New Red‐Emitting Tetrazine‐Phenoxazine Fluorogenic Labels for Live‐Cell Intracellular Bioorthogonal Labeling Schemes

Abstract

The synthesis of a set of tetrazine-bearing fluorogenic dyes suitable for intracellular labeling of proteins in live cells is presented. The red excitability and emission properties ensure minimal autofluorescence, while through-bond energy-transfer-based fluorogenicity reduces nonspecific background fluorescence of unreacted dyes. The tetrazine motif efficiently quenches fluorescence of the phenoxazine core, which can be selectively turned on chemically upon bioorthogonal inverse-electron-demand Diels-Alder reaction with proteins modified genetically with strained trans-cyclooctenes.

Computationally guided discovery of a reactive, hydrophilic trans-5-oxocene dienophile for bioorthogonal labeling

“…Very recently, Lemke, Kele and coworkers reported the genetic incorporation of dioxo-TCO 2 and demonstrated that the lower lipophilicity of this molecule resulted in improved washout times during imaging experiments. In Diels–Alder reactions with tetrazines, the reaction rate with 2 is similar to that with the parent TCO 36,40…”

“…In Diels-Alder reactions with tetrazines, the reaction rate with 2 is similar to that with the parent TCO. 36,40 …”

Green- to far-red-emitting fluorogenic tetrazine probes – synthetic access and no-wash protein imaging inside living cells

“…Tetrazine-based fluorogenic probes 16–20 are particularly interesting as the tetrazine moiety within these molecules serves as reactive group and fluorescence quencher at the same time. Hence, conversion of the tetrazine in DA inv results in the loss of its quenching properties.…”

A Bioorthogonally Applicable, Fluorogenic, Large Stokes-Shift Probe for Intracellular Super-Resolution Imaging of Proteins

“…Such an approach is used routinely in the labeling schemes of various proteins, in cellulo or in vivo [6][7][8][10][11][12].In terms of reaction kinetics, biocompatibility, synthetic accessibility, and compatibility with GCE, strain promoted azide-alkyne cycloaddition (SPAAC) of azides and strained alkynes [13] and the inverse electron demand Diels-Alder (IEDDA) reaction of tetrazines and strained alkenes/alkynes [14] are the most robust bioorthogonal reactions. Besides, as we and others have recently demonstrated, functional groups of the SPAAC and IEDDA reactions, i.e., the azide and the tetrazine moieties, are able to modulate (i.e., quench) fluorescence by various mechanisms (e.g., energy transfer [15][16][17], rotation-induced relaxation [18,19], or photoinduced electron transfer [20]). Upon transformation of the quenching bioorthogonal unit in a specific ligation reaction, the fluorescence reinstates (Scheme 1).…”