A General Strategy to Design Highly Fluorogenic Far‐Red and Near‐Infrared Tetrazine Bioorthogonal Probes

Abstract

Highly fluorogenic tetrazine bioorthogonal probes emitting at near‐infrared wavelengths are in strong demand for biomedical imaging applications. Herein, we have developed a strategy for forming a palette of novel Huaxi‐Fluor probes in situ, whose fluorescence increases hundreds of times upon forming the bioorthogonal reaction product, pyridazine. The resulting probes show large Stokes shifts and high quantum yields. Manipulating the conjugate length and pull–push strength in the fluorophore skeleton allows the emission wavelength to be fine‐tuned from 556 to 728 nm. The highly photo‐stable and biocompatible probes are suitable for visualizing organelles in live cells without a washing step and for imaging of tumors in live small animals to depths of 500 μm by two‐photon excitation.

Metal-free bioorthogonal click chemistry in cancer theranostics

“…HFs were characterized with large Stokes shifts, high S/N ratio and low photo-bleaching. 139 Tumor cells pretreated with BCNmodified conjugates (such as BCN-cetuximab, BCN-morpholine and BCN-triphenylphosphonium) followed by HFs, were all efficiently lit up at their target sites without washing step, such as cell surface (BCN-cetuximab group), lysosomes (BCNmorpholine group) and mitochondria (BCN-triphenylphosphonium group), suggesting these HFs served as excellent NIR probes with high S/N ratio in living systems. The NIR fluorescence of tumor tissues in the HF probe group was significantly stronger than that in control groups.…”

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

“…By further reducing fluorophore‐tetrazine distance and employing the Dexter energy transfer mechanism, Wombacher and co‐workers successfully developed far‐red Si‐rhodamines with fluorescent turn‐on ratios up to 50 times [7] . We recently reported a fluorophore in situ formation strategy, generating a series of far‐red and NIR fluorogenic probes with a novel fluorescent skeleton (namely Huaxi–Fluors), by fusing tetrazine and the fluorophore scaffold into the same conjugation system [3d] . Yet, for more frequently‐used commercially available NIR fluorophores with λ em >700 nm (i.e., Cy7, SiR700), the turn‐on ratio of the resulting tetrazine probe rapidly drops to only a few times despite close tetrazine‐fluorophore distances (Figure 1a) [8] .…”

Dynamic Nucleophilic Aromatic Substitution of Tetrazines

“…[ 52 , 53 , 54 , 55 , 56 , 57 ] They also show an interesting redox performance,[ 58 , 59 , 60 ] and some of them display fluorescence. [ 61 , 62 , 63 , 64 ] Even when it is known that some tetrazines, such as 3,6‐dichloro‐1,2,4,5‐tetrazine ( Cl‐Tz‐Cl ) can undergo S N Ar,[ 65 , 66 ] the reversibility of the substitution is completely unexplored. In this regard, phenols or thiols may act both as good nucleophiles and as efficient leaving groups, and therefore it seems reasonable to focus on them as optimal candidates as substituents of tetrazine to achieve a reversible S N Ar.…”

“…Tetrazine is a valuable scaffold to build stimulating architectures such as corona[ n ]arenes [52–57] . They also show an interesting redox performance, [58–60] and some of them display fluorescence [61–64] . Even when it is known that some tetrazines, such as 3,6‐dichloro‐1,2,4,5‐tetrazine ( Cl‐Tz‐Cl ) can undergo S N Ar, [65, 66] the reversibility of the substitution is completely unexplored.…”