The Research On Photoregulating Artificial Enzyme Based On Azobenzene

A balance between oxidant and antioxidant intracellular systems is vital for cell function, regulation, and adaptation to diverse growth conditions. As one kind of the antioxidative enzyme, the selenium-containing glutathione peroxidase (GPx) represents a cell-specific and tissue-specific mechanism for prevention of oxidative damage in mammalian systems. To elucidate whether the enzyme has a more subtle role in regulation of tissue peroxide levels than a blanket protection against oxidative stress, some methods and strategies of precise manipulation of the GPx activity have been developed. However, they are usually static and unresponsive to the environmental effects. Because of the convenience, expeditiousness, and economy, it will be an alternative method that triggering and control of the enzyme activity with light absorption of bistable photoswitches, for example, azobenzene and spiropyran derivatives.Recently in our group, a series of artificial GPxs have been constructed by monoclonal antibody, bioimprinting, transferring nature enzymes and other techniques etc. Especially, cyclodextrin-based enzyme models have been designed by cooperating both recognition by cyclodextrin and catalysis by telluride containing moieties. Since the first experiment on photoregulation of an enzymic process was performed in 1968, photoswitches have been used in a number of biotechnology applications. However, to our knowledge, there are only several reports about photocontrolled artificial enzyme on cyclodextrin-based biomimetic performance in the field and none about photoregulation of selenoenzyme. In the present study, we have extended our work to the field of controlling the catalytic process of artificial GPx subject to photoregulation for the first time.1. Photoregulating artificial GPx activity by single charged azobenzeneIt has been performed for the first time that photoregulating the activity of artificial glutathione peroxidase (GPx) by using photocontrolled inclusion-exclusion reaction of the azobenzene with two typical GPx mimics, the tellurideβ-Cyclodextrin (β-CD) dimmer and the ditellurideβ-CD dimmer. The activities of both mimics were differently inhibited by either cationic or anionic azobenzene, owing to the catalytic capacity variance of the telluride moieties of the mimics and the different strength of the electrostatic interactions between the charges of the substrates and the azobenzenes. The inclusion of the anionic azobenzene with the ditellurideβ-CD dimmer represented the largest inhibition rate. When the inclusion was irradiated upon UV light, the activity recovered, whereas inhibited again upon visible light. Such process could be repeated many times and a switchable artificial enzyme based on the reaction was proposed.2. The mechanism of the photoregulation of artificial GPx by azobenzene Through the 1H NMR, UV, induced circular dichroism (ICD) spectroscopy, and activity assay, we found that one bridgedβ-CD dimmer can include two azobenzenes. Meanwhile the substrate approaches the inclusion mimic hardly, due to the electrostatic repulsion between the substrate and the azobenzene. As a result, the effective recognition does not happen, and the activity is inhibited reasonably. After irradiation with UV light, the azobenzene leaves away from the cavity behind an appropriate cavity for accommodating a substrate, as a result, the GPx activity recovered.3. The assembly of porphyrin induced by bola-cationic azobenzene.In light of the success in the photoregulation of cyclodextrin-based artificial GPx, we extended the charge of azobenzene to two ones. Bola-ionic azobenzene is a good selection of this goal. By using the electrostatic interaction between the bola-ionic azobenzene and the porphyrin, the ionic complex was constructed, and the structure of the complex was characterized by UV and fluorescence spectroscopy. It proved that the bola-cationic azobenzene and the TCPP forms a 2:1 ionic complex.