The Design, Synthesis Of High-performance Boronate Ligands As Well As The Preparation And Application Of Boronate Affinity Monolithic Columns

Cis-diol biomolecules, such as carbohydrates, glycoproteins, RNA and nucleosides, are an important class of compounds, which have demonstrated important biological significance in many aspects. Many of these biomolecules are the main analytes of the frontiers of life science studies, such as glycoproteins and glycopeptides in proteomics, nucleosides in metabolomics and carbohydrates in glycomics. However, the abundance of these biomolecules in complex biological samples is usually very low while the interfering species are usually present in high abundance. So, specific capture, isolation and enrichment of the target cis-diol biomolecules are the key step in the analysis of these compounds.Boronate affinity monolithic column, which combines the advantages of monolithic column with these of boronate affinity technique, is an important tool for the specific capture and enrichment of cis-diol compounds and has developed quickly in recent years. The binding pH of usual boronate affinity monolithic column is relatively high, requiring alkaline binding pH for strong affinity. However, alkaline condition may cause the degradation of liable compounds. Our group has made many efforts to reduce the binding pH of boronate affinity technique and the binding pH has been reduced to neutral condition piror to this thesis. However, boronate affinity monolithic columns are still encountering two key issues:1) although the binding pH has been reduced to neutral condition, the existing boronate affinity monolithic columns can not function under acidic conditions. Some biological samples, such as urine and saliva, are acidic and it is impossible to directly handle these samples using the existing boronate affinity monolithic columns;2) the binding capacity of the existing boronate affinity monolithic columns are relatively low. Therefore, it is necessary to develop high performance boronate affinity monolithic columns to solve these issues. To solve these issues, we designed and synthesized high-performance boronic acid ligands and prepared novel boronate affinity monolithic columns.First, we designed and synthesized a Wulff type boronate which contained a reactive amino-group. The pKa of Wulff type boronic acid was reported to be6.7and upon immobilization onto the surface of a poly(GMA-co-PEGDA) monolithic column, the boronate affinity binding pH was reduced to5.5. The practicality of this boronate affinity monolithic column towards complex acidic samples was demonstrated by the selective extraction of modified nucleosides in an acidic urine sample.We also prepared a hydrophilic poly(GMA-co-AAM-co-MBAA) monolithic column to reduce the hydrophocity of poly(GMA-co-PEGDA) monolithic column. Upon immobilization of the Wulff type boronate on the surface of the hydrophilic monolithic column, the resulting hydrophilic boronate affinity monolithic column showed improved selectivity towards cis-diol containing biomolecules, such as adenosine because of the suppression of the non-specific interaction from hydrophobicity. The selective enrichment of glycoproteins was basically achieved on this hydrophilic monolithic column, though the tested glycoproteins were not completely captured because of the relative weakness of the specific interaction between boronate affinity monolithic column and the tested glycoproteins.Next, we designed and synthesized an electron-withdrawing group-containing Improved Wulff type boronic acid,3-carboxy-benzoboroxole, to solve the problem of low binding caparity of boronate affinity monolithic columns and the problem of weak binding strength of Wulff type boronic acid towards glycoproteins. The pKa value of the synthesized boronate ligand was about6.98. Upon the immobilization of this ligand on the surface of a hydrophilic poly(GMA-co-MBAA) monolithic column, the resulting boronate affinity monolithic column, on one hand, showed expected properties:1) improved selectivity and affinity towards cis-diol compounds as well as capacity to completely and specifically capture and enrich tested glycoproteins under neutral condition;2) dramatically improved binding capacity relative to the existing boronate affinity monolithic column. On the other hand, it also showed some properties beyond our expection:1) the binding pH was as low as5.0, this was the lowest binding pH in boronic affinity chromatography up to now;2) significant secondary separation capacity under acidic conditions towards nucleosides. These extraordinary properties of this boronate affinity monolithic column, which might mainly derive form the unique structure of the boronic acid ligand, solving the problems encountered by boronate affinity monolithic columns.Finally, to further reduce the binding pH of boronate affinity monolithic column to meet the broad pH range of urine samples (pH4.5-8.0), we designed and synthesized an electron-withdrawing group-containing Wulff-type boronic acid, since Wulff type boronic acid has lower pKa value as compared with other boronic acid ligands while an electron-withdrawing group could reduce the pKa value of the boronic acid ligands. The synthesized boronate ligand exhibited a pKa value of6.5,0.2pH unit lower than the Wulff-type boronic acid. This was due to the electron-withdrawing effect of the carboxyl group.