Studies On The Synthesis Of Phosphorylated Aromatic Compounds And Non-covalent Complexes Formed By Phosphorylated Flavone-Protein Interactions

It is an established fact that esters of phosphoric acid have wide bio-activities and play a vital role in many biological processes. Cellular functions are often triggered by weak non-covalent enzyme-substrate, protein-ligand, protein-protein or antibody-antigen interactions. Since its introduction, ESI-MS has served a powerful tool in providing evidence in support of the existence of non-covalently associated marcromolecular complexes in the gas phase. In the study described in this thesis, aminophenols and dihydroxybenzenes were first phosphorylated by modification of the classical Atheron-Todd procedure in our primary stage; Then the mechanism of the reaction between salicylic acid and dialkyl phosphite was studied by electrospray ionozation mass spectrometry and 3lP NMR; 4-aminoantipine was further phosphorylated by modification of the classical Atheron-Todd procedure; finally, we have been able to enlarge the scope of the Atheron-Todd reaction to flavone and isoflavone phosphorylation. Electrospray ionization results showed that the phosphorylated flavonoids could form non-covalent complexes with many proteins such as lysozyme, myoglobin, bovine insulin and cytochrome c, while non-covalent complexes were not detected with the mixed solution of the chrysin and proteins. The phosphorylated flavonoids possess relatively stronger affinities and form non-covalent complexes with the proteins more easily than the unphosphorylated compounds.1).Aminophenols and dihydroxybenzenes were phosphorylated by modification of the classical Atheron-Todd procedure. Their structures were elucidated by NMR and ESI-MS/MS. Results showed that the cleavage pathways of these phosphorylated compounds were closely related to the relative positions of the phorsporylating groups. Comparatively stable five-membered ring ions were produced when the two phosphorylated functional groups were in the ortho position. These stable five-membered ring ions can be considered as indicators for ortho functional groups of phenols.2).When the Atheron-Todd reaction was employed to phosphorylate salicylicacid, the yield was quite low. But further experiments show that the Atheron-Todd reaction could successfully be applied to phenol and ethyl-salicylate phosphorylation. The reaction between salicylic acid and dialkyl phosphite was then traced by electrospray ionozation mass spectrometry and 31P NMR. The mechanism proposed based on ESI-MS results and 3IP NMR profiles. The results show that both the -COOH and -OH of salicylic acid were phosphorylated, but the -COOH possessed stronger reaction ability from the onset. Finally, most of the DIPPH was translated into tetraisopropyl pyrophosphate through reacting with -COOH. The energy-rich carboxylic-phosphoric mixed anhydrides played important roles in the whole process.3).4-aminoantipine was phosphorylated by our modified classical Atheron-Todd procedure. Their structures were elucidated by NMR, ESI-MS and x-ray data.4).We have been able to enlarge the scope of the Atheron-Todd reaction to flavone phosphorylation. The procedures are practical. 7-hydroxyflvone, chrysin, 4'-chloro-7-hydroxyisoflavone and 4'-fluo-7-hydroxy-isoflavone were phosphorylated by the Atheron-Todd reaction. The expected phosphates were obtained in good yields. All the structures of target products were determined by ESI-MS/MS, NMR and IR for the first time. The structures of three phosphated fiavones were further confirmed by X-ray diffraction.5).The mixed solutions of the five phosphorylated flavonoids and different proteins, such as insulin, lysozyme and cytochrome c were detected by ESI-MS. The results show that all the phosphorylated flavonoids could form non-covalent complexes with proteins mentioned above, while noncovalent complexes were not detected from the mixed solution of the chrysin or 7-hydroxyflavone with proteins. The phosphorylated flavonoids possess relatively stronger affinities and form non-covalent complexes with the proteins more easily than the unphosphorylated compounds. The protonated basic amino...