Strain-Promoted Alkyne-Nitrone Cycloaddition (SPANC)

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Bioorthogonal reactions refer to a class of chemical reactions that can be carried out in biological systems without interfering with natural biochemical processes. The emergence of such reactions has brought revolutionary technology to scientists' in situ research on life processes, has become one of the core directions of the emerging cross-field of chemical biology.

Chemical Reaction Characteristics

Strain-promoted alkyne-nitrone cycloaddition (SPANC) reactions are an important class of bioorthogonal reactions, characterized by fast reaction rates, low reagent concentrations, and stereoelectronic tunability. The nitrones in the reaction can be used to build molecular backbones, such as natural products and biologically active compounds, stable nitro radicals, and some important compounds with special uses, such as spin traps. Among the couplers that react with negative 1,3-dipolar cycloadditions, alkynes are the most used, because the isoxazoles produced by the reaction of alkynes and nitrones are easily rearranged and easily converted into new heterocyclic systems. The SPANC reaction offers high reactivity, (bimolecular rate constant k2 up to 60 M-1s-1) which is usually significantly affected by the determined structure of the strained alkyne. As long as the starting materials are stable, the SPANC reaction can be applied for bioorthogonal labeling studies. Compared with acyclic nitrones, intracyclic nitrone has better stability under acidic and alkaline conditions and is an ideal choice for cell labeling research. The SPANC reaction exhibits a high degree of reaction flexibility and compatibility with biomolecules in different enviroment due to the sensitivity of nitrones to stereoelectronic tuning and the diversity of bioorthogonal cyclooctynes.

Applications of Bioorthogonal SPANC Reactions

Applications of SPANC range from protein and cell surface labeling to materials science.

The most featured applications involve specific N-terminal nitrone functionalization and labeling of peptides or proteins with serine as the first residue. Labeling of N-terminal peptides or proteins can be accomplished using the SPANC reaction. This labeling method ensures single-site modification without disrupting protein structure or function. For example, complexes formed using fluorescent and superparamagnetic cyclooctyne-functionalized nanoparticles bound to correctly folded N-terminal nitrone-containing anti-HER2 antibodies via the SPANC reaction play a key role in targeting HER2-positive breast cancer cells, suggesting that SPANC has potential application in the targeted therapy of HER2-positive breast cancer.

Classical approaches to achieve protein modification, most commonly targeting cysteine thiol or lysine primary amine groups, may result in protein dimerization, poor solubility, or loss of protein function, depending on the position of the modified amino acid. There are several methods for site-selective metabolic integration of azide-functionalized amino acids into proteins, however, they require genetic engineering manipulations and use a type of reporter tag. By contrast, N-terminal SPANC protein tagging is a strategy for direct site-specific binding of functional tags to proteins of interest. This process requires only the conversion of nitrones containing the N-terminal serine and the subsequent SPANC reaction.

The SPANC reaction can also be applied to label on cell membranes. For example, cyclic nitrone-modified epidermal growth factor (EGF) was able to bind human breast cancer cells (MDA-MB-468) with EGF receptors and used in subsequent in situ SPANC reactions. MDA-MB-468 cells were treated with EGF-nitrone, reacted with DIBO-biotin, and then stained with streptavidin-FITC to detect the labeling on the cell membrane.

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