Study Of Glutathione Peroxidase Mimics Based On Cyclodextrin

Oxidative Stress is a kind of unbalance state between oxidation and antioxidation. Reactive oxidative species (ROS) are harmful to cells when oxidative metabolism products increase or the antioxidative defenses lack in the organism. Lots of researches showed that ROS are related to some physiologic and pathological processes and play important role in many diseases. ROS have high activity and very strong oxidative ability and they can attack the molecules by the oxidation, and then make bimolecules denaturate, cross-link and break, and then lead to the damage of cell structure and function and the injury of the tissue.There are two kinds of the scavengers for free radicals in the organism, namely enzymes (Antioxidative enzymes) and non-enzyme compounds. The enzyme scavengers mainly include SOD, CAT and GPX, and they form the defense system in the organism against free radicals. In addition, many small molecular compounds also have strong antioxidative ability. These compounds are vitamin C, vitamin E,α-tocopherol, reduced glutathione,β-carotene, metal chelate complex, calcium ion antagonists, flavonoid, polyphenol compouns and antioxidant peptides, and so on. Together with antioxidant enzymes they make free radicals be in below the normal level of metabolism in organism.GPX are the well-known antioxidant selenoenzymes in organisms which destroy several harmful hydroperoxides and then maintain the metabolic balance of ROS in vivo, thus protecting the biomembranes and other cellular components from oxidative damage. In certain pathogenic states, the production of ROS is enhanced and the excess ROS damage various biomacromolecules including RNA, DNA, protein, sugars and lipids, and therefore result in ROS-mediated diseases. ROS-related diseases include reperfusion injury, inflammatory process, age-related diseases, neuronal apoptosis, cancer and cataract. Therefore, GPX can really act as antioxidant drugs. Unfortunately, scientists can not fully understand the structures of GPX as well as its catalysis mechanism in vivo at the present time. Fabrication of GPX models offers an ideal alternative for elucidating the origin of substrate binding and catalysis of enzyme.Mimicking the natures of molecular recognition and catalysis of enzymes by artificial enzymes is very essential for exploring the evolved biological process of enzymes as well as their properties of structures and functions. On the basis of structural understanding for GPX, we select supramolecular host molecules—cyclodextrins as the scaffolds of enzyme models, obtain systems of cyclodextrin- based GPX models. The substrate specificities of GPX mimic 6-TeCD for structurally distinct hydroperoxides are investigated through molecular dynamics (MD) simulations and enzymatic kinetics. Interaction of 2-TeCD with substrate GSH was studied by SPR sensor. Base on two major theories (Stabilization of intermediate and Substrate binding) 6-CySeCD was designed and synthesized to imitate the antioxidant enzyme GPX. We prepared 6-ImTeCD, which can effectively scavenged·OH, to imitate GPX. Our work not only perfects the development of the research areas of cyclodextrins, but also promotes the understanding of GPX mimics.Ⅰ. Study on Substrate Recognition in Tellurium-Containing Cyclodextrins as GPX Mimics.1. Study of Hydroperoxides Substrate Selectivity of 6-TeCD. The GPX activity of 6-TeCD for the reduction of H2O2, t-BuOOH and CuOOH by GSH is higher than that of Ebselen, respectively. These results show that 6-TeCD has different substrate specificity for hydroperoxides, and the preferred substrate is CuOOH. The results of molecular docking indicate that CuOOH fits well to the size and shape of the cavity ofβ-CD. And the results of MD simulations also show that the favorable total interaction energy of CuOOH-β-CD is higher than that of t-BuOOH-β-CD and H2O2-β-CD. A comparison of kinetic parameters is obtained from kinetic analyss of 6-TeCD using a variety of structurally distinct hydroperoxides, We also find that 2-TeCD shows different substrate selectivity and the CuOOH is the most preferred substrate. In our miniature enzyme model, cavity ofβ-CD as a binding site, that provided maximum hydrophobic interaction with a substrate to form complexes, fits the aryl group of the bound substrate and the CuOOH could take advantage of the binding site of 6-TeCD well. All the above results indicate that two key factors affect hydroperoxides specificity. One is whether size and shape of hydroperoxides match with that of hydrophobic cavity ofβ-CD. Another one is that there is a strong interaction between hydrophobic cavity ofβ-CD and ROOH, or not.2. Study on the Substrates Interaction of 2-TeCD and GSH by Using Surface Plasmon Resonance Biosensor.The surface plasmon resonance (SPR) was designed on the basis of fixing the angle of incidence and measuring the reflected intensities. A charge coupled device (CCD) was used to measure the SPR spectra. Glutathione (GSH) self-assembling in solution is used to form the sensing membrane on the gold substrate. Interaction of 2-TeCD with substrate GSH was studied by this SPR sensor and the kinetic processes of sensing monolayer formation were studied. The kinetic constant of GSH with 2-TeCD is 3.92×102 L /mol. These results validate that the SPR sensor technology can be used to study interaction between the small-molecule GPX mimics and substrate GSH in real time. Under selected experimental condition, simultaneous observation of the interaction between enzyme and substrates can be realized with out sample labeling and, therefore, kinetic constants of reactions can be obtained. Such instrumentation is to be used in fields such as on-line process monitoring.Ⅱ. Design of Selenium-Containing Cyclodextrin as GPX Mimics with High GPX ActivityIn the early efforts to design the enzyme model with GPX-like activity, the main ideas were focused on the imitation of Se-N interaction which was demonstrated important in stabilization and activation of reactive selenol moiety in catalytic cycle. On the basis of the structural understanding for GPX, its nature of molecular recognition and catalysis as well as the early works, we conceive that the generation of specific binding ability for thiol substrate and correct incorporation of the functional selenium group should be critical approaches to construction of an effective GPX model. The previous studies from our group in preparing GPX models by monoclonal antibody technique, bioimprinting, and chemical modification of natutive enzymes had demonstrated this hypothesis.Here we designed and synthesized a new GPX mimic 6-CySeCD, in which cyclohexylamine group was incorporated into the neighborhood of selenium atom and the cavity ofβ-CD supplied a hydrophobic environment for substrate binding. The GPX activity of 6-CySeCD was higher than that of 6-SeCD for the reduction of H2O2, t-BuOOH and CuOOH by GSH, indicating that incorporation of amido group into the neighborhood of selenium atom can increase the stability of selenolate and enhance GPX-like activity of the selenium containing GPX mimics. We also studied the mechanism by steady-state kinetic of 6-CySeCD and investigated the antioxidant ability of 6-CySeCD using mitochondria injury system.Ⅲ. GPX Mimics for Scavenging·OH Free RadicalBase on the Diego's work, we prepared 6-ImTeCD using the cavity ofβ-CD as substrate binding site and ditellurium bond as catalytic group. Also we introduce imidazole group to regulate the hydrophobic microenvironment ofβ-CD. 6-ImTeCD has good GPX activity with different hydroperoxides, and saturation kinetics was observed for both H2O2 and GSH. Double reciprocal plots of the initial velocity versus the concentration of substrates were a family of parallel lines, consistent with a Ping-Pong mechanism. Fenton/Rh-B-induced damage system indicated: 6-ImTeCD can effectively scavenge·OH. We constructed the H2O2-induced culture hepatocyte cells damage model system and demonstrated the hepatocyte cells had great changes in the damage system. We investigate the protective effects of culture hepatocyte cells by 6-ImTeCD using the damage system, indicating that it can inhibit the levels of lipid peroxidation, counteract the cell death. 6-ImTeCD also can prevent the leakage of LDH to sustain the integrity of cell membrane. We found that 6-ImTeCD can penetrate cells, indicating that 6-ImTeCD has great potential value in pharmaceutical application.