Fluorescein conjugates of 9- and 10-hydroxystearic acids: synthetic strategies, photophysical characterization, and confocal microscopy applications.
Different strategies are presented to conjugate a fluorescein moiety to 9- and 10-hydroxystearic acids (HSAs). 5-Amino-fluorescein (5-AF) was used as a starting reagent. When reacted with acyl-chloride-modified HSAs, 5-AF gave rise to stable amide derivatives with a 75% reaction yield. These products exhibited the typical steady-state and time-resolved fluorescence properties of the fluorescein chromophore with absorption at 494 nm and emission at 519 nm. Flow cytometry studies confirmed the distinct proapoptotic effect of underivatized 9-HSA on Jurkat cells and revealed a comparable ability of its amide derivative. Confocal microscopy imaging studies showed that green fluorescence could stain intracellular membranous structures. Moreover, dual-dye labeling with Mito Tracker Red, followed by colocalization analysis, revealed that HSA can move to the mitochondria. Thus, fluorescent derivatives of HSA can be used to monitor the localization of these biologically active molecules in living cells and can provide a useful tool for linking biochemical investigation with optical visualization methods. In contrast, when unmodified HSAs were used, the reaction gave monoesterified and diesterified fluorescein derivatives. These products exhibited unusual steady-state and time-resolved fluorescence properties with the excitation wavelength at 342 nm and the emission wavelength at 432 nm. It is shown that the synthesized HSA amides of fluorescein provide all of the typical photophysical and instrumental advantages of this popular dye, whereas the unusual luminescence and excitation properties of the monoester and diester of the 5-aminofluorescein would make these dyes interesting to explore as potential candidates for two photon excitation applications.
Boga
Carla
C
Department of Organic Chemistry A. Mangini, University of Bologna, Viale Risorgimento 4, 40136 Bologna, Italy.
Puggioli
Silvia
S
Gherpelli
Marica
M
Farruggia
Giovanna
G
Pagnotta
Eleonora
E
Masotti
Lanfranco
L
Neyroz
Paolo
P
D000577:Amides / Q000138:chemical synthesis* / Q000633:toxicity; D017209:Apoptosis / Q000187:drug effects; D005434:Flow Cytometry; D005452:Fluoresceins / Q000737:chemistry*; D005456:Fluorescent Dyes; D006801:Humans; D007202:Indicators and Reagents; D019169:Jurkat Cells; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism; D013229:Stearic Acids / Q000138:chemical synthesis* / Q000737:chemistry*
D000577:Amides; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D013229:Stearic Acids; C030864:5-aminofluorescein; C070375:9-hydroxystearic acid; C070376:10-hydroxystearic acid