Jeroen J. L. M. Cornelissen

A virus-based single-enzyme nanoreactor

Most enzyme studies are carried out in bulk aqueous solution, at the so-called ensemble level, but more recently studies have appeared in which enzyme activity is measured at the level of a single molecule, revealing previously unseen properties. To this end, enzymes have been chemically or physically anchored to a surface, which is often disadvantageous because it may lead to denaturation. In a natural environment, enzymes are present in a confined reaction space, which inspired us to develop a generic method to carry out single-enzyme experiments in the restricted spatial environment of a virus capsid. We report here the incorporation of individual horseradish peroxidase enzymes in the inner cavity of a virus, and describe single-molecule studies on their enzymatic behaviour. These show that the virus capsid is permeable for substrate and product and that this permeability can be altered by changing pH.

Metal-Free Triazole Formation as a Tool for Bioconjugation

The development of selective and site-specific bio-orthogonal conjugation methods is an important topic in chemical biology. A wide range of methods, such as the Staudinger ligation, native chemical ligation, genetic incorporation, expressed-protein ligation, Huisgen azide–alkyne cycloaddition, and the Diels–Alder ligation are currently employed in the selective modification of proteins and other biomolecules. In recent years, the Cu-catalyzed variant of the Huisgen 1,3-dipolar cycloaddition, also referred to as “click reaction”, has been increasingly applied in various fields of chemistry as a versatile and mild ligation method. This method allows for the synthesis of complex materials, which include bioconjugates, glycopeptides, functionalized polymers, virus particles, and therapeutics. However, due to the toxicity of the copper catalyst to both bacterial and mammalian cells applications that involve in vivo ligation are limited. In order to circumvent the use of copper ions, Bertozzi and co-workers have devised a strain-promoted [3+2] cycloaddition reaction that involves azides and a strained cyclooctyne derivative. Recent reports by Ju et al. have also shown successful applications of copper-free 1,3-dipolar cycloaddition by using either elevated temperatures or electron-deficient alkynes. We envisioned that the combination of ring strain and electron deficiency, as occurs in oxa-bridged bicyclic systems 2a and 2b, could also lead to an increased reactivity toward [3+2] cycloaddition reactions. Here, we report a spontaneous tandem [3+2] cycloaddition–retro-Diels–Alder ligation method that results in a stable 1,2,3-triazole linkage. This methodology can be applied to biomacromolecules that contain various functional groups under physiological conditions. The oxabridged bicyclic systems 2a and 2b were prepared by a Diels– Alder reaction of substituted propiolates with furan (Scheme 1). Subsequent hydrolysis provided the desired carboxylic acid derivatives 3a and 3b, in excellent yield. To compare the reactivity of Diels–Alder products 2a and b with the corresponding alkynes, [3+2] cycloaddition reactions were performed under ambient conditions by using benzyl azide, and monitored over time with H NMR spectroscopy (Figure 1). The oxanorbornadienes 2a and 2b and their respective alkynes provided identical 1,4,5-substituted triazoles to the products.

Virus-like Particles Templated by DNA Micelles: A General Method for Loading Virus Nanocarriers

DNA amphiphile particles template formation of virus capsids and enable their loading.

Viruses and protein cages as nanocontainers and nanoreactors

In this highlight, recent studies related to the use of viral capsids and other protein cages as nanocontainers and nanoreactors are discussed. The templating of chemical reactions within these biological hollow scaffolds is an emerging area which has allowed the synthesis of inorganic materials with nanoscale dimensions. Encapsulation of synthetic polymers, enzymes working within viral capsids, and the solubilisation of protein cages in organic solvents are some other examples of research that is currently being performed and reviewed in this article.

Monodisperse polymer–virus hybrid nanoparticles

Self-assembly of polystyrene sulfonate and modified cowpea chlorotic mottle virus protein yields monodisperse icosahedral nanoparticles of 16 nm size.

Block copolymer vesicles

Amphiphilic block copolymers have the ability to assemble into multiple morphologies in solution. Depending on the length of the hydrophilic block, the morphology can vary from spherical micelles, rods, and vesicles to large compound micelles (LCMs). Vesicle formation is favored upon an increase in total molecular weight of the block copolymer, that is, an increasing bending modulus (K). Owing to the polymeric character of this type of vesicle (also called polymersomes), they possess remarkable properties. The diffusion of (polymeric) amphiphiles in these vesicles is very low compared to liposomes and for high-molecular-weight chain entanglements even lead to reptation-type motions, which make it possible to trap near-equilibrium and metastable morphologies. Additionally, in contrast to liposomes, membrane thicknesses can exceed 200 nm. As a consequence, this increased membrane thickness, in combination with the conformational freedom of the polymer chains, leads to a much lower permeability for water of block copolymer vesicles compared to liposomes. The enhanced toughness and reduced permeability of polymersomes makes them, therefore, very suitable as stable nanocontainers, which can be used, for example, as reactors or drug delivery vehicles.

Protein-polymer hybrid amphiphiles

Synthesis of 1: 350 mg (0.036 mmol) of carboxy terminated polystyrene, 20 mg (0.053 mmol) of biotinyl-3,6-dioxaoctanediamine and 0.06 ml Et3N were dissolved in 15 ml destilled DMF. Subsequently, 22 mg (0.050 mmol) of (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate was added. After stirring for 16 hrs at room temperature under a nitrogen atmosphere the solution was precipitated in 600 ml MeOH. The crude product was further purified by column chromatography (MeOH/CH2Cl2, 2:98 v/v); yield 142 mg (0.015 mmol; 43%) of 1. Mn = 9147; Mw/Mn = 1.03;

Reactions inside nanoscale protein cages

Chemical reactions are traditionally carried out in bulk solution, but in nature confined spaces, like cell organelles, are used to obtain control in time and space of conversion. One way of studying these reactions in confinement is the development and use of small reaction vessels dispersed in solution, such as vesicles and micelles. The utilization of protein cages as reaction vessels is a relatively new field and very promising as these capsules are inherently monodisperse, in that way providing uniform reaction conditions, and are readily accessible to both chemical and genetic modifications. In this review, we aim to give an overview of the different kinds of nanoscale protein cages that have been employed as confined reaction spaces.

Cascade Reactions in an All‐Enzyme Nanoreactor

Good things come in small packages! Giant amphiphiles, consisting of a polymeric hydrophobic tail and a horseradish peroxidase head, were simultaneously synthesized and self-assembled into vesicles. During the self-assembly process, glucose oxidase was encapsulated to create enzymatic cascade nanoreactors (see picture; ABTS= 2,2′-azido-bis(3-ethylbenzthiazoline-6-sulfonic acid)).

Encapsulation of DNA-templated chromophore assemblies within virus protein nanotubes

A beneficial virus: The hierarchical self-assembly of a three-component system consisting of single-stranded DNA (oligothymines; Tq), chromophores (G), and virus coat proteins (CP) leads to the formation of micrometer-long nanotubes (see picture). Tuning the interaction between the three components leads to the formation of structures with different length scales, and the chromophores within the nanotubes maintain the helical arrangement of the Tq–G template