions, we found that the combination of CuSO4/sodium ascorbate (NaAsc) gave the best results. Whereas it is destructive to most other protein complex systems, ascorbate is evidently benign to TMV and has no impact on its structural integrity. 3-Azido-7-hydroxy-coumarin a was first employed as the ACHTUNGTRENNUNGazido counterpart in the reaction, which could be easily monitored by UV-visible absorption at 340 nm (Figure 1B). As a general protocol, 2 (2 mgmL ) and a (3 mm) were added to a solution of CuSO4 (1 mm) and NaAsc (2 mm) in Tris buffer (10 mm, pH 7.8) with 20% DMSO (used to increase the solubility of the azide component). After incubation for 18 h at room temperature, the viral particles were separated from the small molecules by sucrose gradient sedimentation. The integrity of TMV was confirmed by TEM and size-exclusion chromatography (SEC) analysis (data not shown). A strong absorption at 340 nm indicated the successful attachment of coumarin motifs (Figure 1B). MALDI-TOF MS analysis indicated a near quantitative transformation of surface alkynes to triazoles as shown in Figure 1A. [a] M. A. Bruckman, G. Kaur, L. A. Lee, F. Xie, J. Sepulveda, Dr. Q. Wang Department of Chemistry and Biochemistry and Nanocenter University of South Carolina 631 Sumter Street, Columbia, South Carolina 29208 (USA) Fax: (+1)803-777-9521 E-mail : wang@mail.chem.sc.edu [b] R. Breitenkamp, X. Zhang, M. Joralemon, Dr. T. P. Russell, Dr. T. Emrick Polymer Science and Engineering Department, University of Massachusetts Conte Center for Polymer Research, Massachusetts 01003 (USA)

Electrospinning fabrication, structural and mechanical characterization of rod-like virus-based composite nanofibers

Tobacco mosaic virus (TMV) was electrospun with polyvinyl alcohol (PVA) into continuous TMV–PVA composite nanofibers to form a biodegradable nonwoven fibrous mat as an extracellular matrix (ECM) mimetic. Morphological characterizations by electron microscopy showed that the addition of varying amounts of TMV resulted in homogeneous nanofibers without phase separation and did not change the diameter of the composite nanofibers. The orientation of TMV in as-spun fibers could be readily controlled and post-processing of the nonwoven TMV–PVA mat significantly improved its water resistance. In addition, tensile tests were performed on individual nanofibers, which revealed that the TMV–PVA composite nanofibers achieved a comparable Youngs modulus as PVA nanofibers. Since the modification of TMV is readily achieved via genetic or chemical methods, this process offers a facile way to incorporate a variety of functionalities into polymer nanofibers. As a demonstration of its potential as ECM mimetic, a mutant TMV containing RGD peptide was co-spun with PVA and the resulting fibrous substrates were used to promote cell growth.

Multivalent ligand displayed on plant virus induces rapid onset of bone differentiation.

Viruses are monodispersed biomacromolecules with well-defined 3-D structures at the nanometer level. The relative ease to manipulate viral coat protein gene to display numerous functional groups affords an attractive feature for these nanomaterials, and the inability of plant viruses to infect mammalian hosts poses little or no cytotoxic concerns. As such, these nanosized molecular tools serve as powerful templates for many pharmacological applications ranging as multifunctional theranostic agents with tissue targeting motifs and imaging agents, potent vaccine scaffolds to induce cellular immunity and for probing cellular functions as synthetic biomaterials. The results herein show that combination of serum-free, chemically defined media with genetically modified plant virus induces rapid onset of key bone differentiation markers for bone marrow derived mesenchymal stem cells within two days. The xeno-free culture is often a key step toward development of ex vivo implants, and the early onset of osteocalcin, BMP-2 and calcium sequestration are some of the key molecular markers in the progression toward bone formation. The results herein will provide some key insights to engineering functional materials for rapid bone repair.

RGD-conjugated rod-like viral nanoparticles on 2D scaffold improve bone differentiation of mesenchymal stem cells.

Viral nanoparticles have uniform and well-defined nano-structures and can be produced in large quantities. Several plant viral nanoparticles have been tested in biomedical applications due to the lack of mammalian cell infectivity. We are particularly interested in using Tobacco mosaic virus (TMV), which has been demonstrated to enhance bone tissue regeneration, as a tunable nanoscale building block for biomaterials development. Unmodified TMV particles have been shown to accelerate osteogenic differentiation of adult stem cells by synergistically upregulating bone morphogenetic protein 2 (BMP2) and integrin-binding bone sialoprotein (IBSP) expression with dexamethasone. However, their lack of affinity to mammalian cell surface resulted in low initial cell adhesion. In this study, to increase cell binding capacity of TMV based material the chemical functionalization of TMV with arginine-glycine-aspartic acid (RGD) peptide was explored. An azide-derivatized RGD peptide was “clicked” to tyrosine residues on TMV outer surface via an efficient copper(I) catalyzed azide-alkyne cycloaddition (CuAAC) reaction. The ligand spacing is calculated to be 2–4 nm, which could offer a polyvalent ligand clustering effect for enhanced cell receptor signaling, further promoting the proliferation and osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs).

Dynamic 3D Patterning of Biochemical Cues by using Photoinduced Bioorthogonal Reactions

Recreating an appropriate microenvironment for the purpose of regenerating tissues or organs poses clinically relevant issues that intrigue every mind, regardless of background. Living tissue, however, is an enigmatic amalgamation of cells that is surrounded by the extracellular matrix, a hierarchically organized hydrogel that is made of polysaccharides and proteins with specific biochemical and biomechanical functions. Controlling the 3D microenvironment has been an important aspect, not only for tissue engineering, but also to study the cells in more biologically relevant systems. Researchers have approached this problem with clever designs by combining materials with concepts from cell biology to associate specific cellular functions with controllable cues. These cues range from cell adhesion ligands, soluble cytokines/growth factors, mechanical properties of the substrate (that is, stiffness, porosity, etc.), 2D versus 3D architectures, and many more variations that are still being explored with diverse cell types. Whereas the advent of some earlier 3D materials forged a path towards a greater understanding of the cells, the limitations of these materials became quite apparent. The typical platform consists of a static architecture with fixed cues, like a mashed network of electrospun fibers or a porous hydrogel that contains randomly embedded bioactive molecules. In most cases, these previous systems possess poorly defined spatial patterns of biological moieties. Alternatively, an ideal scaffold should address the dynamic nature of the cells and their microenvironment. Therefore, a platform that possesses both high spatial resolutions and precise temporal control of the signaling motifs would indeed be useful for studying how cells work in dynamic networks. As demonstrated by DeForest and Anseth, 3D spatial programming of a hydrogel platform can be achieved through bioorthogonal photochemistries to attach or remove functional groups by using multiphoton light, which allows dynamic control of the pericellular region to test how cells would respond to biochemical changes in their local environment. Although using multiphoton chemistry to achieve a 3D immobilization of proteins or other signaling motifs in hydrogels has been reported by a number of groups, DeForest and Anseth further advance this technology by employing a combination of three bioorthogonal chemical processes (Figure 1). In the first step, the strain-promoted azide–alkyne cycloaddition (SPAAC) reaction forms the hydrogel. The bis-azido crosslinker contains an alkenyl tag which can undergo a photocontrolled thiol-ene “click” reaction to pin down the cell-binding peptide within the hydrogel network. Each peptide contains a photocleavable onitrobenzyl ether linker that can be removed to provide spatiotemporal control over the ligands. The application of two click processes, that is, the SPAAC and the thiol-ene reactions, with multifunctional molecular designs provide a clever approach to explore a broad array of bioactive motifs with good spatiotemporal control. Furthermore, this process is used to present gradients of cell-binding moieties, either linear or exponential, by applying a user-defined gradient of light exposure. Such functionally graded materials with spatiotemporal control provide powerful methods to study many biological processes, which include directionality for neuron outgrowth from the mammalian retina, and bone–muscle transitions with tendon/ ligament regeneration. The concept of positional information, that a cell knows its position in the gradient and determines its developmental fate accordingly, could be further elaborated with the 3D model reported by DeForest and Anseth. For example, as the cell transitions from one position to another (that is, as the ligament transitions from bone to muscle), the surrounding signaling moieties follow specific gradients and patterns in the natural tissue growth or even embryo development. The use of static 3D matrices would limit the cells to one artificial stage of development unless bioactive signals can be altered in time and space. With the spatiotemporal control, molecules would be presented at the “right time and right place”, and possibly with the proper concentrations to recognize the threshold levels that are required to initiate signal recognition. A platform that has an added level of sophistication takes in vitro experiments one step closer towards physiologically relevant systems. The beauty of the system reported by DeForest and Anseth comes from the simple chemistries that can be performed even in a living cell system. These [*] Dr. L. A. Lee, Prof. Q. Wang Department of Chemistry & Biochemistry, University of South Carolina, Columbia, SC 29208 (USA) E-mail: wang@mail.chem.sc.edu

Increased activity of β-glucuronidase variants produced by site-directed mutagenesis

β-glucuronidase (BGus) is an essential glycosyl hydrolase which has been widely used in biological and biomedical applications. In this paper, we report the construction and screening of nineteen Escherichia coli BGus (EBGus) mutants using site-directed mutagenesis. The mutants G559N, G559S and G559T showed a 3-5 fold increase in enzyme activity in comparison to wild type EBGus. In particular, G559S, with the highest activity, showed 2-6 fold enhanced activity compared to abalone and snail BGus extracts. Moreover, the glycine to serine mutagenesis for the same site in Staphylococcus sp. RLH1 BGus (StBGus) exhibited significantly enhanced activity, which indicated the importance of the G559→S mutation on BGus function. Based on this structural analysis, we postulate that the mutation at G559 plays an important role in the stabilization of the enzyme conformation, and thereby facilitates the effective binding of substrate.