A Bifunctional Fluorogenic Rhodamine Probe for Proximity‐Induced Bioorthogonal Chemistry

Abstract

Bioorthogonal reactions have emerged as a versatile tool in life sciences. The inverse electron demand Diels-Alder reaction (DA ) stands out due to the availability of reactants with very fast kinetics. However, highly reactive dienophiles suffer the disadvantage of being less stable and prone to side reactions. Herein, we evaluate the extent of acceleration of rather unreactive but highly stable dienophiles by DNA-templated proximity. To this end, we developed a modular synthetic route for a novel bifunctional fluorogenic tetrazine rhodamine probe that we used to determine the reaction kinetics of various dienophiles in a fluorescence assay. Under proximity-driven conditions the reaction was found to be several orders of magnitude faster, and we observed almost no background reaction when proximity was not induced. This fundamental study identifies a minimally sized fluorogenic tetrazine dienophile reactant pair that has potential to be generally used for the visualization of biomolecular interactions with temporal and spatial resolution in living systems.

Bioorthogonal red and far-red fluorogenic probes for wash-free live-cell and super-resolution microscopy

“…While we used BCN as dienophile for in vitro characterization due to its formation of a single product, we tested both, BCN and trans-cyclooctene (TCO) as dienophiles in live cells. TCO derivatives have been reported to possess superior kinetics over BCN [37][38][39] and it is further known that the choice of the DAinv-dienophile can influence the fluorescence turn-on of tetrazine dyes 26,40,41 .…”

Quantum Dot-PNA Conjugates for Target-Catalyzed RNA Detection

“…The chemistries that have been successfully used for template-directed ligations include native chemical ligation, [37] selenocystine-selenoester ligation, [38] phosphorothioate-based ligation, [39][40][41] electrophilic phosphorothioester-based ligation, [42] tetrazine-based ligation, [43][44] and others (Scheme 2.4). These techniques have been used for RNA imaging in bacterial cells [40] , human cells [41,44] , and cell lysates.…”

“…[38] However, product inhibition of turnover in template remains a major challenge, despite the successful attempts to alleviate it by the introduction of destabilizing linker at the ligation site. [37,[45][46] the thioester and iso-cysteine [37] ; b) selenocystine-selenoester ligation [38] ; c) phosporothioate-based ligation [39][40] ; d) electrophilic prosphorothioester-based ligation [42] ; e) tetrazine-based ligation reported by Seckute et al [44] and f) tetrazinebased ligation reported by Werther et al [43] Transfer-based OTR relocates a functional unit such as a quencher [15,[47][48] or a fluorophore [49] from one reactive probe to another (Scheme 2.5a and a`).…”

“…Examples of ligation-based oligonucleotide-templated reactions (OTRs). a) Native chemical ligation between the thioester and iso-cysteine[37] ; b) selenocystine-selenoester ligation[38] ; c) phosporothioate-based ligation[39][40] ; d) electrophilic prosphorothioester-based ligation[42] ; e) tetrazine-based ligation reported by Seckute et al[44] and f) tetrazinebased ligation reported by Werther et al[43]…”

Emerging Applications of Fluorogenic and Non‐fluorogenic Bifunctional Linkers

“…SCF8 was coupled to amine‐terminated ODN1 (NH 2 ‐ODN1) to afford nonfluorescent SCF8‐ODN1, which was then utilized in an ODN‐templated fluorogenic Heck reaction with vinyl‐functionalized ODN2 (ODN2‐vinyl; Figure ) . Other types of hetero‐bifunctional SCF linkers (SCF9‐SCF12) were also developed for ODN detection through template‐directed fluorogenic ODN ligation reaction (Figure ) –…”