Fluorogenic Sydnones for Bioorthogonal Labeling of DNA

ABSTRACT

Five cyanine‐styryl dyes were synthetically conjugated to sydnones combining fluorogenicity with bioorthogonal reactivity. The synthesis was based on synthetic modules and building blocks using Heck couplings as central conjugation steps. The novel cyanine‐styryl dyes show significant Stokes shifts to well separate excitation and emission for fluorescent cell imaging. Reaction kinetics and fluorogenicity of the strain‐promoted sydnone‐alkyne cycloaddition (SPSAC) with 7‐deazadenosine, 7‐deaza‐2’‐deoxyadenosine and DNA modified by bicyclo[6.1.0]non‐4‐yne (BCN) were investigated by spectroscopic means. The determined second‐order rate constants k ₂ with nucleoside and 2‐deoxynucleoside were found in a range from k ₂ = 0.4±0.05 M ⁻¹ s ⁻¹ to 7.0±0.3 M ⁻¹ s ⁻¹ . The reaction is accompanied by fluorescence turn‐on up to 8. SPSAC with BCN‐modified DNA showed significantly enlarged fluorescence turn‐on of up to 17 and significant acceleration of reaction kinetics up to k ₂ = 2,300 M ⁻¹ s ⁻¹ due to the DNA template effect. The bioorthogonality of fluorogenic SPSAC was evidenced by fluorescence labeling of HeLa cells transfected by BCN‐modified DNA. The fluorescence of one SPSAC product showed a red‐shift of 123 nm which is a potentially useful for metabolic labeling of RNA and DNA because it helps to distinguish the labeling of the modified nucleosides from the labeling of modified DNA by the fluorescence color as readout inside cells.