THESIS
2003
xiii, 107 leaves : ill. ; 30 cm
Abstract
Nowadays, the widespread antibiotic drug resistance has become a serious healthy problem to human beings. With more and more microbe hold resistant ability, it seems that we will lose this war against microbe. People have to find new antibiotic drugs from diverse resources. Macrocyclic peptide antibiotic gramicdidin S and tyrocidine A show light on possible resolution to this problem due to their potent antibiotic activity and unique action mode. Although the detailed action mode of these peptide antibiotics has not been thoroughly revealed, it is believed that the bacteria cell membrane is their primary target where accumulation resultes in the disruption of its normal barrier function. It is unlikely to provoke the resistance to these peptide antibiotics because it requires significan...[
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Nowadays, the widespread antibiotic drug resistance has become a serious healthy problem to human beings. With more and more microbe hold resistant ability, it seems that we will lose this war against microbe. People have to find new antibiotic drugs from diverse resources. Macrocyclic peptide antibiotic gramicdidin S and tyrocidine A show light on possible resolution to this problem due to their potent antibiotic activity and unique action mode. Although the detailed action mode of these peptide antibiotics has not been thoroughly revealed, it is believed that the bacteria cell membrane is their primary target where accumulation resultes in the disruption of its normal barrier function. It is unlikely to provoke the resistance to these peptide antibiotics because it requires significant alternation of the lipid composition of the cell wall. But the disadvantages of these peptide antibiotics such as low interaction specificity and high haemolytic activity prevent them from practical application. Our research work focuses on functional optimization of macrocyclic peptide antibiotic gramicidin S for leads discovery. In order to find leads based on gramicidin S scaffold, it is highly desirable of us to develop a facile approach suitable for combinatorial synthesis because conventional chemical and biosynthesis approaches obviously can not provide convenient access to large quantity gramicidin analogues.
Inspired by the biosynthesis of these peptides, we wanted to develop a biomimetic approach utilizing putative thioesterase domain (TE). In order to realize this goal, we choose TentaGel resin to implement the solid phase peptide synthesis (SPPS) of linear precursor peptides. Using Fmoc SPPS and an optimized Fmoc deprotection method, the fully deprotected linear precursor peptide thioester of gramicidin S was successfully synthesized on solid support. The following enzymatic cyclization test proved the putative gramicidin thioesterase (GrsB TE) was active to this on-resin linear precursor. The structure characterization proved the cleavage product was consistent with wild type gramicidin S reported before. In this process, we also developed a facile synthetic method using aqueous ammonia solution to cleave fully deprotected linear precursor peptide immobilized on solid support. Using this method, gramicidin S and tyrocidine A were successfully obtained. A possible explanation to this specificity and quantitive conversion of linear precursor to cyclic product was that linear precursors of gramicidin S and tyrocidine A formed a pre-organized conformation which favored the ring closure. The CD analysis proved above hypothesis. Unfortunately, it was found that above approaches were not suitable for combinatorial synthesis due to enzyme’s substrate specificity and the enzymatic cyclization was incomplete.
Inspired by the conformation property of linear precursors of gramicidin S and tyrocidine A, a conformation-dependent chemical approach was developed and utilized to synthesize cyclic peptides. Furthermore, the alanine-scanning experiment proved this approach could be readily adapted to combinatorial synthesis of cyclic peptides. Considering former structure-activity clues, we designed and synthesized a 64-membered library based on gramicidin S scaffold using above conformation-dependent approach. 20 Compounds were selected randomly for characterizing the library. Then we examined the selectivity of these compounds through the determination of their therapeutic index (MHC/MIC). 18 Compounds were found to show apparent improvement in selectivity. In order to confirm above screening result, these hit compounds were re-synthesized and purified for selectivity determination. 4 Compounds were found to remain high antibiotic activity and at the same show greatly reduced haemolytic activity. These compounds may be used to assay against a series of pathogenic bacteria.
In summary, in this thesis, 3 concise biomimetic approaches were developed to synthesize gramicidin S and it analogues. Furthermore, a -64-membered library based on gramicidin S scaffold was successfully synthesized utilizing the conformation-dependent chemical approach. Through the determination of their therapeutic index (MHC/MIC), 4 compounds were found to show great improvement in selectivity. All these result provided lots of information for further functional optimization of cyclic peptide antibiotic gramicidin S.
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