Azadioxatriangulenium: a long fluorescence lifetime fluorophore for large biomolecule binding assay

Abstract

Of the many optical bioassays available, sensing by fluorescence anisotropy have great advantages as it provides a sensitive, instrumentally simple, ratiometric method of detection. However, it is hampered by a severe limitation as the emission lifetime of the label needs to be comparable to the correlation lifetime (tumbling time) of the biomolecule which is labelled. For proteins of moderate size this is in the order of 20–200 ns, which due to practical issues currently limits the choice of labels to the dansyl-type dyes and certain aromatics dyes. These have the significant drawback of UV/blue absorption and emission as well as an often significant solvent sensitivity. Here, we report the synthesis and characterization of a new fluorescent label for high molecular weight biomolecules assay based on the azadioxatriangulenium motif. The NHS ester of the long fluorescence lifetime, red emitting fluorophore: azadioxatriangulenium (ADOTA-NHS) was conjugated to anti-rabbit Immunoglobulin G (antiIgG). The long fluorescence lifetime was exploited to determine the correlation time of the high molecular weight antibody and its complex with rabbit Immuniglobulin G (IgG) with steady-state fluorescence anisotropy and time-resolved methods: solution phase immuno-assay was performed following either steady-state or time-resolved fluorescence anisotropy. By performing a variable temperature experiment it was determined that the binding of the ligand resulted in an increase in correlation time by more than 75 %, and a change in the steady-state anisotropy increase of 18%. The results show that the triangulenium class of dyes can be used in anisotropy assay for detecting binding events involving biomolecules of far larger size than what is possible with the other red emitting organic dyes.

Azadioxatriangulenium: Synthesis and Photophysical Properties of Reactive Dyes for Bioconjugation

“…While these compounds were previously reported in account reporting of bio-relevant experiments, [10a, 12] , the synthesis was repeated and optimized as part of this work. The first reaction is between 5 ·BF 4 and the methyl ester of 4-aminobutanoic acid.…”

“…[11] The electronic transitions in the azaoxatriangulenium dyes are highly polarized, [8] which in combination with the long fluorescence lifetime make the dyes ideally suited for fluorescence polarization (FP) or fluorescence anisotropy (FA) assays, or simply ideally suited to monitor rotational motion of large biomolecules. [12] In order to fully explore these applications of ADOTA dyes, reactive derivatives for bioconjugation have to be readily available. We have previously reported an amino-reactive NHS ester of ADOTA, with the reactive NHS group linked via a flexible propyl linker, 1-NHS (Scheme 1).…”

“…We have previously reported an amino-reactive NHS ester of ADOTA, with the reactive NHS group linked via a flexible propyl linker, 1-NHS (Scheme 1). [10a, 12] Here, we report the synthesis of three new reactive derivatives, two amino-reactive NHS esters with semi-rigid and rigid linkers and one derivative with a rigid linked thiol-reactive maleimide group. In particular for FP and FA assays rigidity of the linker may play a critical role, since flexible linkers may cause loss of anisotropy due to fast local motions.…”

Sandwich type plasmonic platform for MEF using silver fractals

“…The water used for all solutions and washings was Millipore grade with > 18.2 MΩ resistivity. Me-ADOTA.Cl was prepared as previously described [15] while ADOTA-NHS (butyric acid derivative) was synthesised accordingly to established procedures[16]. …”

Fluorescence anisotropy (polarization): from drug screening to precision medicine

“…The search for efficient fluorophores with long (>20 ns) lifetimes and emitting in the red part of the spectrum has recently produced an interesting compound, ADOTA+, with a lifetime of 25 ns. 44 Furthermore, for really large targets (MW>1,000 kDa), fluorophores with long lifetimes, such as Ru or Re-based emitters with lifetimes in the range of few hundred ns to microseconds, have been identified, thus extending the theoretically accessible molecular weight range. 45 …”