On-line electrochemistry–bioaffinity screening with parallel HR-LC-MS for the generation and characterization of modified p38α kinase inhibitors

Abstract

In this study, an integrated approach is developed for the formation, identification and biological characterization of electrochemical conversion products of p38α mitogen-activated protein kinase inhibitors. This work demonstrates the hyphenation of an electrochemical reaction cell with a continuous-flow bioaffinity assay and parallel LC-HR-MS. Competition of the formed products with a tracer (SKF-86002) that shows fluorescence enhancement in the orthosteric binding site of the p38α kinase is the readout for bioaffinity. Parallel HR-MSn experiments provided information on the identity of binders and non-binders. Finally, the data produced with this on-line system were compared to electrochemical conversion products generated off-line. The electrochemical conversion of 1-{6-chloro-5-[(2R,5S)-4-(4-fluorobenzyl)-2,5-dimethylpiperazine-1-carbonyl]-3aH-indol-3-yl}-2-morpholinoethane-1,2-dione resulted in eight products, three of which showed bioaffinity in the continuous-flow p38α bioaffinity assay used. Electrochemical conversion of BIRB796 resulted, amongst others, in the formation of the reactive quinoneimine structure and its corresponding hydroquinone. Both products were detected in the p38α bioaffinity assay, which indicates binding to the p38α kinase.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-011-5663-2) contains supplementary material, which is available to authorized users.

Solution-phase electrochemistry-nuclear magnetic resonance of small organic molecules

“…This molecule had not been studied extensively by EC before and NMR spectra of its quinone imine and hydroquinone had not been published. Presence of both reactive EC products, whose structures had previously been based solely on EC-LC-MS and EC-LC-MS n data [37], could be confirmed. However, independent structure elucidation was not yet possible due largely to an unexplained mismatch between conversion efficiency off-line and EC-NMR experiments.…”

Tandem mass spectrometry study of p38α kinase inhibitors and related substances

“…The minor product (CP501, m / z 501.170, C 25 H 27 ClFN 4 O 4 + ) was also observed in EC experiments and was identified as resulting from the loss of C 3 H 4 from the dimethylpiperazine group …”

“…In general, only a few of the previously identified fragments of DMPIP are actually observed for the CPs, especially the CD ion. CPs generated by EC are discussed in more detail elsewhere …”

“…Each of these methods is discussed briefly but has been described in detail elsewhere. Electrochemistry adds (reduction) or removes (oxidation) electrons from the substrate on a surface electrode, which results in further reaction for example with the solvent . During incubation with (human) liver microsomes or with mutants of cytochrome P450 BM3, the enzymes present catalyse specific reactions such as hydroxylation.…”

Development of On-line Liquid Chromatography-Biochemical Detection for Soluble Epoxide Hydrolase Inhibitors in Mixtures

“…Both reactors were kept at 37 °C by a Grant Instruments (Shepreth, UK) water bath. The enzyme and substrate solutions were delivered by in-house built superloops which were kept on ice [25]. The fluorescence was measured at excitation of 320 ± 10 nm and emission of 460 ± 10 nm.…”