Synthesis Of Carbamates Capable Of Self-Assembly On Gold And Their Interaction With Acetylcholinesterase

Drug discovery is the preliminary stage of drug research and development, including drug molecular design, synthesis, target discovery and confirmation, lead compounds screening and optimization, and so on. In nowadays, rational design and synthesis of lead compounds are the most important method for target directed drug development. The so-called rational drug design, lies on the study research results of biochemistry, enzymology, molecular biology, genetics, informatics, as well as computation chemistry.The acetylcholinesterase (AChE) is one kind of serine hydrolases, which is the essential for normal nerve conduction activity of vertebra and the non- vertebra. Carbamates are the inhibitors of AChE, which have been used pesticides widely in agriculture. Investigation of the interaction of carbamates and AChE, using new techniques of molecular recognition and interaction, is important for the development of novel carbamate pesticides, as well as the establishment of the basement for other kind of new drugs research.The self-assembled monolayers (SAMs) modified electrodes have important applications in the reaearch of the interaction of large biomolecules with large biomolecules as well as small organic molecules. In recent years, biological activity molecules, such as enzyme, the immune bodies, even cells have been constructed on gold electrodes to make various kinds of biosensors using the self-assembly monolayer technology. The biosensors have been applied widely in chemistry, biology, clinical diagnosis and environment examination, as well as the molecular interaction and recognition, and the last is the keypoint for new drug development.In this work, based on the molecular structures of AChE inhibitor carbamates, we designed and synthesized four kinds of carbamates (schem 1) capable of self-assembly on the gold surface and inhibitive effect on AChE. The carbamate self assembled monolayers were constructed on gold electrodes, and their structures as well as the ability to bind with AChE were investigated by surface plasmon resonance biosensors (SPR) and electrochemical methods. Thus a kind drug discovery experimental model method was established: 1) if the target enzyme is known, lead compounds can be screened and their binding kinetics can be investigated for further structural optimization and resynthesis; 2) for compounds with known bioactivity, they can be employed to form self assemble monolayers on electrodes, and their targets can be screened, which is important for the finding of new targets.The main contents are listed as following:1: Four kinds of cabamates, three of them containing disulfide linkage and one containing biotin as the self-assembly functionalities, were synthesized. The molecular structures of the intermediates and the target compounds were confirmed by HNMR, MS, and IR etc.2: Regarding the three kinds of carbamates with disulfide linkage were constructed on gold electrodes as the slef assembled monolayers, whereas the carbamate with biotin group was constructed on gold electrode by stepwise self-assembly technology via avidin linkage. The electrochemical cyclic voltammetry and impendence spectra were employed to characterize the structures. The results showed that all the carbamates with self-assembly ability could be constructed on gold electrodes successfully for the mation of the corresponding self assembled monolayers. The surface coverage of the stepwise constructed SAM was higher than that of pure SAMs with the disulfide linkage. In pure SAMs with the disulfide linkage, the coverage was increased with the increase of the polyester chain length between disulfide group and cabamate groups.3: The in vivo bioactivities of the four cabamates are tested and their interactions as well as specific binding kinetics were invevestigated by SPR technology for the carbamates on SAMs and AChE in solution in realtime. The results show that all the four cabamates have in vivo pesticidal activities, and amone them, the compounds (15) has the highest bioactivity, it reaches to 73.3% to aphid, which is comparable to the results from the SPR in vitro kinetic measurements. For the compounds (4,8,11) modified electrodes,SPR results show that compound (8) has the highest binding ability to AChE, which is constant to the results of in vivo bioactivity measurements.4: The specific binding of surface confined cabamates to AChE was tested with the discrimination of AChE from bovine serum albumin (BSA). From this result, and their target can definitely determined to be AChE. This may be a general method for the determination of the targets for various inhibitive drugs.