Studies on Proximity-Induced Cycloaddition for Bioconjugation
Authors:   , Preux Yoan 
Published: October 2022

<p><b>Labelling a biomolecule in vivo or building a bioconjugate in vitro requires the use of very selective chemical reactions. Such reactions, classified as “bioorthogonal”, involve reaction partners and products which are inert to the functional groups of the biomolecules involved and to the reaction medium. The partners should react selectively with each other at low concentrations with good kinetics under mild conditions, without requiring high temperatures or unstable reagents. These challenging demands often lead to a trade-off and, as such, there is no single bioorthogonal reaction that meets the requirements of every application. Thus, there is a need to have a toolbox of bioorthogonal reactions at disposal and an incentive to develop new ones. </b></p>
<p>The aim of this thesis was to develop a bioorthogonal reaction relying on a concept called “proximity-induced conjugation”. This methodology relied on a two-step protocol where the first step involved a reversible reaction that brings a second reaction pair into proximity such that they could react in an irreversible, permanent manner. Three model reactions were explored in order to validate and develop this concept. </p>
<p>In Chapter 1, a brief overview of classic bioorthogonal reactions as well as some applications is provided. </p>
<p>Chapter 2 and Chapter 3 focus on the first model reaction studied, the reaction between α-azido ketones and amino alkynes. For this model, the reaction partners would react through an imine/enamine formation, facilitating an intramolecular azide-alkyne cycloaddition. Chapter 2 covers the synthesis of the α-azido ketones and amino alkynes to be tested, and Chapter 3 covers the conjugation tests between them. While the reaction could proceed as intended when using specific α-azido ketones, producing a triazolo-pyrazine product, the model reaction was deemed unsuitable as a bioorthogonal reaction due to its slow kinetics, instability of α-azido ketones and the failure of the reaction to proceed in solvents relevant for bioconjugation.</p>
<p>Nonetheless, triazolo-pyrazine compounds could be synthesized from this model reaction under mild and straightforward conditions. Chapter 4 covers the work conducted to optimize the reaction conditions and expand the range of triazolo-pyrazine derivatives accessible. </p>
<p>Chapter 5 covers the second model reaction studied, the reaction between an electron-deficient aryl azido substrate fitted with a secondary amino group and various enolizable carbonyl compounds. For this model, the two reacting partners would react through a fast enamine formation. The enamine functional group formed in proximity to the electron-deficient azide would react with it through an intramolecular cycloaddition, generating a 1,2,3-triazoline intermediate and, after an elimination step, a 1,2,3-triazole conjugate. Most of the test reactions were unsuccessful as the intermediates decomposed. However, one reaction conducted with an aliphatic aldehyde led to the synthesis of two products, but the yields remained low and as such, this model was also deemed non-viable as a bioorthogonal reaction.</p>
<p>Chapter 6 covers the third model reaction studied which was the reaction between an aryl azido aldehyde and several amino alkynes, also relying on an imine formation bringing into proximity the azide and alkyne functional groups for an enhanced intramolecular cycloaddition. The test reactions using primary amino alkynes were unsuccessful, the imine intermediates being of an E-configuration, unsuitable to bring into proximity the azide and alkyne functional groups. Test reactions with secondary amino alkynes produced aminal intermediates (containing two equivalents of the secondary amine), allowing a successful intramolecular cycloaddition. Upon treatment with TFA, the aminal collapsed to an iminium ion, which was intercepted by various nucleophiles to give 1,2,3-triazole-fused 1,4-benzodiazepines with decent yields. The reaction was not suitable as a bioorthogonal reaction due to the low kinetics of the aminal formation as well as the aryl azido aldehyde reacting preferentially with primary amines when present as competitors. Nonetheless, the reaction was a straightforward way to produce under mild conditions 1,2,3-triazole-fused 1,4-benzodiazepine derivatives, this class of compounds being considered a “privileged scaffold” due to some triazole-fused 1,4-benzodiazepines possessing strong biological activities. As such, the reaction scope was expanded to make several 1,2,3-triazole-fused 1,4-benzodiazepine derivatives.</p>
<p>Finally, a brief summary of the work conducted in this thesis is given in Chapter 7, as well as potential future studies.</p>