Bioorthogonally Applicable Fluorogenic Cyanine-Tetrazines for No-Wash Super-Resolution Imaging

The synthesis, fluorogenic characterization, and labeling application of four tetrazine-quenched cyanine probes with emission maxima in the red–far red range is reported. Fluorescence of the cyanine-cores is quenched via <i>through-bond-energy-transfer</i> (TBET) exerted by a bioorthogonal tetrazine unit. Upon bioorthogonal labeling reaction with cyclooctyne tagged proteins, the quenching effect ceases, and thus the fluorescence reinstates, resulting in an increase in fluorescence intensity. As a rare example among indocyanines, one of our new probes was found suitable in STED-based super-resolution imaging. The applicability of this fluorogenic Tet-Cy3 probe was therefore further demonstrated in the bioorthogonal labeling of cytoskeletal protein, actin, with subsequent super-resolution microscopy (STED) imaging even under no-wash conditions

New Generation of Bioorthogonally Applicable Fluorogenic Dyes with Visible Excitations and Large Stokes Shifts

Synthesis of a set of new, azide bearing, biorthogonally applicable fluorogenic dyes with large Stokes shifts is presented herein. To assess the fluorogenic performance of these new dyes we have labeled a genetically modulated, cyclooctyne-bearing protein in lysate medium. Studies showed that the labels produce specific signal with minimal background fluorescence. We also provide theoretical insights into the design of such fluorogenic labels

Scope and limitations of typical copper-free bioorthogonal reactions with DNA: Reactive 2’-deoxyuridine triphosphates for postsynthetic labeling

Four triphosphates of 2′-deoxyuridine that carried the following bioorthogonally reactive groups were synthesized by organic–chemical methods. Two triphosphates with tetrazines and one with a cyclopropene moiety were designed for Diels–Alder reactions with inverse electron demand, and one triphosphate with a tetrazole core was designed for the “photoclick” cycloaddition. These triphosphates were not only successfully applied for oligonucleotide preparation by standard DNA polymerases, including Hemo KlenTaq, Vent, and Deep Vent, but also bypassed for full length primer extension products. Fluorescent labeling of the primer extension products was achieved by fluorophores with reactive counterparts and analyzed by polyacrylamide gel electrophoresis mobility shifts. The tetrazine–oligonucleotide conjugates were reacted with carboxymethylmonobenzocyclooctyne- and bicyclononyne-modified fluorophores. The yield of these postsynthetic reactions could significantly be improved by a more stable but still reactive nicotinic acid-derived tetrazine and by changing the key experimental conditions, mainly the pH of 7.2 and the temperature of 45–55 °C. The cyclopropene–oligonucleotide conjugate could be successfully labeled with a tetrazine-modified rhodamine in very good yields. The “photoclick” cycloaddition between tetrazole–oligonucleotide conjugates and a maleimide-modified dye worked quantitatively. The combination of primer extension, bypass, and bioorthogonal modification works also for double and triple labeling using the cyclopropene-modified 2′-deoxyuridine triphosphate

Bisazide Cyanine Dyes as Fluorogenic Probes for Bis-cyclooctynylated Peptide Tags and as Fluorogenic Crosslinkers of Cyclooctynylated Proteins

Herein we present the synthesis and fl uoro- genic characterization of a series of double-quenched bisazide cyanine probes with emission maxima between 565 and 580 nm that can participate in covalent, two-point binding bioorthogonal tagging schemes in combination with bis- cyclooctynylated peptides. Compared to other fl uorogenic cyanines, these double-quenched systems showed remarkable fl uorescence intensity increase upon formation of cyclic dye − peptide conjugates. Furthermore, we also demonstrated that these bisazides are useful fl uorogenic cross-linking platforms that are able to form a covalent linkage between mono- cyclooctynylated proteins