Development And Application Of A New Strain-promoted Bioorthogonal Reaction

Development of bioorthogonal reactions showing fast reaction rate,excellent stability,and spatiotemporal control has been an intense pursuit of chemical biology research.Herein we report an unprecedented pH-triggered reverse electronic demand Diels-Alder(IEDDA)cycloaddition between trans-cycloheptene(TCH)and tetrazine.Distinct from the classic strain-promoted IEDDA reactions that entail building strained alkenes as reactants,the current scenario adopts a “trigger-release-conjugate” reaction model,in which the TCH species are released in situ from a latent N-bicyclo [4.1.0] hept-7-yl-N-nitrosourea precursor upon treatment of an external pH stimulus,and then react with tetrazine derivatives.According to the synthesis of bicyclo [4.2.1]-1(8)-nonylene and bicyclo [3.3.1] non-1-ene,we successfully synthesized N-bicyclo [4.1.0] hept-7-yl-N-nitrosourea.N-bicyclo [4.1.0] hept-7-yl-N-nitrosourea as a new biorthogonal chemical tool is demonstrated by the relative stability and reactivity.The pH-triggered IEDDA reaction showed fast reaction rate and excellent biocompatibility,which has been successfully applied to in vitro protein labeling,live cell imaging and tissue imaging.It is indicated from in vitro labeling that the new strain-promoted bioorthogonal reaction has stronger fluorescence in a neutral environment,and the reaction is concentration-and time-dependent.More appealingly,its potential for in vivo application has also been demonstrated by the pretargeted tumor imaging in living mice.The biggest highlight in this paper lies in the introduction of the "bicyclic nitrosourea" switch structure,which releases trans-cyclohept-ene in the presence of pH as the only triggered condition.The stability of trans-cycloheptene greatly improved with high reactivity still,the new strain-promoted bioorthogonal reaction could evolve into a bioorthogonal reaction with higher stability,rapid reactivity and real-time controllability,which represents a valuable addition to the existing strain-promoted bioorthogonal chemistry.