A Novel Visible Range FRET Probe for Monitoring Acid Sphingomyelinase Activity in Living Cells

Abstract

Supporting information and the ORCID identification number(s) for the author(s) of this articlecan be found under: https://doi.Scheme1.Synthesis of probes 4 and 5.Reagents and conditions:a )H 4 N 2 ·H 2 O, MeOH,5 .5 h, r.t.,84%;b )5(6)-carboxyfluorescein, N-hydroxysuccinimide, DCC, CH 2 Cl 2 ,4h, r.t.,then NEt 3 ,DMF,2 0h,r .t.,6 6%;c )4m HCl dioxane/iso-PrOH,4h, 70 8C, then 1.3 equiv. BODIPY TR-X SE, NEt 3 ,pyridine/DMF,10h,r .t.,59% over 2s teps. d) 4 m HCl dioxane/iso-PrOH, 3.5 h, 70 8C, then 1.4 equiv.C 11 H 23 COOSu, NEt 3 ,iso-PrOH/THF,30h,r .t.,78% over2steps or 1.2 equiv.C 15 H 32 COCl, DIPEA, pyridine/CH 2 Cl 2 ,5 .5 h, 0 8C, 12 %o ver 2s teps.

Liposomal FRET Assay Identifies Potent Drug‐Like Inhibitors of the Ceramide Transport Protein (CERT)

“…We therefore aimed at developing as creening assayt hat should be as realistic as possible in order to be able to detect compounds with completely new binding modes. We reasoned that in anyc ase, at ransfer assay would be more predictive than ab inding assay.B eing inspiredb yo ur recent success in developing assayso fs phingolipid metabolizing enzymes based on Fçrster Resonance Energy Transfer (FRET), [18] we wanted to apply this principle to aC ERT-mediated lipid transfer assay.T he goal was to monitort he activity of CERT in ah omogeneous assay system in multi-well format and making use of the inherentn ormalization of fluorescenceb yd ual wavelength readout. [19] The idea wast hat perhaps ab i-directional transfer of dye-labelled ceramides coulda chieve the necessary transfer rate.…”

BRED: bioluminescence energy transfer to dye for monitoring ceramide trafficking in cell

“…Bioluminescence resonance energy transfer (BRET) [2][3][4][5] and fluorescence resonance energy transfer (FRET) [6][7][8] have been frequently used for this purpose. However, despite numerous examples, these techniques are limited by low brightness and different maturation speed of RET donors and acceptors [9], poor spectral overlap [10], and a low dynamic range of resonance energy transfer (RET) from a donor to an acceptor within a distance of 2−10 nm [11].…”

Application of combinatorial optimization strategies in synthetic biology

“…A ligand-binding peptide is sandwiched between a pair of donor and acceptor fluorophores, and ligand binding is observed via the FRET change. A set of FRET biosensors, based on three pairs of donor/acceptor, carboxyfluorescein (FAM)/Boron dipyrromethene (BODIPY) 85 , Nitrobenzoxadiazole (NBD)/nonsteroidal dye (Nile red) 86 , and coumarine/NBD 86 , was developed for real time monitoring of acid sphingomyelinase at high sensitivity and with high spatial resolution. The FRET biosensor is selectively cleaved by sphingomyelinase that leads to significant increase in fluorescence of the fluorescein FRET donor 85 .…”