Readily Accessible Bicyclononynes for Bioorthogonal Labeling and Three-Dimensional Imaging of Living Cells**

The advent of chemical biology tools for imaging and tracking of biomolecules (proteins, lipids, glycans) in their native environment is providing unique insights into cellular processes that are not achievable with traditional biochemical or molecular biology tools. Bioorthogonal labeling of biomolecules has proven particularly useful for the detection and study of glycans and lipids, based on a highly selective reaction between an abiotic functional tag and a designed chemical probe.With respect to the abiotic tag, azide has been used extensively because of its straightforward chemical introduction, small size, and relative inertness. The finding that azides react rapidly and cleanly with terminal acetylenes in the presence of copper(I), the quintessential “click” reaction, has found tremendous application in life and material sciences. However, because up to 20 mol% of copper(I) species is typically used, such click chemistry is not suitable for labeling of living systems without compromising cell function. Apart from that, the presence of copper may induce oligonucleotide and polysaccharide degradation. To avoid the use of toxic metals, several metal-free bioorthogonal labeling reaction have been developed. In particular, phosphines have been used for covalent ligation to azides, a procedure known as Staudinger ligation. However, owing to the oxygen sensitivity of phosphines, recent focus of chemical ligation is shifting towards strain-promoted cycloaddition reactions with cyclooctynes (Scheme 1a). Most prominently, azides were find to react with cyclooctynes with high reaction rates in a so-called strain-promoted alkyne–azide cycloaddition (SPAAC). The toolbox of metal-free bioorthogonal reactions was most recently further expanded by our research group and others, by demonstrating that cyclooctynes undergo even more rapid strainpromoted cycloaddition with nitrones (SPANC), a procedure that was found suitable for dual, irreversible, and site-specific N-terminal modification of proteins. The broad application of metal-free cycloaddition in life sciences is, however, hampered by the limited commercial availability and lengthy synthetic routes for preparation of the most common cyclooctynes (Scheme 1b). For example, eight synthetic steps are required to generate second-generation DIFO (1), nine steps for DIBAC (3), and seven steps for BARAC (4), while yields are usually low (10% for 2, 16% for 4). Additional modifications, such as dibenzoannulation (compounds 2–4), increase lipophilicity and may, therefore lead to non-specific binding to proteins. Here we report bicyclo[6.1.0]nonyne (BCN) as a novel ring-strained alkyne for metal-free cycloaddition reactions with azides and nitrones. Bicyclononyne derivatives, which were obtained in a highly straightforward process through cyclopropanation of 1,5-cyclooctadiene, are Cs symmetrical and display excellent reaction kinetics in strain-promoted cycloaddition reactions. Functionalized derivatives of BCN were applied in the labeling of proteins and glycans, as well as in the three-dimensional visualization of living melanoma cells. Based on the known reactivity enhancement of cyclopropane fusion, we speculated that analogues of bicycloScheme 1. Reactions and structures of cyclooctyne compounds for strain-promoted cycloaddition. a) Cycloaddition with azide (SPAAC) or nitrone (SPANC). b) Structures of the most commonly employed cyclooctynes.