ABSTRACT
The novel cysteine-cholamine (Cys-chol) trapping reagent was synthesized by coupling N-(tertbutoxycarbonyl)- S-trityl-L-cysteine with cholamine in the presence of HBTU (O-(benzotriazol-1-yl)- N,N,N',N'-tetramethyluronium hexafluorophosphate), and then deprotecting by trifluoroacetic acid. Cys-chol reagent enhanced the sensitivity of reactive metabolite screening 4 to 20 times without introducing additional sample preparation or derivatization steps. Retention of Cys-chol conjugates on reversed-phase column is higher than for respective GSH conjugates which helps in reduction of background interference. The use of Cys-chol trapping reagent can potentially improve sensitivity and specificity of routine reactive metabolite screening assay in drug discovery.
Subject(s)
Clozapine/metabolism , Cysteine/chemical synthesis , Drug Discovery/methods , High-Throughput Screening Assays , Microsomes, Liver/enzymology , Trimethyl Ammonium Compounds/chemical synthesis , Biotransformation , Chromatography, Liquid , Clozapine/chemistry , Cysteine/analogs & derivatives , Glutathione/metabolism , Humans , Tandem Mass SpectrometryABSTRACT
Binary stable isotope labeling couple with LC-ESI-MS has been used as a powerful non-targeted approach for the relative quantification of lipids, amino acids, and many other important metabolite classes. A multiplexed approach using three or more isotopic labeling reagents greatly reduces analytical run-time while maintaining excellent sensitivity and reproducibility. Three isotopic cholamine labeling reagents have been developed to take advantage of the pre-ionized character of cholamine, for ESI, and the ease by which stable isotopes can be incorporated into the cholamine structure. These three cholamine labeling reagents have been used to relatively quantify three fatty acid samples simultaneously. The quantification resulted in the observation of 12 fatty acids that had an average absolute error of 0.9% and an average coefficient of variation of 6.1%. Caged versus cage-free isotope labeling experiments showed that cage-free eggs have an increased level of omega-3 fatty acids as compared to caged eggs. This multiplexed fatty acid analysis provides an inexpensive and expedited tool for broad-based lipid profiling that will further aid discoveries in the mechanisms of fatty acid action in cells.
Subject(s)
Eggs/analysis , Fatty Acids, Nonesterified/chemistry , Isotope Labeling , Mass Spectrometry , Animals , Chickens , Egg Yolk/chemistry , Fatty Acids, Nonesterified/analysis , Lauric Acids/analysis , Lauric Acids/chemistry , Nuclear Magnetic Resonance, Biomolecular , Trimethyl Ammonium Compounds/chemical synthesis , Trimethyl Ammonium Compounds/chemistryABSTRACT
Blocking open ion channels provides a promising way to modulate synaptic transmission. Using the muscle-type acetylcholine receptor (AChR) as a test system, we seek to develop blockers that have blockade kinetics tunable via structural modifications. Here we investigate whether the blockade kinetics can be modulated by specifying the length of a poly(ethylene glycol) (PEG) spacer incorporated into the blocker. Single-channel electrophysiological experiments show that simple bis(trimethylammonium) compounds ( 1a- 3) both activate the AChR and block the open channel. The blockade kinetics are found to depend on spacer length: both the association and dissociation rate constants decrease with increasing spacer length. The decrease in the association rate constant can be quantitatively explained by the entropic cost of polymer confinement in the transmembrane lumen, while the decrease in the dissociation rate constant is consistent with weak, additive noncovalent interactions between the channel and the spacer. These results provide useful insights into the future design of kinetically tunable open-channel blockers for the AChR.
