A Double‐Clicking Bis‐Azide Fluorogenic Dye for Bioorthogonal Self‐Labeling Peptide Tags

Abstract

Herein, we give the very first example for the development of a fluorogenic molecular probe that combines the two-point binding specificity of biarsenical-based dyes with the robustness of bioorthogonal click-chemistry. This proof-of-principle study reports on the synthesis and fluorogenic characterization of a new, double-quenched, bis-azide fluorogenic probe suitable for bioorthogonal two-point tagging of small peptide tags by double strain-promoted azide-alkyne cycloaddition. The presented probe exhibits remarkable increase in fluorescence intensity when reacted with bis-cyclooctynylated peptide sequences, which could also serve as possible self-labeling small peptide tag motifs.

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.…”

Modern Synthetic Avenues for the Preparation of Functional Fluorophores

“…This work has later been extended to other functional groups, such as the phenylsydnone 1,3‐dipole and bicyclononyne pair, for strain‐promoted reactions under physiological conditions 69. Kele and co‐workers recently published strain‐promoted azide–alkyne cycloadditions in peptide sequences 70. The authors synthesized a quenched bis(azide) fluorogenic probe for two‐point binding tagging of bis‐cyclooctynylated short hexapeptides in the pursuit of self‐labeling small peptide tag motifs.…”

Emerging Applications of Fluorogenic and Non‐fluorogenic Bifunctional Linkers

“…Doubly caged bis‐azide cyanine linkers (DCF1–DCF5) and coumarin linker (DCF6) were utilized for one‐pot covalent dimerization of GFP‐BCN and intramolecular cyclization of dicyclooctyne‐modified peptides (BCN‐POI‐BCN). These DCF linkers exhibited remarkable emission enhancement upon consumption of both azide moieties, which enabled optical tracking of the conjugation process …”

“…These DCF linkers exhibited remarkable emission enhancement upon consumption of both azide moieties, which enabledo ptical tracking of the conjugation process. [72,73] whereas the single addition product remained weakly fluorescent (Figure 19 a). [58a] This strategy was successfully utilized to design the SCF2l inker [58a] for the fabrication of polypeptidepolymer conjugates (Figure 8b).…”