A cell-permeable fluorescent prochelator responds to hydrogen peroxide and metal ions by decreasing fluorescence

Abstract

Described here is the development of two boronic ester-based fluorescent prochelators, FloB (2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-4(5)-[2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzylidene-hydrazinocarbonyl]-benzoic acid) and FloB-SI (2-(6-hydroxy-3-oxo-3Hxanthen-9-yl)-4(5)-[2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyloxy)-benzylidene-hydrazinocarbonyl]-benzoic acid) that show a fluorescence response to a variety of transition metal ions only after reaction with H2O2. Both prochelators’ boronic ester masks are oxidized by H2O2 to reveal a fluorescein-tagged metal chelator, FloS (4(5)-(2-hydroxy-benzylidenehydrazinocarbonyl)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid). Chelation of Fe3+ or Cu2+ elicits a 70% decrease in the emission signal of FloS, while Zn2+, Ni2+, and Co2+ produce a more modest fluorescence decrease. The conversion of FloB to FloS proceeds in organic solvents, but hydrolytic decomposition of its hydrazone backbone is observed in aqueous solution. However, FloB-SI oxidizes cleanly with H2O2 within 1 h in aqueous solutions to produce FloS. Fluorescence microscopy studies in HeLa cells with FloB-SI show that the sensor’s fluorescence intensity remains unchanged until incubation with exogenous H2O2, which results in a decreased fluorescent signal. Incubation with a competitive chelator restores the emission response, thus suggesting that FloB-SI can effectively report on a H2O2-induced increase in intracellular labilized metal.

Copper Toxicity Links to Pathogenesis of Alzheimer’s Disease and Therapeutics Approaches

“…Various analogs of boronic ester, such as boronic esters (BSIH, BSBH) and acids (BASIH), have been investigated to check their performance as metal chelators [ 297 ]. Currently, many promising molecular scaffolds using the same strategy are exploring their effect on the multifactorial AD [ 298 , 299 , 300 , 301 ]. Choi (2011) and Hindo (2009) [ 302 , 303 ] reported the derived compounds and analyzed their impact on metal-binding properties, β-amyloid deposition both in deficiency and the presence of metal ions.…”

Probing oxidative stress: Small molecule fluorescent sensors of metal ions, reactive oxygen species, and thiols

“…The final example in this class of multifunctional sensors is FloB-SI [200]. We recently reported that this fluorescein-appended prochelator is stable to hydrolysis in aqueous solution and cell culture media, and undergoes fast deprotection with H 2 O 2 through loss of its self-immolative pinacol boronic ester protecting group.…”

“…Fluorescence microscopy studies in HeLa cells with FloB-SI showed that the sensor’s fluorescence intensity remained unchanged until incubation with exogenous H 2 O 2 , which caused the cellular fluorescence to dim. Incubation with a competitive chelator, SIH, restored the emission response, suggesting that FloB-SI was effectively reporting on an H 2 O 2 -induced increase in intracellular labilized metal, the most likely candidate being iron [200]. While these results are encouraging, the fluorescencequenching response mechanism and the poor photostability of the compounds indicate that more work remains to be done to optimize this class of multifunctional probes and use them to study the role of metal-mediated oxidative stress in neurodegenerative disease.…”

Prochelator strategies for site-selective activation of metal chelators

“…The uptake of a fluorescent analogue of this prochelator by various hepatic cell lines unequivocally demonstrated the crucial role of the targeting unit to efficiently deliver the system to the hepatocytes. 66 Among the structures proposed for optimal ASGP-R recognition, there are tripods bearing on each arm a GalNAc moiety that is kept 20 Å away from the branching point of the tripod by an ethylene glycol spacer (53). Mintz and Delangle have exploited the tripodal architecture of a ligand to design a triantennary glycoside cluster system.…”