A new macrocyclic dansyl-cyclen fluorescent probe for the identification of mildly acidic intracellular compartments

A new dansyl-based fluorescent probe containing the dimethylcyclen macrocycle scaffold (1,7-dimethyl-4,10-bis(dansyl)-1,4,7,10-tetraazacyclododecane, AJ2DAN) has been synthesized and fully characterized; it was designed based on a previous similar molecule (AJ2NBD), featuring two NBD (4-amino-7-nitrobenzo[1,2,5]oxadiazole) fluorophores in place of dansyl groups. Absorption and fluorescence emission spectra of AJ2DAN were recorded in both aqueous solution and organic solvents, always showing large Stokes shifts. In acidic solution the compound is very weakly fluorescent, while the emission increased by moving towards neutral pH. Moreover, a very high emission was detected in 1,4-dioxane, a solvent able to mimic the cell membrane polarity. The chemical response is reflected by the intracellular behaviour: by coupling AJ2DAN and LTDR labelling, it is possible to observe the lack of colocalization of “green” AJ2DAN positive vesicles with LTDR positive lysosomes, attesting a specificity for vesicular structures with mild acidic environment, such as autophagosomes and amphisomes. The autophagy inhibitor Chloroquine (CQ) has been used to prevent the fusion of autophagosomes with lysosomes and their consequent lysosomal degradation, thereby increasing the accumulation of autophagosomes: this drug enabled to set-up the protocol used in the present study. The new AJ2DAN molecule shows a high and pH-dependent quantum yield thus it lies in the field of fluorescent probes. Here, it has been demonstrated its possible use in the biological environment not only for cell imaging but also as pH-probe inside the biological compartments. • A new macrocyclic dansyl-cyclen (AJ2DAN) has been designed and synthesized. • AJ2DAN proven as a fluorescent pH probe in aqueous solution with large Stokes shift. • AJ2DAN shows specificity for vesicular structures with mild acidic environment. • AJ2DAN can detect processes involving autophagic and vesicular trafficking. • Chloroquine was used to set-up the analysis protocol.