New Noninvasive Methodology for Real-Time Monitoring of Lipid Flip

Abstract

A new methodology for the detection of lipid flip was developed. This methodology relies on the quenching of the fluorescence of the cascade-blue-labeled lipid through complex formation with a membrane-impermeable cyclen-tetranaphthalenethiourea synthetic receptor for this dye. The high affinity of the receptor to cascade-blue label allows the use of micromolar concentrations of this receptor during the experiment. At these low concentrations, the receptor does not interfere with the membrane integrity and, therefore, renders this new methodology less invasive to the model and cell membranes than commonly utilized 7-nitro-1,2,3-benzoxadiazol-4-yl (NBD)-dithionite methodology. Unlike with the NBD-dithionite assay, where the fluorescence quenching of the NBD group is achieved through its chemical modification, this new assay relies on the noncovalent interactions between cascade-blue label and the receptor. Therefore, the quenching can be reverted by either competitive displacement of the lipid-attached label with a water-soluble substrate or by enzymatic degradation of the receptor leading to the label release and fluorescence dequenching. We demonstrate that this new methodology is suitable for the study of lipid flip in both model spherical bilayer membranes and in-vitro experiments.

A Controlled Cyclization of Functionalized Thioureas and Unprecedented Termolecular Decyclization of Iminothiazolidinones

“…Thiourea function incorporated into complex molecules was shown to serve as the basic unit in recognition of anionic substrates . The latter compounds were used for the detection of membrane leakage and lipid flip in cellular assays, separation of isomeric saccharides and photosensing . Similar compounds facilitated development of a drug delivery system potentially useful for the treatment of type 1 diabetes …”

Chemoselective precipitation of lactose from a lactose/sucrose mixture: proof of concept for a new separation methodology

“…Recently, we have identified a family of artificial cyclen-thiourea receptors that form complexes with the pyrene-based dyes under near-physiological conditions (26,27). These complexes feature micromolar stability and diminished solubility in water and methanol compared to the intrinsic solubility of the dye and receptor alone (26).…”