Bistetrazine‐Cyanines as Double‐Clicking Fluorogenic Two‐Point Binder or Crosslinker Probes

Abstract

Fluorogenic probes can be used to minimize the background fluorescence of unreacted and nonspecifically adsorbed reagents. The preceding years have brought substantial developments in the design and synthesis of bioorthogonally applicable fluorogenic systems mainly based on the quenching effects of azide and tetrazine moieties. The modulation power exerted by these bioorthogonal motifs typically becomes less efficient on more conjugated systems; that is, on probes with redshifted emission wavelength. To reach efficient quenching, that is, fluorogenicity, even in the red range of the spectrum, we present the synthesis, fluorogenic, and conjugation characterization of bistetrazine-cyanine probes with emission maxima between 600 and 620 nm. The probes can bind to genetically altered proteins harboring an 11-amino acid peptide tag with two appending cyclooctyne motifs. Moreover, we also demonstrate the use of these bistetrazines as fluorogenic, covalent cross-linkers between monocyclooctynylated proteins.

Fluorogenic probes for super-resolution microscopy

“…We attempted to address this problem by introducing more than one quencher to cyanine cores and synthesized bisazides and bistetrazines. [91][92][93] It was expected that multiple quenching i.e. more quenchers together with the formation of a constrained cyclic product with biscyclooctynylated targets enhances fluorogenicity.…”

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

“…Analytical reverse-phase highperformance liquid chromatography with ultra-violet/visible and mass spectrometry detection (RP- To optimize probes for biological applications, it would be highly desirable to create fluorogenic dyes that are either excitable towards the red range or possess large Stokes-shift, while keeping the excellent fluorogenicity of UV/blue excitable systems. Our continuing efforts to address this problem resulted in the development of multiple fluorogenicity, as demonstrated on orange/far red excitable advanced probes [24,25]. Though these double fluorogenic probes solved the above-mentioned problems, their applicability is limited either to special sequences (i.e., double genetic incorporation of two non-canonical-cyclooctynylated-amino acids) [24] or microenvironments (i.e., the polar environment in the proximity of ligation) [25].…”

“…COS-7 cells were transferred into ibidi µ-slide 8-well glass-bottom plates (15,000 cell/well) and incubated for 20-24 h at 37 • C in a 5% CO 2 atmosphere. Cells were transfected with 0.25 µg Vimentin N116TAG -PSmOrange and tRNA Pyl /NES-PylRS AF plasmids [24,30] (kind gift of Edward Lemke's lab) with Jetprime transfection agent according to its protocol. Cells were incubated in 250 µM BCN-lysine (Sichem, Bremen, Germany, SC-8014) containing complete medium during the transfection and the following 24 h period.…”

“…Our continuing efforts to address this problem resulted in the development of multiple fluorogenicity, as demonstrated on orange/far red excitable advanced probes [24,25]. Though these double fluorogenic probes solved the above-mentioned problems, their applicability is limited either to special sequences (i.e., double genetic incorporation of two non-canonical-cyclooctynylated-amino acids) [24] or microenvironments (i.e., the polar environment in the proximity of ligation) [25]. Thus, new approaches that could result in generally applicable, advanced probes are still needed.…”

Water-soluble, stable and azide-reactive strained dialkynes for biocompatible double strain-promoted click chemistry

“…5,6 The use of a double-strained dialkyne reagent enables efficient conjugation of two azide containing molecules in a metal-free environment. The Sondheimer dialkyne has been used for a range of applications, including the macrocyclisation of bis-azide functionalised peptides, [7][8][9][10][11][12] chemical modification of azidoproteins in cells, 5 post-synthetic modification of metal-organic framework thin films, 13 tetramerisation of HIV-related peptides, 14 and for the preparation of fluorescent dyes. 15 Notably, these dialkynes are fundamentally limited in their biological application due to inherently poor water-solubility and aqueous stability (τ 1/2 ∼ 10 min at pH = 7.4).…”