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

这项研究的目的是为了使功能分子的设计和合成，这些分子会影响活有机体中的谷胱甘肽水平。谷氨酰胺合成酶（γ-GCS）和γ-谷氨酰胺转肽酶（GGT）介导的生物合成和降解，负责。基于这些酶的反应机制，这两个酶的特定抑制剂是以抑制潜能和概况的术语设计、合成和评估的。γ-GCS催化反应通过磷酸化激活γ-碳基组的L-Glu，并通过与L-Cys的核生理替代物跟踪。作为替代步骤、四氢磷酸盐和硫氧胺模拟的过渡状态模拟是合成的。每个化合物都被发现有一个潜在的时间--γ-GCS的ATP依赖抑制剂。在特定的情况下，硫氧胺被用作一种非常强有力的低结合抑制剂，在硫氧胺S=NH硝基被磷酸化在基于机制的人体中 ... More r形成一种磷酸化的硫二胺，其中对可选择性过渡状态具有高度模拟性。两种具有对chiral sulfur atom的尊重的二次淀粉样硫酸盐原子被合成和评价为γ-GCS的抑制剂。具有（R）-硫原子的代谢物被用作高潜力ATP依赖抑制剂（KI-D2 i-D2=39 nM），而（S）-代谢物被用作较弱的可逆抑制剂（KI-D2 i-D2=12微米）。因此，酶被识别为硫基原子的手性和磷酸化了（R）-硫基胺的手性。（R）-硫氧胺抑制剂γGCS超过1000次，比丁二烯硫氧胺更强，是γ-GCS的著名抑制剂。在一项尝试中发现γ-GCS的酶-自杀基底，其对L-谷氨酸γ-羟基的影响已被测试。这一化合物发现了抑制γ-GCS非常缓慢，但不可逆地在ATP的存在中，并且抑制概况建议，羟基化酶通过ATP通过Rossen型重新排列形成异硫氰酸。一个过渡-状态模拟抑制剂的GGT也被合成。一种γ-磷酸盐氟氯化物模拟方法已被合成，并已找到用于服务的是一种基于机制的标记试剂，将其转化为磷酸化的活性站点催化核。标记的GGT离子喷雾MS揭示了N-终端Thr-391在较小的子单元中是GGT的催化核物质。Less（低）

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