Overcoming Spectral Dependence: A General Strategy for Developing Far‐Red and Near‐Infrared Ultra‐Fluorogenic Tetrazine Bioorthogonal Probes

Abstract

Bioorthogonal fluorogenic dyes are indispensable tools in wash-free bioimaging of specific biological targets. However, the fluorogenicity of existing tetrazine-based bioorthogonal probes deteriorates as the emission wavelength shifts towards the NIR window, greatly limiting their applications in live cells and tissues. Herein, we report a generalizable molecular design strategy to construct ultra-fluorogenic dyes via a simple substitution at the meso-positions of various far-red and NIR fluorophores. Our probes demonstrate significant fluorescence turn-on ratios (10 2 -10 3 -fold) in the range 586-806 nm. These results will greatly expand the applications of bioorthogonal chemistry in NIR bioimaging and biosensing.

Preparation of 3‐Bromo‐1,2,4,5‐tetrazine

“…Novel Far-Red and Near-Infrared Dyes have been enabled through the corresponding methylamine tetrazine. [22] The exceptional rate acceleration of mono-substituted tetrazine amino acids derived from the title compound 7 has been corroborated. [23] Such tetrazines could be utilized for drug release based on dynamic chemistry.…”

Supramolecular ‘catch-and-release’ strategy for bioorthogonal fluorogenic imaging across the visible spectrum

“…[28][29][30][31][32][33][34][35][36][37][38] Tz has been shown to quench fluorescence via various photophysical mechanisms, including Forster resonance energy transfer (FRET), 39,40 through-bond energy transfer (TBET), 26,27 Dexter-type electron exchange, 41 and photoinduced electron transfer (PET). 42 Over the last few years, a range of design strategies has been investigated in collaboration with different fluorophore scaffolds to harness various Tz-mediated quenching mechanisms and achieve fluorogenic tetrazine probes extending up to the far-red/NIR range. 26,27,30,[33][34][35][36][37][38][39][40][41][42] While such fluorogenic probes have been promising, the quenching strategies are not generalizable to diverse libraries of fluorophore families, requiring extensive design efforts to customize quenching mechanisms for each fluorophore scaffold.…”

“…42 Over the last few years, a range of design strategies has been investigated in collaboration with different fluorophore scaffolds to harness various Tz-mediated quenching mechanisms and achieve fluorogenic tetrazine probes extending up to the far-red/NIR range. 26,27,30,[33][34][35][36][37][38][39][40][41][42] While such fluorogenic probes have been promising, the quenching strategies are not generalizable to diverse libraries of fluorophore families, requiring extensive design efforts to customize quenching mechanisms for each fluorophore scaffold. In addition, access to these fluorogenic probes often requires direct alteration of the core fluorophore skeleton, demanding re-optimization of the laborious fluorophore synthesis scheme and tackling critical synthetic challenges.…”

Dynamic Bioorthogonal Imaging Using a Tetrazine Nir-Aza Fluorogenic Probe

“…[5][6][7][8] Despite the numerous advantages of tetrazine fluorogenic probes, there are still relatively few near-infrared (NIR) tetrazine fluorogenic probes available that can exhibit emission in the NIR spectral region following cycloaddition, especially over 700 nm, because it has minimal background noise, high penetration depth and low phototoxicity in the NIR region. [9][10][11] The class of BF2-chelated tetraarylazadipyrromethenes (NIR-AZA) is widely used in the 700-900 nm spectral region due to the relative ease of synthesis of the core fluorophore scaffold (Figure 1, dashed box), which allows for customization of their properties as needed. [12][13][14][15][16][17] In our group, we reported on the development of a fluorogenic NIR probe based on 1,2,4,5tetrazine Tz-NIR-AZA 1 (Figure 2).…”