A Redox-Mediated Negative Feedback Loop Based on Tetrazine-Thiol Exchange

Abstract Feedback loops are critical components for rationalizing the out-of-equilibrium behavior ubiquitous in life. Despite the significant advances in systems chemistry, strategies to rationally expand the complexity of chemical feedback loops in a defined and modular way remain scarce. Here, we designed a negative feedback loop based on thiol-tetrazine exchange (TeTEx). Negative feedback is broadly defined as a set of regulatory steps that feed the output signal, inverted, back to the input. In our case, the inputs are thiols, which react with tetrazines exhibiting only limited redox activity, and the outputs are redox-activated tetrazines, i.e., TeTEx products. More specifically, we demonstrated that tetrazines could become redox-activated upon TeTEx, after which then become dihydrotetrazines via two distinct reduction pathways. The reduction pathways directly compete with the exchange process, as they deplete the source of thiols, thereby constituting a negative feedback mechanism at the molecular level. A microfluidic flow setup was developed to show that the chemical environment of the thiol-containing substrates─particularly when additional thiols or carboxylic acids are present as neighboring groups─could enhance the redox activity of tetrazines. The method to create a negative feedback loop, integrating thiol-mediated exchange and redox reactivity within the same system, will be applicable to many other dynamic covalent reactions, paving the way to advance our synthetic toolbox for creating out-of-equilibrium reaction networks with ever-increasing complexity.