New Dual Fluorescent Probe for Simultaneous Biothiol and Phosphate Bioimaging

Abstract

The simultaneous detection of relevant metabolites in living organisms by using one molecule introduces an approach to understanding the relationships between these metabolites in healthy and deregulated cells. Fluorescent probes of low toxicity are remarkable tools for this type of analysis of biological systems in vivo. As a proof of concept, different naturally occurring compounds, such as biothiols and phosphate anions, were the focus for this work. The 2,4-dinitrobenzenesulfinate (DNBS) derivative of 9-[1-(4-tert-butyl-2-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (Granada Green; GG) were designed and synthesized. This new sulfinyl xanthene derivative can act as a dual sensor for the aforementioned analytes simultaneously. The mechanism of action of this derivative implies thiolysis of the sulfinyl group of the weakly fluorescent DNBS-GG by biological thiols at near-neutral pH values, thus releasing the fluorescent GG moiety, which simultaneously responds to phosphate anions through its fluorescence-decay time. The new dual probe was tested in solution by using steady-state and time-resolved fluorescence and intracellularly by using fluorescence-lifetime imaging microscopy (FLIM) in human epithelioid cervix carcinoma (HeLa) cells.

Fluorescent probes for the simultaneous detection of multiple analytes in biology

“…39 Motivated by reports that intracellular phosphate and biothiol levels could enable study of altered bone metabolism in disease, 61 Resa et al reported a fluorescent probe, 23, which responds to biothiols followed by phosphate. 40 23 bears a thiol-cleavable group that releases the fluoresceinderivative Granada green, which has previously been shown to report on changes in phosphate levels through an excited-state proton transfer (ESPT)-induced decrease in fluorescence lifetime. 62 The probe does not show complete selectivity towards thiols, as the protecting group can be cleaved by other reactants including H2O2 and alanine.…”

Design, Synthesis and Photophysical Studies of Improved Xanthene Dye to Detect Acetate

“…Iso-PG was prepared based on a synthetic strategy previously described by our group [5,18]. Thus, organilithium derivative of 1-bromo-4-isopropyl-2-methoxybenzene [19] was added to previously described MOM-protected ketone, [5] affording after acidic hydrolysis iso-PG in good yield (See Scheme 1 and SI for more details).…”

Tetrazine as a general phototrigger to turn on fluorophores

“…Photoactivatable uorophores, also called turn-on uorophores, [1][2][3][4][5][6][7] have numerous advanced biological applications, including detection and release of ions [8][9][10][11] and metabolites, [12][13][14][15] monitoring of enzyme activity, [16][17][18][19] and multiple types of specialized microscopy. 7,[20][21][22][23][24][25] Most of these photoactivatable uorophores are masked by a reactive "cage" group intimately attached to the uorophore to alter its photophysical properties or to serve as an energy-transfer agent for use in a uorescence resonance energy transfer (FRET) pair.…”