Comprehensive Characterization of an “Off/On” Rhodol‐Based Lysosomal Tracker for Orthogonal Cellular Analysis by Confocal Imaging

A microwave-assisted reaction was used for the preparation of cyanamido-amide lysotrackers based on fluorescein and Rhodol scaffolds. We were able to identify and fully characterize the compounds in their two forms spirolactam “off” and amide “on” form, the latter showing strong fluorescence with emission wavelength of ∼525 nm. Both molecules were used to create a map of MC3T3-E1 cells.

Abstract

Two florescent xanthene-cyanamide lysosomal trackers emitting strongly at ∼525 nm were prepared from fluorescein and rhodol methyl esters in microwave-assisted reactions. Both forms named “off” (nonfluorescent lactam) and “on” (strongly fluorescent ring-opened amide) have been comprehensively characterized out by using a combination of NMR spectroscopy, X-ray analysis, fluorimetry and confocal microscopy. Known rhodamines bearing electron-withdrawing groups (EWGs) exhibit an equilibrium between non-fluorescent (off) and fluorescent (on) depending on the dielectric constant of the medium. Here, cyanamide was introduced as EWG amine into the fluorescein and rhodol framework. Unlike rhodamine-type dyes, the ring-opened forms of fluorescein- and rhodol-cyanamides are stable in protic solvents under circumneutral and basic pH conditions. The osteoblastic cell line MC3T3-E1 from C57BL/6 mouse calvaria was used for confocal imaging where the different organelles and nuclei were distinguished by using an orthogonal combination of fluorescent dyes.

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