The Design, Catalytic Mechanism And Biological Activities Of Artificial Glutathione Peroxidase Based On Cyclodextrin

Glutathione peroxidase is an important mammalian selenoenzyme that function in cellularredox reactions and plays an essential role in the detoxification of hydroperoxides in vivo, therebyscavenging active oxygen, protecting biomembranes from oxidative stress. It is related to manydiseases and it is regarded as one of the most important antioxidant enzymes in living organisms.However, native GPx has some shortcomings, such as instability, antigenicity and pooravailability, which have limited its therapeutic use. In addition, it is extremely difficult tosynthesize selenium-containing proteins by traditional recombinant DNA methods; thereforeconsiderable effort has been spent to find other routes to compounds capable of imitating theproperties of GPx.On the basis of structure and catalytic mechanism understanding for GPX, we choosecyclodextrins as the scaffolds of enzyme models to establish the systems of GPX models based oncyclodextrin.1. Cyclodextrin-Derived Chalcogenides as Glutathione Peroxidase MimicsTo elucidate the importance of the goodness of fit in complexes between substrates andglutathione peroxidise （GPX） mimics, we examined the decomposition of a variety of structurallydistinct hydroperoxides at the expense of glutathione （GSH） catalyzed by2,2’-ditellurobis（2-deoxy-γ-cyclodextrin）（2-Te-γ-CD） and by the corresponding derivatives ofβ-cyclodextrin （β-CD） and α-cyclodextrin. Hydroperoxides decomposing capacity of2-Te-γ-CDwas determined to be80.5,109.8,149.6U/μmol, respectively, with H₂O₂, t-BuOOH and CuOOH,which was almost80.5,333.3,118.3-fold than that of Ebselen. Furthermore, the catalytic constantand the combination with the best binding also exhibited the highest regioselectivity in thesubstrate decomposition. Saturation kinetics was observed and the catalytic reaction agreed with aping-pong mechanism, in analogy with natural GPX.2. Studies on the Molecular Recognition and Related Catalytic Mechanism of BridgedBis-cyclodextrinsAs the substitutes of glutathione （GSH）, S-substituted dinitrophenyl glutathione（GSH-S-DNP） are used in the present study to explore the recognition mechanism between twoglutathione peroxidase （GPX） mimics and GSH. The effect of the addition of γ-CD, or2-Te-γ-CDto the phosphate buffer solutions of GSH-S-DNP has been fully investigated by means ofUltraviolet-visible （UV-vis） absorption. The stoichiometry of the inclusion complex wasdetermined to be of2:1host-to-guest. The value of stability constant Kcfor （2-Te-γ-CD）₂/GSH atroom temperature was calculated to be3.815×104M-2, which suggested that2-Te-γ-CD had amoderate ability to bind GSH. Importantly, the proposed mode of the （2-TeCD）2/GSH complexwas the possible important noncovalent interactions between enzymes and substrates ininfluencing catalysis and binding. Using electrospray ionization mass （and tandem） spectrometry（ESI-MS and ESI-MS/MS） spectrometric experiments, the decomposition of hydrogen peroxide atthe expense of GSH catalyzed by2-TeCD was monitored on-line. The key intermediates weresuccessfully intercepted and structurally characterized for the first time by coupling a microreactoron-line to the ESI ion source, which permitted the fast screening of intermediates directly fromsolution. The catalytic mechanism of2-TeCD catalysis has been elaborated based on massspectrometric data and exerted its peroxidase activity via tellurol, tellurenic acid, andtellurosulfide, in analogy with natural GPX.3. Gamma-Cyclodextrin-Derived Chalcogenides as Glutathione Peroxidase MimicsA series of novel glutathione peroxidase （GPx） mimics based on organochalcogencyclodextrin dimer were synthesized. Their GPx-like antioxidant activities were studied usingH₂O₂, t-BuOOH, and CuOOH as substrates and glutathione as thiol co-substrate. The resultsshowed that6A,6B-ditelluronic acid-A’,6B’-tellurium bridged γ-cyclodextrin （6-diTe-γ-CD） hadthe highest peroxidase activity, which was approximately670-fold higher than Ebselen, awell-known GPx mimic. Reduction of lipophilic CuOOH often proceeded much faster thanreduction of the more hydrophilic H₂O₂or t-BuOOH, which cannot bind into the hydrophobicinterior of the cyclodextrin. Saturation kinetics was observed and the catalytic reaction agreedwith a ping-pong mechanism, in analogy with natural GPX.4. Cyclodextrin Derivatives as Glutathione Peroxidase Mimics and Their Protection ofMitochondria against Oxidative DamageThe biological activities were also evaluated for their capacity to protect mitochondriaagainst Ferrous sulfate/ascorbate-induced oxidative damage.6-diTe-γ-CD was the best inhibitor which significantly suppressed Ferrous sulfate/ascorbate-induced cytotoxicity as determined byswelling of mitochondria, lipid peroxidation and cytochrome c oxidase activity. Our data suggeststhat6-diTe-γ-CD and2-Te-γ-CD has potential pharmaceutical application in the treatment ofROS-mediated diseases.