A fluorogenic probe for the catalyst-free detection of azide-tagged molecules

Abstract

Fluorogenic reactions in which non- or weakly fluorescent reagents produce highly fluorescent products can be exploited to detect a broad range of compounds including biomolecules and materials. We describe a modified dibenzocyclooctyne that under catalyst-free conditions undergoes fast strain-promoted cycloadditions with azides to yield strongly fluorescent triazoles. The cycloaddition products are more than 1000-fold brighter compared to the starting cyclooctyne, exhibit large Stokes shift, and can be excited above 350 nm, which is required for many applications. Quantum mechanical calculations indicate that the fluorescence increase upon triazole formation is due to large differences in oscillator strengths of the S(0) ↔ S(1) transitions in the planar C(2v)-symmetric starting material compared to the symmetry-broken and nonplanar cycloaddition products. The new fluorogenic probe was successfully employed for labeling of proteins modified by an azide moiety.

Figure 1

a) SPAAC between Fl-DIBO probe (1) and various azides; b) Various triazole products generated from the corresponding azides with Fl-DIBO probe; Absorption (solid blue trace) and emission (solid green trace, λ_exc=420 nm) spectra of Fl-DIBO and absorption (dashed blue trace) and emission (dashed green trace, λ_exc=370 nm) spectra of triethyleneglycol triazole 8a recorded in MeOH. Inset images compare the visible fluorescence emission of the Fl-DIBO probe (left cuvette) to the triazole product (8a) (right cuvette) under UV excitation (365 nm).

Figure 2

Formation of azido-BSA and its labeling with Fl-DIBO probe. Purified azido-BSA (50 μM) and native BSA (50 μM) were labeled with 250 μM of Fl-DIBO (1) or DIBO-FITC for 18 h at 37 °C in PBS containing 10% EtOH and 1% SDS. The crude reaction mixtures were separated by SDS-PAGE and the gel was analyzed by fluorescence imaging (top row; λ_exc=365 nm; λ_detec=480 nm) and by Coomassie Blue stain to reveal total protein content (bottom row).

Figure 3

Excited state manifold for A) Fl-DIBO (1) and B) the triazole 8f illustrating the relative energies of the (π-π*) and (n-π*) singlet states in methanol (TD-DFT at the CAM-B3LYP/6-31+G(d)//B3LYP/6-31G(d) level of theory, including PCM solvent correction). The color plots illustrate the total electron density difference between the respective ground and excited states (decreasing density shown in blue, increasing density red; GS = ground state; S₁, S₂ = first and second excited singlet state).

Scheme 1

Synthesis of Fl-DIBO. Reagents and conditions: a) Pd(PPh₃)₄, CuI, (ⁱPr)₂NEt, THF, reflux, 18 h, 97%; b) H₂, Lindlar’s catalyst, quinoline, hexane, rt, 20 min, 89%; c) AlCl₃, tetrachlorocyclopropene, CH₂Cl₂, −20 °C to rt, 4 h, H₂O, 62%; d) Br₂, CH₂Cl₂, 0 °C, 2 h, 87%; e) KOH, EtOH, rt, 18 h, 76%.