Development of Bioorthogonal Reactions Using 3-Oxidopyridiniums and Expanding the Biological Applications of Cyclic Nitrones

Abstract

Bioorthogonal chemistry is a rapidly growing field that enables spatiotemporal monitoring of biomolecules using targeted probes. The development of bioorthogonal reactions therefore requires several criteria to be met. Reactions need to be selective, and fast enough so lower concentrations of reagents are used to mitigate toxicity, and they need to be stable in biological environments. 3-oxidopyridiniums are a class of water stable six-membered heteroaromatic latent dipoles, and have been previously reported to undergo [3+2] cycloadditions with electron deficient dipolarophiles, but have yet to be investigated for bioorthogonal use. To test their applicability as a bioorthogonal reagent, a series of N-methyl-3-oxidopyridiniums were synthesized with varying substituents on the 5 position and were reacted with DIBO (dibenzocyclooctyne). Electron donating 5-substituents have been shown to significantly increase the rate of the reaction, with bimolecular rate constants ranging from 3.31 x 10⁻⁴ with 5-trifluoromethyl-N-methyl-3-oxidopyridinium to 1.07 M⁻¹ s⁻¹ with 5-amino-N-methyl-3-oxidopyridinium, putting the faster reactions on par with commonly used bioorthogonal reactions for cell labelling. Strain-promoted alkyne-nitrone cycloadditions (SPANC) are a class of [3+2] cycloadditions that are commonly used for bioorthogonal reactions. In comparison to their predecessor SPAAC (strain promoted azide alkyne cycloadditions), SPANC offers much better reaction tunability. icSHAPE (in vivo click selective hydroxyl acylation analyzed by primer extension) is a labelling technique used to determine RNA structure. This is done by selectively targeting the 2' hydroxyl on the ribose sugar of RNA that is structurally available in regions of RNA that are single stranded, and the use of SPAAC allows for an improved signal to noise ratio. Herein, DMImO (1-[(p-methoxycarbonylphenyl)methyl]-2,2-dimethyl-5-oxo-3-imidazolin-3-ium-3-olate), a previously used nitrone in SPANC reactions, has been modified to include an electron deficient carbonyl imidazole to allow for a nucleophilic attack from the 2' hydroxyl of the RNA. Hydrolysis of the nitrone probe is on par with previous SHAPE reagents that are used for in vivo labelling and is able to label 5S rRNA for structure determination as effectively as previously used SHAPE reagents.