| The Krebs cycle, a central metabolic pathway which is conserved in all living organisms, provides both energy and precursors for synthesis of various biomolecules. One of its key intermediates,2-oxoglutaric acid (2-OG in Scheme 1), occupies a strategically important position:it provides the carbon skeleton required for ammonium (the reduced form of nitrogen) to be assimilated into amino acids.2-OG may therefore be the link required between the carbon metabolism and nitrogen assimilation in order to properly balance the carbon/nitrogen metabolisms. Based on in vitro study,2-OG was suggested to be a metabolic signal that regulates the coordination of carbon/nitrogen metabolism in plants and bacteria. It is only recently, in collaboration with Prof. Cheng-Cai Zhang at University of Aix-Marseilleâ…¡in France, we have demonstrated for the first time in vivo that 2-OG serves as a signal molecule by using a non-metabolizable fluorinated analogue of 2-OG, DFPA (Scheme 1) as a chemical probe and a cyanobacterium Anabaena as a biological model. By employing another 2-OG analogue,2-MPA (Scheme 1), we confirmed that it is the keto form rather than the ketal form of 2-OG that plays the signaling role.In our continuing effort to investigate the signaling role of 2-OG, we attempt to further identify 2-OG receptors in order to study the corresponding signaling pathways of 2-OG. For this purpose, we propose to use affinity column and photoaffinity labeling approaches. Affinity column can be harnessed to separate and purify putative 2-OG receptors or 2-OG binding biomacromolecules, whereas photoaffinity labeling approach can be used to study the interaction between 2-OG and its corresponding receptors at the binding site. In both approaches, it is very important to have suitable affinity probes which have minimal structural deviation while retaining the biological activity of 2-OG. To design the affinity probes, it is necessary for us to have information on the structure/activity relationship of 2-OG. Therefore, the first part of my Ph.D thesis deals with the structure/activity relationship study of 2-OG, and the second part describes the design and synthesis of 2-OG probes for affinity column and photolabeling study.Four different families of 2-OG analogues (D, E, F and G in Scheme 2) have been designed and synthesized for the purpose of structure/activity relationship studies.Family DFamily EFamily FFamily G All these analogues have the vinyl group to replace the keto function of 2-OG in order to be stable against metabolization in vivo. Further structural modifications have been introduced, respectively, at the carboxylic acid functionalities (family D), at the linkage between the two carboxylic acid terminals (family E) and at the C3 or C4 position respectively (family F and G). Consequently, these analogues offer us the possibility to investigate the influence of two carboxylic acid terminals, the distance between them and structural modifications at C3 or C4 position on the corresponding signaling role of 2-OG, allowing us to perform an insightful structure/activity relationship analysis.Most probes in Scheme 2 were synthesized successfully using methods either reported in the literature or developed during my thesis, except compounds D-2 to D-4 and E-2, which are commercially available. Unfortunately, we were not able to obtain F-1 and F-2 due to the unstable intermediates.S AR study with these analogues were carried out to assess their ability to induce heterocyst formation in Anabaena, their promotion on DNA binding affinity of NtcA, a 2-OG receptor, using DNA mobility shift assay, and their cellular uptake via recognition by 2-OG permease incorporated in the cell membrane of KGTP strain using HRMAS NMR (High Resolution Magical Spinning NMR). Results from these studies showed that DF-MPA and DMPA could mimic 2-OG to play the signaling role for heterocyst formation in Anabaena. Similar to 2-OG, DF-MPA and DMPA could be recognized by 2-OG permease and efficiently taken up in KGTP strain. Furthermore, both DF-MPA and DMPA promoted the DNA binding affinity of NtcA. In addition, neither DF-MPA nor DMAP was able to induce heterocyst formation in NtcA-knock-out mutant. These findings indicated that the differentiation process triggered by DF-MPA or DMPA in Anabaena requires the normal pathway regulating heterocyst development, as in the case of 2-OG. Meanwhile, the inactive analogues hardly promoted the DNA binding affinity of NtcA, implying that they could not be recognized by NtcA due to their structural deviation, and consequently could not mimic 2-OG for in the differentiation process. Altogether, these findings suggest that limited and restricted modification could be introduced at C4 position, whereas the two carboxylic groups and their distance are very important for retaining the signaling role of 2-OG, no structural modification could be tolerated at these positions.In line with the obtained SAR results, we have designed photoaffinity probes H-1 to H-6 (Scheme 3). Probes H-1 to H-3 have the keto moiety of 2-OG replaced by the photoaffinity labeling groups, whereas H-4 to H-6 bear a vinyl group to mimic the keto function at the C2 position and a photolabeling group at the C4 position. Only probes H-1 and H-4 have been successfully synthesized, whereas the other probes could not be obtained in pure and stable form. Unfortunately, both H-1 and H-4 were not able to enhance the DNA binding of NtcA as 2-OG, probably due to the bulky azido group, and consequently they may not resemble 2-OG closely.We further designed probes H-7 and H-8 as affinity tags for affinity column (Scheme 4). Both H-7 and H-8 contain a structural motif of DMPA to mimic 2-OG for binding with 2-OG receptors, a terminal amine functionality for attachment on the resin of affinity column, and a linker bridging the above two entities with a reasonable distance. H-7 has a hydrophobic linker, whereas H-8 has a hydrophilic linker. Both of them have been successfully synthesized and attached to the solid affinity matrix. They are ready for further use in the separation and purification of 2-OG receptors. In summary, various 2-OG analogues were designed and synthesized, and a study on structure/activity relationship has been carried out. Among them, DM-FPA and DMPA could mimic 2-OG for its signaling role in heterocyst formation in Anabaena, similarly as previously developed non-metabolizable 2-OG analogues, DFPA and 2-MPA. The structural scaffold of DMPA was further used for the design of affinity probes and photoaffinity probes. Two photoaffinity probes bearing azido functionality were obtained, and two further affinity probes were synthesized and attached covalently to the affinity column resin, which are ready for separation and purification of 2-OG receptors. Their utility in identifying 2-OG receptors and studying the 2-OG signaling pathways is under the way.
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