Generation of functional molecules which regulate the glutathione level in living organisms-Design and synthesis of specific inhibitors of γ-glutamylcysteine synthetase-

The purpose of this research is to design and synthesis of functional molecules which affects the glutathione level in living organisms。The biosynthesis and degradation of glutathione are mediated byγ-glutamylcysteine synthetase(γ-GCS)andγ-glutamyltranspeptidase(GGT)，respectively.Based on the reaction mechanisms of these enzymes，specific inhibitors of these two enzymes were designed，synthesized and evaluated in terms of inhibition potency and profile。γ-GCS catalyzes the reaction by activating theγ-carboxyl group of L-Glu by phosphorylation，followed by nucleophilic substitution with L-Cys.As a transition-state analogue of the substitution step，tetrahedral phosphinic acid and sulfoximine analogues were synthesized。Each compound was found to be a potent time-and ATP-dependent inactivator ofγ-GCS.In particular，the sulfoximine served as an extremely powerful slow-binding inhibitor，in which the sulfoximine S=NH nitrogen was phosphorylated by ATP in a mechanism-based manne…More r to form a phosphorylated sulfoximine which is highly analogous to the putative transition state.Two diastereomeric sulfoximines with respect to the chiral sulfur atom were synthesized and evaluated as an inhibitor ofγ-GCS。The diastereomer with(R)-sulfur atom served as a highly potent ATP-dependent inactivator(K I D2=39nM)，but the(S)-diastereomer was a rather weak reversible inhibitor(K I D2=12µM)。Thus，the enzyme recognized the chirality of the sulfur atom and phosphorylated the(R)-sulfoximine solely。The(R)-sulfoximine inhibitedγGCS1000times more strongly than buthionine sulfoximine，a famous inhibitor ofγ-GCS。In an attempt to obtain an enzyme-suicidal substrate ofγ-GCS，the effect of L-glutamic acidγ-hydroxamate was examined.This compound was found to inhibitγ-GCS very slowly，but irreversibly in the presence of ATP，and the inhibition profile suggested that the hydroxamate was phosphorylated enzymatically by ATP to form an isocyanate via a Rossen-type rearrangement。A transition-state analogue inhibitor of GGT was also synthesized.Aγ-phosphonofluoridate analogue of Glu was synthesized and was found to serve as a mechanism-based labeling agent of GGT to phosphonylate the active site catalytic nucleophile.Ion-spray MS of the labeled GGT revealed that the N-terminal Thr-391in the small subunit was the catalytic nucleophile of GGT.Less：Less

The purpose of this research is to design and synthesis of functional molecules which affects the glutathione level in living organisms. The biosynthesis and degradation of glutathione are mediated by γ-glutamylcysteine synthetase (γ-GCS) and γ-glutamyltranspeptidase (GGT), respectively. Based on the reaction mechanisms of these enzymes, specific inhibitors of these two enzymes were designed, synthesized and evaluated in terms of inhibition potency and profile. γ-GCS catalyzes the reaction by activating the γ-carboxyl group of L-Glu by phosphorylation, followed by nucleophilic substitution with L-Cys. As a transition-state analogue of the substitution step, tetrahedral phosphinic acid and sulfoximine analogues were synthesized. Each compound was found to be a potent time-and ATP-dependent inactivator of γ-GCS. In particular, the sulfoximine served as an extremely powerful slow-binding inhibitor, in which the sulfoximine S=NH nitrogen was phosphorylated by ATP in a mechanism-based manne … More r to form a phosphorylated sulfoximine which is highly analogous to the putative transition state. Two diastereomeric sulfoximines with respect to the chiral sulfur atom were synthesized and evaluated as an inhibitor of γ-GCS. The diastereomer with (R)-sulfur atom served as a highly potent ATP-dependent inactivator (KィイD2iィエD2=39 nM), but the (S)-diastereomer was a rather weak reversible inhibitor (KィイD2iィエD2=12μM). Thus, the enzyme recognized the chirality of the sulfur atom and phosphorylated the (R)-sulfoximine solely. The (R)-sulfoximine inhibited γGCS 1000 times more strongly than buthionine sulfoximine, a famous inhibitor of γ-GCS. In an attempt to obtain an enzyme-suicidal substrate of γ-GCS, the effect of L-glutamic acid γ-hydroxamate was examined. This compound was found to inhibit γ-GCS very slowly, but irreversibly in the presence of ATP, and the inhibition profile suggested that the hydroxamate was phosphorylated enzymatically by ATP to form an isocyanate via a Rossen-type rearrangement. A transition-state analogue inhibitor of GGT was also synthesized. A γ-phosphonofluoridate analogue of Glu was synthesized and was found to serve as a mechanism-based labeling agent of GGT to phosphonylate the active site catalytic nucleophile. Ion-spray MS of the labeled GGT revealed that the N-terminal Thr-391 in the small subunit was the catalytic nucleophile of GGT. Less