Construction Of Selenium-containing Artificial Enzymes Based On The Host-Guest Interaction

Enzymes, as biological catalysts with high catalytic efficiency, exhibit thousands of folds in activities than normal chemical catalysts, so scientists have shown great interest in researches on enzymes. While because enzymes, whose nature is a kind of proteins, have some defects as natural proteins, such as low yield, strongly conditional reliability, so researches on artificial enzymes emerged widely since last century. Natural enzymes are outcomes of biological evolution which has lasted for more than four billion years, so researches on artificial enzymes are based on structures of natural enzymes and micro environments.Glutathione Peroxidase (abbreviation GPx) was first discovered on 1957, and it associated with many kinds of oxidative injuries, such as arthritis, heart disease, carcinoma, etc, which interests scientists. There are so many reports on GPx mimics by biological or chemical methods after the relationship between the element selenium and the catalytic mechanism of GPx was discovered. By doing research on bovine's and human's GPx, scientists found that there are three features in the catalytic pocket of GPx. Three features are followings: (1).There is an element selenium in the catalytic center. (2).There is a hydrophobic environment around the selenium. (3).There are some amino acid residues in the hydrophobic environment which function by inducing the Glutathione (abbreviation GSH) to the right position. All of these features in natural catalytic pocket show us a definite direction to design artificial enzymes.Supramolecular chemistry, which has developed fast in recent years, has penetrated in many kinds of natural sciences, including biology. Natural enzymes, which exhibit as biological macromolecules, are just based on the supramolecular interaction when forming transition states with low activation energy after combine with substrates, and this is the essential reason why the catalytic ability of natural enzyme exceed many folds compared with normal chemical catalysts, so finding the junction between supramolecular chemistry and science of enzyme will certainly do an important job for constructing artificial enzyme with high catalytic efficiency.β-cyclodextrin (abbreviationβ-CD), which has been most widely investigated as a host molecule in the supramolecular chemistry, has a perfect dimensional matching with the adamantyl group, so it could form a host-guest inclusion complex by utilizing supramolecular interaction between the hydrophobic cave ofβ-CD and the hydrophobic adamantyl group, which exhibits a good flexibility based on the non-valent interaction, similar to flexible pocket of natural enzyme. Further more, by modifying guanidyl group onβ-CD to identify the carboxyl group of substrate and introducing hydrophobic part and catalytic center element selenium to mimicking the pocket of natural GPx, we expect to obtain artificial enzymes by host-guest interaction with an efficient GPx activity.We designed and synthesized guest molecules 4-(adamantylcarbamoyl) benzeneselenonic acid (abbreviation AdPhSeO2OH), 4,4'- selenoxybis(N-adamantyl- benzamide) (abbreviation (AdPh)2SeO), and host molecule mono-6-deoxy-6-guanidino-cycloheptaamylose bicarbonate (abbreviationβ-GCD). Further more, we prepared water-soluble AdPhSeO2OH-β-CD,AdPhSeO2OH-β-GCD,(AdPh)2SeO-β-CD,(AdPh)2SeO-β-GCD four inclusion complexes. By utilizing the element selenium as a catalytic center, phenyl group as a hydrophobic pocket, host-guest interaction as a flexible pocket of natural GPx, and guanidyl group as amino acid residues around the catalytic center which poessess the inducing function, we expect to obtain efficient artificial enzymes. All above molecules were characterized by NMR (1H, 13C), ESI-MS and HPLC, indicating the terminal molecules were successful obtained.We made detailed tests on the enzyme's activity of all inclusion complexes above by UV. Firstly, we discovered AdPhSeO2OH and its inclusion complexes exhibited GSH oxidase activity, which is similar to that from Hilvert etc's reports, so we concluded that maybe there exists a–SeH group which is easy to be oxided by oxygen during the catalytic process. Secondly, when we tests AdPhSeO2OH and its inclusion complexes for GSH oxidase activity and GPx activity, the cation NH4+ in the solution of gltutathione reductase (abbreviation GR) shields the anion COO- of GSH that the inducing interaction between guanidyl group ofβ-GCD and carboxyl group of GSH won't function.So we chose 3-carboxy-4-nitrobenzenethiol (abbreviation TNB) system to make detailed activity tests of AdPhSeO2OH,AdPhSeO2OH-β-CD,AdPhSeO2OH-β-GCD,(AdPh)2SeO -β-CD,(AdPh)2SeO-β-GCD. According to tests we found that without the negative effect of cation NH4+, inclusion complexes with guanidyl group all exhibit certain degree enhancement compared with inclusion complexes without guanidyl group. Specially, because the negative charge of AdPhSeO2OH is a disadvantage to the negative substrate TNB, there is only a limit enhancement when AdPhSeO2OH-β-GCD's activity compares with AdPhSeO2OH-β-CD's. While there is a great enhancement in the activity which is about fifty percent when (AdPh)2SeO-β-GCD's activity compares with (AdPh)2SeO-β-CD's, so it is sufficiently indicated that the guanidyl group play a very important role in enhancing the activity when inducing the substrate TNB to the right position. This research provides a new idea for constructions of GPx mimics with high efficiency.In addition, we proposed the possible catalytic mechanism of (AdPh)2SeO's inclusion complex, and we did a research on the kinetic research of (AdPh)2SeO-β-GCD which exhibit the highest catalytic activity. By this research, we confirmed that (AdPh)2SeO-β-GCD is real artificial enzyme further and many kinetic data were obtained. The second-order rate constant of (AdPh)2SeO-β-GCD is 3 magnitudes.This method of constrcting novel artificial enzyme based on the host-guest interaction not only absorb merits of chemical enzyme, such as high yield, easy preparement and stability, and also merits of biological enzyme, such as flexibility, catalytic microenvironment and good solubility in water, hence it will lay the foundation of constructing GPx mimics with high efficiency in the future.