Kinetic Investigation of Nitrone Cycloaddition Reactions for Bioorthogonal Labelling

Abstract

Bioorthogonal chemistry involves the development of chemical reactions that can be used to monitor, quantify and manipulate the roles of biological molecules in their native environments. Although there is a continuous demand to develop reactions with fast kinetics, developing reactions that make use of small “mini tag” functional groups are necessary to avoid perturbing function and to cross cellular membranes. Nitrones are small functional groups that have been shown to be excellent alternative 1,3-dipoles to azides in strain-promoted alkyne-nitrone cycloaddition reactions (SPANC). To further expand on the use of nitrones as reacting partners with other dipolarophiles, the kinetics of a variety of acyclic and cyclic nitrones were evaluated with strained- trans-cyclooctene. The reactions of acyclic nitrones were conducted under pseudo first-order conditions using UV-visible spectroscopy. By changing the α-aryl- substituent on acyclic nitrones, a linear free energy relationship shows increased reactivity with electron deficient nitrones. Second order rate constants of cyclic nitrones were found to be similar to those of acyclic nitrones, achieving 95% conversion in less than 20 minutes. Using unnatural D-amino acids tagged with nitrones, and s-TCO-Alexa488, labelling of the bacterial peptidoglycan layer was demonstrated. These new findings expand the bioorthogonal toolbox and allow for TCO reagents to be used in bioorthogonal applications beyond tetrazine ligations.

The optimization of click reactions for biological settings has substantially increased the versatility of these reactions for applications beyond biological labelling such as polymer synthesis. A strained cyclootadiyne, namely sym-dibenzo- 1,5-cyclooctadiene-3,7-diyne (diyne) is a benzylated- strained di-alkyne primed for strained- double-click chemistry for conjugation of molecules containing 1,3-dipoles. The second order rate constants for the consecutive additions of a variety of nitrones onto diyne were established via pseudo first-order kinetics by UV-visible spectroscopy. The kinetics of the double addition of acyclic nitrones onto this diyne show that the second addition occurs with second order rate constants more than 2-fold greater, and rates of the second addition can be increased by using electron deficient nitrones. Mechanistic investigation failed to observe the mono-intermediate, and experiments are outlined for future investigation of this phenomenon. Using the kinetic analysis results, di-nitrone monomers containing cyclic and diaryl-nitrones were designed for use in polymer applications. Based on the reports herein, we anticipate the application of this new double-SPANC reaction for peptide cross-linking, polymer synthesis and conjugation of biological molecules.