Sau.1c00025 JACS Au 2021, 1, 669-JACS Aupubs.acs.org/jacsauArticleFigure 7. Probe 9 cross-links to hGR (A) MS/MS fragmentation pattern of identified peptides of hGR photoMMP list reaction mixture with probe 9. Left panel – peptide cross-linked at K397 (2893.23 Da = Y393-K416 + 9 – 18 Da; dehydration is popular for benzophenone adducts). Right panel – peptide cross-linked at C234 (2874.23 + 9-BX[NH2] Da). Red circles indicate identified cross-linking sight. (B) Left panel – position of K256-7, K397 (blue), C234 (orange), Tyr197 (pink), and FAD (yellow) have been marked on the previously reported hGR dimer structure cross-linked to menadione analogue (red). The substrate binding cleft leading for the catalytic disulfide bridge is visible amongst K397 and menadione core (orange triangle). (C) Magnification on C234 (yellow) containing the binding pocket with indicated water molecules (violet balls). Surface of A241 (blue) and H374 (pink) at pocked opening and V200 (green) in cavity is visible.pathways: the probe, the benzophenone-like adduct, the two(SG-methyl)-probe adduct, the cyclized probe-BX, the probeBX-derived enone, as well as the probe-BX insertion adduct.Probe Cycling with Glutathione Reductase Generates BenzoxanthoneAs for photoreduction, the benzoxanthone formation has been postulated to take place for the duration of several cycles of enzymatic (GR) 1e-RelB site reduction of PDOox (Figure 1A). However, the metabolite was only indirectly detected by electrochemical measurements of PDO derivatives due to its minor quantity.31 To prove definitively that PDO-BX is generated by continuous redoxcycling in the drug beneath hGR catalysis, we analyzed such reaction by LC-MS/MS soon after 6 h of frequent addition of NADPH. The BX-derived enone could be discovered in reactions in open air (Figures S21B, S21C) but not inside the deoxidized control where redox-cycling was not attainable as a consequence of the absence of oxidants like oxygen (Figure S21A). This clearly demonstrates that PDO-BX is indeed a solution of PD metabolite redox-cycling (Figure 1A). Moreover, we investigated similarities in the BX formation for the duration of the redox-cycling processes in the course of photoreduction and GR catalysis. For this, we irradiated hGR with probe 9 in oxygen-free conditions. Interestingly, in spite of the lack of oxygen, we were capable to receive 9-BX from probe 9 aftermin of UV-irradiation with hGR (Figure S21D), though this was not probable in a comparable period of time when GSH was acting as a nucleophile. This demonstrates that the presence in the enzyme is enough to accelerate light-induced formation of 9-BX. Certainly, the UV-photoreduction process can mimic the reduction of naphthoquinone by NADPHreduced enzyme within this pathway, indicating that both processes may well share similarities. Generation of BX from PDOox or probe 9 was also attainable inside the presence of thiophenol following ten min of photoirradiation. Nonetheless, the cysteine thiol group in GSH only led to minor formation of PDO-BX even soon after overnight UV-irradiation (Figure S22 compared to S26). The outcomes obtained with hGR upon irradiation imply that the protein cysteines may possibly be far more reactive than GSH. Alternatively, the entropic interaction between the naphthoquinone as well as the enzyme could play a mutual influence on every single other upon transferring electrons and kinetically favor pathways 2-3 following probe binding to a cavity exactly where the molecular environment favors BX formation.Making use of Glutathione Reductase as a Model for PhotoreactionTo test the capability in the probes to interact with protei.
