Combination of biotransformation by P450 BM3 mutants with on-line post-column bioaffinity and mass spectrometric profiling as a novel strategy to diversify and characterize p38α kinase inhibitors

Tandem mass spectrometry study of p38α kinase inhibitors and related substances

“…Data on relative retention time (RRT) were used to assist in identifying the modification, e.g., to differentiate between hydroxylation and N ‐oxide formation . In the case of five BM3‐generated CPs of TAK‐715, the fourth step involved purification of the CPs by preparative LC and subsequent acquisition and interpretation of the 1 H NMR spectra, guided by the knowledge obtained from the MS n data . The data are summarized in Tables .…”

“…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. Oxidation with hydrogen peroxide occurs mainly via reactive oxygen species, whereas photochemical conversion is the result of the excitation of electrons in the substrate by intense visible light irradiation …”

“…For example, the use of electrochemistry in an on‐line combination with the HRS platform resulted in a number of related substances for p38α kinase inhibitors, including unstable and reactive conversion products (CPs) . Mixtures of related substances can also be generated enzymatically, e.g., biosynthetically using (human) microsomal incubations or by incubation with specific bacterial mutants (BM3) of cytochrome P450s . Alternatively, other (chemical) means such as photochemistry or oxidative reagents can be applied.…”

Selective whole-cell biosynthesis of the designer drug metabolites 15- or 16-betahydroxynorethisterone by engineered Cytochrome P450 BM3 mutants

“…Also within our own lab, isolated CYP BM3 mutants have previously been successfully applied for biosynthesis of drug metabolites on a preparative scale [32][33][34][35][36]39,47,48]. During our in vitro experiment, bioconversion was monitored in time (see Fig.…”

“…The 40 other previously described mutants have been based upon these four templates [28][29][30][31][32][33][34]. The 44 mutants have been shown to metabolize a wide range of substrates, including marketed drugs [28,30,35,36], steroids [29,32,34], ionones [33], kinase inhibitors [47], alkoxyresorufins [29,31], and endocrine disrupting chemicals [31,48], with varying catalytic activities while also displaying significant differences in the metabolic profiles generated. The 22 mutants that have not been described before were all created inhouse by site-directed mutagenesis using the appropriate mutant templates (see the Supporting Information, Table S2).…”