Developing Novel Bioorthogonal Chemistry Employing Nitrone Dipoles

Abstract

The introduction of the concept of Click chemistry by Barry Sharpless has inspired countless chemists to further develop traditional chemical transformations into powerful tools for a number of applications that require extremely fast, efficient, selective and robust reactions. Recently, these concepts have been extended to reactions amenable to complex biological systems allowing researchers to interrogate them with ever greater precision. The field of bioorthogonal chemistry has rapidly been developed to give access to a plethora of robust and powerful tools allowing for the labelling and tracking of biomolecules in living systems. The development of new bioorthogonal reactions displaying faster kinetics and the use of alternative reagents, allows for more complex biological systems to be interrogated, allows for applications requiring multiplex labelling where multiple biomolecules can be simultaneously functionalized through applying orthogonal bioorthogonal reactions, and hopefully allow for the minimal perturbation to biomolecules’ native functions. Towards these goals, we have focused efforts towards the development of reactions employing nitrone dipoles, which can act as alternative dipoles in a multitude of bioorthogonal cycloaddition reactions. In the context of this thesis, work involving the optimization of the copper-catalyzed alkyne-nitrone cycloaddition involving rearrangement (CuANCR), the strain-promoted nitrone-cyclooctadiyne cycloaddition, and other exploratory nitrone cycloaddition chemistry will be explored. The optimization of CuANCR, also known as the Kinugasa reaction, follows previous efforts by our research group towards the application of micelles to allow for faster and more efficient reactions under aqueous conditions. Building upon these initial studies, we were able to further optimize the reaction to apply activated alkynes, propiolamides and propiolic esters, to obtain better product yields under micellar conditions. The development of a fluorescence magnetic bead assay to allow for rapid development of reaction conditions is also presented, thus allowing for rapid screening of biomimetic lipids forming micelles and studying the efficiency of applying amino acids as copper ligands. The applicability of micelle-assisted CuANCR towards bioorthogonal applications was demonstrated by the functionalization of cell membrane-associated peptides. Initial development of dual labelling protocols involving CuANCR, followed by thiol addition to the formed β-lactam products is also presented. Following CuANCR development, the study of strain-promoted nitrone-alkyne cycloadditions (SPANC) between nitrones and cyclic diynes (molecules containing two alkyne moieties, cyclooctadiynes) is presented. These cyclooctadiyne scaffolds allow for the cycloaddition of two separate dipoles, with great rate accelerations observed for the second cycloaddition. Kinetic and computational investgations of this bioorthogonal transformation have allowed for a better understanding of the parameters which influence and accelerate the reaction. Applicability of these SPANC reactions was demonstrated through the synthesis of oligomer materials and through the labelling of bacterial L. innocua cells using a sequential protocol of metabolic incorporation of nitrone amino acids to cells surfaces, followed by treatment with cyclooctadiyne and finally labelling with a fluorophore azide dipole. Finally, further development of SPANC chemistry is presented with an analysis of reactions between phenanthridine nitrone scaffolds and strained cyclic alkynes. Kinetic evaluation of the reaction showed that these nitrones scaffold show fast reaction kinetics with second-order rate constants (k2) in the range of 101 M-1s-1, which while slower than previously examine acyclic nitrones and other small five-membered endocyclic nitrones, represent faster reactions than with analogous azide dipoles. Analysis of the fluorescence properties of the reaction and its fluorogenic potential uncovered a small FRET effect with strained cyclic alkyne fluorophores, which shows the potential for further development of fluorogenic transformations involving nitrone dipoles. Initial development of the reactions between nitrones and cyclopropenes and cyclopropenone-caged cyclooctadiyne are also presented, further demonstrating the wide applicability of this versatile class of bioorthogonal dipoles.