Sydnone-coumarins as clickable turn-on fluorescent sensors for molecular imaging
Résumé
Copper-catalyzed and copper-free sydnone-alkyne cycloaddition reactions have emerged as complementary click tools for chemical biology but their use in bioorthogonal labeling is still in its infancy. Herein, combinations of alkynes and coumarin-sydnones were screened for their ability to generate pyrazole products displaying strong fluoroscence enhancement compared to reactants. One sydnone was identified as a particularly suitable new turn-on probe for protein labeling. Clickable fluorogenic probes are important chemical tools to label, visualize and study biomolecules. 1 They are typically designed with a profluorophore bearing a clickable function that both suppresses the fluorescence and allows the ligation to the corresponding click partner. 2 The structural changes accompanying the click reaction lead to unquenching of the profluorophore and result in the enhancement of the fluorescence. Many of these probes are based on the Cu(I)-catalyzed azide-alkyne [3+2] cycloaddition reaction (CuAAC), which can be considered as the archetype of click chemistry , and its copper free version using strained cycloalkynes. 3 Over the last decade, a series of azides 4 and alkynes 5 profluorophores have been developed and successfully used for biological imaging. Inspired by this remarkable amount of work, we wondered if sydnones might be used to expand the toolbox of clickable fluoro-genic probes. Our group identified sydnones as an interesting dipole partner for the Cu-catalyzed cycloaddition reaction with terminal alkynes, leading to 1,4-pyrazoles. This reaction was called CuSAC for Cu-catalyzed Sydnone Alkyne Cycloaddition. 6 Following this work, the copper free version (SPSAC, Strained Promoted Sydnone Alkyne Cycloaddition) was developed by the group of J. W. Chin, 7 our group 8 and by the group of J. M. Murphy. 9 Later, we showed that sydnones can be tuned to provide highly reactive clickable reagents for both SPSAC and CuSAC reactions, displaying higher kinetic properties than reactions involving azides. 10 Very recently, sydnone-based luminophores were developed and used for the labeling of proteins and living cells using SPSAC. 11 An example of fluorogenic photoligation reaction using diarylsydnones was also recently disclosed and applied to protein labeling. 12 In this contribution, we designed a series of sydnone-coumarins and evaluated their ability to behave as effective fluorogenic clickable turn-on probes, both in CuSAC and SPSAC reactions. The enhancement of the fluorescence results from the suppression of internal quenching and the structural modifications triggered by the transformations (Scheme 1). Coumarins were chosen as profluorophore candidates because they are stable, small in size and routinely used for molecular imaging. Substitutions in position 3-and 7-of coumarin dyes are well known to have a strong impact on their fluorescence properties. We reasoned that the mesoionic ring could be installed on these positions and synthetized a series of coumarin derivatives , as depicted in Scheme 2. Though the synthesis of coumarins is well established, and the insertion of a variety of heterocycles has been reported, sydnone-coumarin scaffolds are still unexplored. 13 7-Sydnone-coumarins 1 and 2 were obtained from commercially available 7-amino-coumarins through a standard reductive amination step in presence of glyoxylic acid and sodium cyanoborohydride as a reducing agent. The glycine intermediates were nitrosylated in presence of tert-butyl nitrite, and subsequent cyclodehydration Scheme 1 Schematic principle of turn-on sydnones probes.