A small azide-modified thiazole-based reporter molecule for fluorescence and mass spectrometric detection

Abstract

Molecular probes are widely used tools in chemical biology that allow tracing of bioactive metabolites and selective labeling of proteins and other biomacromolecules. A common structural motif for such probes consists of a reporter that can be attached by copper(I)-catalyzed 1,2,3-triazole formation between terminal alkynes and azides to a reactive headgroup. Here we introduce the synthesis and application of the new thiazole-based, azide-tagged reporter 4-(3-azidopropoxy)-5-(4-bromophenyl)-2-(pyridin-2-yl)thiazole for fluorescence, UV and mass spectrometry (MS) detection. This small fluorescent reporter bears a bromine functionalization facilitating the automated data mining of electrospray ionization MS runs by monitoring for its characteristic isotope signature. We demonstrate the universal utility of the reporter for the detection of an alkyne-modified small molecule by LC-MS and for the visualization of a model protein by in-gel fluorescence. The novel probe advantageously compares with commercially available azide-modified fluorophores and a brominated one. The ease of synthesis, small size, stability, and the universal detection possibilities make it an ideal reporter for activity-based protein profiling and functional metabolic profiling.

Keywords: activity-based protein profiling (ABPP); bioorthogonal; click chemistry; mass defect; molecular probe.

Figure 1

Structure of the reporter molecule BPT (1).

Scheme 1

Synthesis of the azide-bearing 4-hydroxythiazole derivative 1.

Figure 2

Structure of the tested azide-modified standard fluorophores DNS (8) and NBD (9) and the bromine modified DNS system 6.

Figure 3

UV–vis spectra of 20 µM solutions of the azide modified fluorophores BPT (1), DNS (8), NBD (9) and BNS (6) in THF/water (20:80; v/v).

Figure 4

Normalized absorbance and fluorescence of BPT (1) in 20% THF/80% water (v/v), excitation at 374 nm.

Figure 5

Peak area of 100 pmol BPT (1), DNS (8), NBD (9) and BNS (6) measured with (A) C18-UPLC coupled to a photodiode array detector at their absorption maxima or (B) C18-UPLC–ESIMS in positive ionization mode.

Figure 6

Procedure of the model reaction between L-lysine and DDY (10) to form an imine (only one of two possible reactions shown) followed by CuAAC with the azide-modified fluorophores. The rectangle represents the respective reporter unit. For clarity only reactions with the terminal lysine are depicted.

Figure 7

Mass spectra of labeled L-lysine/DDY (10)/fluorophore conjugates 11 (containing BPT), 12 (containing DNS), 13 (containing NBD) and 14 (containing BNS) measured in ESI positive mode (S/N: signal to noise ratio after reaction of equal molar amounts of reagents).

Figure 8

Fluorescent labeling of catalase treated with DDY (10) followed by CuAAC with all four reporter molecules and in-gel fluorescence detection at 365 nm. Equivalent amounts of protein were labeled and loaded on the gel. (Protein ladder masses in kDa).