Strain-promoted alkyne-nitrone cycloaddition (SPANC)

SPAAC can be carried out efficiently. On the one hand, the chemical potential energy of azide and alkyne substrates as reactants is very high. Diaryl-strained-cyclooctynes, such as dibenzylcyclooctyne (DIBO), have found application in reacting with 1,3-nitrones in strain-promoted alkyne-nitrone cycloadditions (SPANC). This process results in the formation of N-alkylated isoxazolines.[1]

Due to its metal-free nature and rapid kinetics (with a rate constant k2 as high as 60 1/Ms, surpassing both CuAAC and SPAAC), SPANC emerges as a viable option for live cell labeling. Furthermore, it exhibits tolerance towards substitutions on both the carbon and nitrogen atoms of the nitrone dipole, accommodating acyclic and endocyclic nitrones. This substantial flexibility allows for the versatile incorporation of nitrone handles or probes.[2]

Fig 1. The SPAAC vs SPANC reaction

Nevertheless, the isoxazoline product lacks the stability observed in the triazole product of CuAAC and SPAAC, making it susceptible to rearrangements under biological conditions. Despite this limitation, the reaction remains highly valuable due to its notably rapid reaction kinetics.[1]

This reaction finds applications in labeling proteins that feature serine as the initial residue. The process involves oxidizing serine to aldehyde using NaIO4, followed by conversion to a nitrone using p-methoxybenzenethiol, N-methylhydroxylamine, and p-ansidine. The resulting product is then incubated with cyclooctyne to yield a click product. Additionally, SPANC enables multiplex labeling.[3,4]

Application

SPANC finds diverse applications ranging from the labeling of proteins and cell surfaces to its utilization in materials science.

Prominent applications of SPANC include the specific functionalization of N-terminal nitrone and the labeling of peptides or proteins featuring serine as the initial residue. This versatile labeling technique enables the modification of N-terminal peptides or proteins, ensuring single-site customization without compromising the structural integrity or functionality of the proteins. An illustrative example involves the formation of complexes using fluorescent and superparamagnetic cyclooctyne-functionalized nanoparticles, which bind selectively to correctly folded N-terminal nitrone-containing anti-HER2 antibodies through the SPANC reaction. This strategic application plays a crucial role in targeting HER2-positive breast cancer cells, suggesting potential utility for SPANC in the targeted therapy of HER2-positive breast cancer.

In contrast to classical approaches targeting cysteine thiol or lysine primary amine groups for protein modification, which may lead to dimerization, poor solubility, or loss of function, SPANC offers a site-selective and direct strategy for binding functional tags to proteins. Unlike methods requiring genetic engineering manipulations and reporter tags for site-selective metabolic integration of azide-functionalized amino acids, N-terminal SPANC protein tagging involves the conversion of nitrones containing N-terminal serine and subsequent SPANC reaction, simplifying the process.

The versatility of SPANC extends to cell membrane labeling, as demonstrated by the application of cyclic nitrone-modified epidermal growth factor (EGF) binding to human breast cancer cells (MDA-MB-468) with EGF receptors. In situ SPANC reactions were employed, where MDA-MB-468 cells treated with EGF-nitrone were reacted with DIBO-biotin and stained with streptavidin-FITC to detect membrane labeling. This showcases the potential of SPANC for diverse applications, including bioimaging and cellular targeting.

Reference

1. MacKenzie, DA; Sherratt, AR; Chigrinova, M; Cheung, LL; Pezacki, JP (Aug 2014). "Strain-promoted cycloadditions involving nitrones and alkynes—rapid tunable reactions for bioorthogonal labeling". Curr Opin Chem Biol. 21: 81–8. https://doi.org/10.1016/j.cbpa.2014.05.023

2. (64) (a) Ning, X.; Temming, R. P.; Dommerholt, J.; Guo, J.; Ania, D.B.; Debets, M. F.; Wolfert, M. A.; Boons, G.-J.; van Delft, F. L" Angew. Chem. Int. Ed. 2010; 49, 3065. (b) McKay, C. S.; Moran, J.; Pezacki, J. P. Chem. Commun. (Cambridge, U. K.) 2010, 46, 931. (c) Debets, M. F.; van Berkel, S. S.; Dommerholt, J.; Dirks, A. T. J.; Rutjes, F. P. J. T.; van Delft, F. L. Acc. Chem. Res. 2011, 44, 805. (d) McKay, C. S.; Chigrinova, M.; Blake, J. A.; Pezacki, J. P. Org. Biomol. Chem. 2012, 10, 3066.

3. Lang, K.; Chin, J. (2014). "Bioorthogonal Reactions for Labeling Proteins". ACS Chem. Biol. 9 (1): 16–20. https://doi.org/10.1021/cb4009292

MacKenzie, DA; Pezacki, JP (2014). "Kinetics studies of rapid strain- promoted [3+2] cycloadditions of nitrones with bicyclo[6.1.0]nonyne". Can J Chem. 92 (4): 337–340. https://doi.org/10.1139/cjc-2013-0577

标签:

查看更多