Design of peptide substrates for nanosecond time-resolved fluorescence assays of proteases: 2,3-Diazabicyclo[2.2.2]oct-2-ene as a noninvasive fluorophore

Squeezing Fluorescent Dyes into Nanoscale Containers—The Supramolecular Approach to Radiative Decay Engineering

“…Owing to the availability of functionalized DBO derivatives (Scheme 1), new opportunities for substrate design arise, in particular due to the compatibility of the purely organic chromophore during solid-phase peptide and oligonucleotide synthesis. Because the time-gate is applied in the nanosecond rather than in the millisecond range, we have referred to this new technique as Nano-TRF [82]. As an additional asset, Nano-TRF facilitates the use of high-energy lasers or lasers with higher repetition rates.…”

“…They were employed, among others, for the sensitive detection of DNA and proteins [78], and for a variety of enzyme assays [79][80][81]. Recently, a novel type of TRF assay based on DBO as a truly fluorescent (rather than phosphorescent) label has been introduced, thereby by-passing the need for rare earth metal ions [82]. Owing to the availability of functionalized DBO derivatives (Scheme 1), new opportunities for substrate design arise, in particular due to the compatibility of the purely organic chromophore during solid-phase peptide and oligonucleotide synthesis.…”

“…In fact, the signal to background ratio in Nano-TRF 204 W.M. Nau et al measurements, facilitated by the use of cucurbiturils as fluorescence lifetimeenhancement agents, can be increased by several orders of magnitude [82].…”

“…It originates from scattered light, biological autofluorescence, sample container materials, as well as additives and potential inhibitors. In TRF measurements, the short-lived emission is not suppressed, but electronically gated out, which increases the signal to background ratio drastically compared to steady-state detection, owing to the exponential fluorescence decay characteristics [82].…”

Nanocrystals and Nanoparticles Versus Molecular Fluorescent Labels as Reporters for Bioanalysis and the Life Sciences: A Critical Comparison

“…440-500 nm) [66] and the only recently reported UVabsorber and vis-emitter DBO (2,3-diazabicyclo[2.2.2]oct-2-ene) with a lifetime of ca. 300 ns in aerated water [67]. Due to the forbidden nature of the electronic transitions involved, in addition to its short wavelength absorption and emission (absorption and emission maximum at ca.…”