Structure–activity relationship study of ProxyPhos chemosensors for the detection of proximal phosphorylation and other phosphate species

Abstract

Chemosensors for the detection of phosphate-containing biological species are in high need. Detection of proximally phosphorylated sites of PP and those found in peptides and proteins has been demonstrated using chemosensors containing pyrene, as a fluorescent reporter, and a Zn-chelate, as a phosphate-binding group. Using these sensors, detection of proximal phosphate groups is afforded by binding of at least two of the sensor molecules to the adjacent phosphates, via the Zn centres, leading to excimer formation between the pyrene groups and the corresponding shift in emission from 376 to 476 nm. Although several reports of this chemosensor class have been made, no detailed studies of selectivity of these sensors among major phosphate targets have been reported. In this study, a library of this class of chemosensors, termed ProxyPhos, which contained various linkers and Zn-chelating groups (i.e. DPA, cyclen and cyclam), was prepared and the effects of structural variation on the sensing efficiency and selectivity were evaluated among proximally phosphorylated peptides, proteins, nucleotides, P and PP. As a result of this study, we have identified ProxyPhos library members that are most suitable for the detection of proximally phosphorylated peptides, PP, UTP, and a DpYD peptide motif, and have generally provided a foundation for the selection of ProxyPhos chemosensors for further development of specific biologically relevant assays. The broad utility of ProxyPhos is further supported by the demonstrated lack of these sensors' cytotoxicity, ability to rapidly permeate into live and fixed cells and compatibility with gel staining methods.

Sensitive Detection of Broad-Spectrum Bacteria with Small-Molecule Fluorescent Excimer Chemosensors

“…ProxyPhos sensors consist of three components: a pyrene fluorophore for excimer emission, a Zn­(II)-chelate for phosphate binding, and an alkane linker. Configurations of this scaffold have been shown to effectively detect phosphorylated biological analytes, such as peptides, proteins, nucleotides, and phospholipid vesicles, through a two-component binding system. − The electrostatic and coordinative interactions between cationic zinc and anionic phosphates allow for the intramolecular stacking of proximal pyrene groups through hydrophobic and π–π interactions. This binding mechanism was hypothesized to be similar for bacteria, which contain several phosphorylated biomolecular components within their membranes, discussed previously (Figure S1).…”

“…In this work, we demonstrate rapid, potent, and facile broad-spectrum detection of bacteria by select receptors derived from the ProxyPhos class of fluorescent chemosensors (Figure A) in sterile physiological fluids. ProxyPhos turn-on chemosensors were previously shown to detect membrane-embedded negatively charged phospholipids (MENCPs), as well as proximally phosphorylated biomolecules such as proteins and phospholipids. , Within the ProxyPhos library, a unique family of cyclen and cyclam-based chemosensors are shown to have high sensitivity and selectivity toward bacterial cells. We hypothesize that the ProxyPhos sensors operate, at least partially, by recognizing the negatively charged bacterial surfaces and a detectable excimer signal produced by the proximity-based stacking of multiple pyrene units leading to a red-shifted emission to an excimer wavelength (λ em = 476 nm; Figure B).…”

Pyrophosphate Prompted Aggregation‐Induced Emission: Chemosensor Studies, Cell Imaging, Cytotoxicity, and Hydrolysis of the Phosphoester Bond with Alkaline Phosphatase

“…Pyrophosphate is one of the important biological anions, as it demonstrates an intense role in many cellular processes , . Therefore, it has led to the remarkable interest of analytical chemist to synthesize such sensor which can detect PPi in the biological system.…”

Optical chemosensors for the detection of proximally phosphorylated peptides and proteins

“…Further improvements on the chemosensor 8 scaffold were identified through a structure–activity relationship (SAR) study. 72 The sensor structure ( 9 ) was modified at the three components: fluorophore, receptor, and linker. Initially, polybenzene aromatic fluorophores were evaluated for excimer capabilities, but neither anthracene nor naphthalene were able to elicit suitable excimer responses.…”