Temporal Control of Efficient <i>In Vivo</i> Bioconjugation Using a Genetically Encoded Tetrazine-Mediated Inverse-Electron-Demand Diels–Alder Reaction

Abstract

An inverse-electron-demand Diels-Alder (IEDDA) reaction using genetically encoded tetrazine variants enables rapid bioconjugation for diverse applications <i>in vitro</i> and <i>in cellulo</i>. However, <i>in vivo</i> bioconjugation using genetically encoded tetrazine variants is challenging, because the IEDDA coupling reaction competes with rapid elimination of reaction partners <i>in vivo</i>. Here, we tested the hypothesis that a genetically encoded phenylalanine analogue containing a hydrogen-substituted tetrazine (frTet) would increase the IEDDA reaction rate, thereby allowing for successful bioconjugation <i>in vivo</i>. We found that the <i>in vitro</i> IEDDA reaction rate of superfolder green fluorescent protein (sfGFP) containing frTet (sfGFP-frTet) was 12-fold greater than that of sfGFP containing methyl-substituted tetrazine (sfGFP-Tet_v2.0). Additionally, sfGFP variants encapsulated with chitosan-modified, pluronic-based nanocarriers were delivered into nude mice or tumor-bearing mice for <i>in vivo</i> imaging. The <i>in vivo</i>-delivered sfGFP-frTet exhibited almost complete fluorescence recovery upon addition of trans-cyclooctene via the IEDDA reaction within 2 h, whereas sfGFP-Tet_v2.0 did not show substantial fluorescence recovery. These results demonstrated that the genetically encoded frTet allows an almost complete IEDDA reaction <i>in vivo</i> upon addition of trans-cyclooctene, enabling temporal control of <i>in vivo</i> bioconjugation in a very high yield.