Marjoke F. Debets

Bioconjugation with Strained Alkenes and Alkynes

The structural complexity of molecules isolated from biological sources has always served as an inspiration for organic chemists. Since the first synthesis of a natural product, urea, chemists have been challenged to prepare exact copies of natural structures in the laboratory. As a result, a broad repertoire of synthetic transformations has been developed over the years. It is now feasible to synthesize organic molecules of enormous complexity, and also molecules with less structural complexity but prodigious societal impact, such as nylon, TNT, polystyrene, statins, estradiol, XTC, and many more. Unfortunately, only a few chemical transformations are so mild and precise that they can be used to selectively modify biochemical structures, such as proteins or nucleic acids; these are the so-called bioconjugation strategies. Even more challenging is to apply a chemical reaction on or in living cells or whole organisms; these are the so-called bioorthogonal reactions. These fields of research are of particular importance because they not only pose a worthy challenge for chemists but also offer unprecedented possibilities for studying biological systems, especially in areas in which traditional biochemistry and molecular biology tools fall short. Recent years have seen tremendous growth in the chemical biology toolbox. In particular, a rapidly increasing number of bioorthogonal reactions has been developed based on chemistry involving strained alkenes or strained alkynes. Such strained unsaturated systems have the unique ability to undergo (3 + 2) and (4 + 2) cycloadditions with a diverse set of complementary reaction partners. Accordingly, chemistry centered around strain-promoted cycloadditions has been exploited to precisely modify biopolymers, ranging from nucleic acids to proteins to glycans. In this Account, we describe progress in bioconjugation centered around cycloadditions of these strained unsaturated systems. Being among the first to recognize the utility of strain-promoted cycloadditions between alkenes and dipoles, we highlight our report in 2007 of the reaction of oxanobornadienes with azides, which occurs through a sequential cycloaddition and retro Diels-Alder reaction. We further consider the subsequent refinement of this reaction as a valuable tool in chemical biology. We also examine the development of the reaction of cyclooctyne, the smallest isolable cyclic alkyne, with a range of substrates. Owing to severe deformation of the triple bond from ideal linear geometry, the cyclooctynes show high reactivity toward dienes, 1,3-dipoles, and other molecular systems. In the search for bioorthogonal reactions, cycloadditions of cyclic alkenes and alkynes have now established themselves as powerful tools in reagent-free bioconjugations.

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.

Azide: A Unique Dipole for Metal-Free Bioorthogonal Ligations

Covalently bound azide on a (small) organic molecule or a (large) biomolecular structure has proven an important handle for bioconjugation. Azides are readily introduced, small, and stable, yet undergo smooth ligation with a range of reactive probes under mild conditions. In particular, the potential of azides to undergo metal‐free reactions with strained unsaturated systems has inspired the development of an increasing number of reactive probes, which are comprehensively summarized here. For each individual probe, the synthetic preparation is described, together with reaction kinetics and the full range of applications, from materials science to glycoprofiling. Finally, a qualitative and quantitative comparison of azido‐reactive probes is provided.

Protein Modification by Strain-Promoted Alkyne-Nitrone Cycloaddition

The bioorthogonal chemical reporter strategy is emerging as a versatile method for the labeling of biomolecules, such as nucleic acids, lipids, carbohydrates, and proteins.1 In this approach, an abiotic chemical functionality (reporter) is incorporated into a target biomolecule and can then react with a complementary bioorthogonal functional group linked to one of a diverse set of probes.

Synthesis and self-assembly of well-defined elastin-like polypeptide–poly(ethylene glycol) conjugates

A series of stimulus-responsive elastin-like polypeptide-poly(ethylene glycol) (ELP-PEG) block copolymers was synthesized. The polymeric building blocks were conjugated via the efficient and specific strain-promoted alkyne-azide cycloaddition (SPAAC). For this purpose, ELP and PEG blocks were functionalized with azide and cyclooctyne moieties, respectively. Azides were introduced by applying a recently developed pH-controlled diazotransfer reaction on the primary amines present in ELP (N-terminus and lysine side chains). By varying pH, ELP-blocks with one or two azides were obtained, which subsequently allowed us to synthesize both ELP-PEG diblock copolymers and miktoarm star polymers. Triggering the phase transition of the ELP-block resulted in the formation of an amphiphilic block copolymer, which self-assembled into micelles. This is the first example of an ELP-containing hybrid block copolymer in which PEG as the hydrophilic corona-forming domain is combined with a stimulus-responsive ELP-block. The encapsulation of a hydrophobic fluorescent dye was shown to exemplify the potential of the micelles to serve as nanocarriers for hydrophobic drugs, with the PEG corona providing stealth and steric protection of encapsulated materials.

Construction of a Multifunctional Enzyme Complex via the Strain-Promoted Azide-Alkyne Cycloaddition

Inspired by the multienzyme complexes occurring in nature, enzymes have been brought together in vitro as well. We report a co-localization strategy milder than nonspecific cross-linking, and free of any scaffold and affinity tags. Using non-natural amino acid incorporation, two heterobifunctional linkers, and the strain-promoted azide-alkyne cycloaddition as conjugation reaction, three metabolic enzymes are linked together in a controlled manner. Conjugate formation was demonstrated by size-exclusion chromatography and gel electrophoresis. The multienzyme complexes were further characterized by native mass spectrometry. It was shown that the complexes catalyzed the three-step biosynthesis of piceid in vitro with comparable kinetic behavior to the uncoupled enzymes. The approach is envisioned to have high potential for various biotechnological applications, in which multiple biocatalysts collaborate at low concentrations, in which diffusion may be limited and/or side-reactions are prone to occur.

Nanobody-functionalized polymersomes for tumor-vessel targeting.

Targeted carrier systems (e.g., liposomes or nanoparticles) are used to specifically deliver drugs to a site of interest. Site-direction can be achieved by attachment of targeting molecules, such as peptides, DNA/RNA, or antibodies, to the surface of the carrier. Here, the formation of polymersomes with tumor-targeting potential is described. A single-domain antibody (A12) that specifically targets PlexinD1 (a transmembrane protein overexpressed in tumor vasculature) is equipped with an azide-functionality using expressed protein ligation. This azide-containing A12 can subsequently be attached to BCN-functionalized polymersomes using a strain-promoted azide alkyne cycloaddition, thereby forming polymersomes with tumor-targeting potential.

Copper-free click chemistry on polymersomes: pre- vs. post-self-assembly functionalisation

The optimal accessibility of functional groups on polymeric nanosized vesicles was investigated with copper-free clickable probes as a model system. Cu-free clickable polymersomes were developed either through co-assembly of end group modified amphiphilic block copolymers or by introduction of the reactive moieties on preformed vesicles. For the co-assembly approach, the highest degree of availability was obtained for the most hydrophilic functional group, whereas hydrophobic species were unable to react as efficiently since they were seemingly buried in the membrane. Post-self-assembly introduction led to good results for all three examined moieties whereby surface saturation was reached above a certain percentage of immobilised probes. Finally, we demonstrated that protrusion of functional entities from the membrane corona via a longer hydrophilic segment of the block-copolymer significantly enhances the accessibility.

Detection of transglutaminase activity using click chemistry

Transglutaminase 2 (TG2) is a Ca2+-dependent enzyme able to catalyze the formation of ε(γ-glutamyl)-lysine crosslinks between polypeptides, resulting in high molecular mass multimers. We have developed a bioorthogonal chemical method for the labeling of TG2 glutamine-donor proteins. As amine-donor substrates we used a set of azide- and alkyne-containing primary alkylamines that allow, after being crosslinked to glutamine-donor proteins, specific labeling of these proteins via the azide-alkyne cycloaddition. We demonstrate that these azide- and alkyne-functionalized TG2 substrates are cell permeable and suitable for specific labeling of TG2 glutamine-donor substrates in HeLa and Movas cells. Both the Cu(I)-catalyzed and strain promoted azide-alkyne cycloaddition proved applicable for subsequent derivatization of the TG2 substrate proteins with the desired probe. This new method for labeling TG2 substrate proteins introduces flexibility in the detection and/or purification of crosslinked proteins, allowing differential labeling of cellular proteins.