Selective On/Off‐Nitroxides as Radical Probes to Investigate Non‐radical Enzymatic Activity by Electron Paramagnetic Resonance

Abstract

A nitroxide carrying a peptide specific to the binding pocket of the serine proteases chymotrypsin and cathepsin G is prepared. This peptide is attached as an enol ester to the nitroxide. Upon enzymatic hydrolysis of the peptide, the enol ester moiety is transformed into a ketone moiety. This transformation affords a difference of 5 G in phosphorus hyperfine coupling constant between the electronic paramagnetic resonance (EPR) signals of each nitroxide. This property is used to monitor the enzymatic activity of chymotrypsin and cathepsin G by EPR. Michaelis constants were determined and match those reported for conventional optical probes.

An elastase activity reporter for Electronic Paramagnetic Resonance (EPR) and Overhauser-enhanced Magnetic Resonance Imaging (OMRI) as a line-shifting nitroxide

“…Data are presented as follows: chemical shift (in ppm), integration, multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet, br = broad, dd = doublet of doublets), coupling constant (J in Hz) and integration. 31 P NMR spectra were recorded on a Bruker AC 300 (122 MHz) and on a Bruker AC 400 (162 MHz) spectrometers with complete proton decoupling. Chemical shifts (δ) were reported in ppm using TMS as internal reference for 1 H and 13 C NMR spectra, and 85% H3PO4 for 31 P NMR spectra.…”

“…As mentioned above, nitroxides 1•/2• exhibit high potential to investigate proteolysis both by EPR and OMRI. Recently, enantiomers of 1• were separated, identified and, then used for the preparation of the peptide-nitroxide substrate (reported elsewhere [31]).…”

Chemical Probes for Human UDP‐Glucuronosyltransferases: A Comprehensive Review

“…Although many drugs and endogenous substances have been identified as probe substrates for a target UGT enzyme, the glucuronidation rates of these UGT probe substrates were routinely detected by LC‐UV or LC‐MS detectors, requiring relative long time for sample preparation and analysis. By contrast, fluorescence‐based biochemical assays have attracted great attention in biological and medical related fields, due to their inherent advantages including non‐destructive, highly sensitive, and applicable to HTS assays . Over the past decade, a wide range of fluorescence probes for serine hydrolases and other phase I metabolizing enzymes have been developed and widely used for evaluating enzyme activities and screening of enzyme modulators .…”

“…By contrast, fluorescence-based biochemical assays have attracted great attention in biological and medical related fields, due to their inherent advantages including non-destructive, highly sensitive, and applicable to HTS assays. [157][158][159][160][161][162][163][164][165][166][167][168] Over the past decade, a wide range of fluorescence probes for serine hydrolases and other phase I metabolizing enzymes have been developed and widely used for evaluating enzyme activities and screening of enzyme modulators. [47,48,50,51,[169][170][171][172][173] However, the design and development of fluorescence probes for UGTs is a very difficult task, due to the following three reasons.…”

Imaging of Enzyme Activity by Electron Paramagnetic Resonance: Concept and Experiment Using a Paramagnetic Substrate of Alkaline Phosphatase

“…Recent work demonstrated the detection of proteases using a β-phosphorylated NR-substrate prototype [11] and an NR-labeled peptide specific to chymotrypsin and cathepsin G. [12] Upon enzymatic hydrolysis of the peptide, the enol ester moiety of the NR is converted into a ketone moiety accompanied by a large 4 to 5 G change of the phosphorus hyperfine splitting (hfs) constant. Note that the measurement of hfs changes provides higher specificity than changes in correlation time or spin exchange, as the latter are sensitive to the local environment.…”