Glycosylation And Application Of Fluorescent Probes Containing Hydroxylamine Functional Group

Carbohydrate is an important class of organic compounds widely distributed in nature and has important roles in many fields.Therefore,the detection and analysis of carbohydrates has become one of the research hotspots of chemists and biologists.High performance liquid chromatography?HPLC?is an effective method for the detection and analysis of carbohydrates.Precolumn derivatization to incorporate fluorophores into carbohydrates and then detect the derivatives by fluorescence detectors is widely used.This method has the advantages of high separation efficiency,short analysis time,high sensitivity,low detection limit,etc.It also overcomes the difficulty in the detection of carbohydrates due to lack of chromophores or fluorophores.Mass spectrometry technology also plays an important role in the structural analysis of carbohydrates.The combination of mass spectrometry and stable isotope reagents has become one of the most feasible strategies for the quantitative glycomic analysis.The combination of high-performance liquid chromatography based on precolumn fluorescence derivatization and mass spectrometry based on stable isotope labeling will greatly enhance our ability to resolve complex carbohydrates.This requires us to design and synthesize probes containing fluorophore and functional group able to react with carbohydrates.Another important property the probes must have is the introduction of stable isotope into them should be easy.Coumarin has been selected as the fluorophore in the probe because of its excellent fluorescence properties.The hydroxylamine functional group in the probe can react with the carbonyl group in the carbohydrate to form a covalent linkage.The introduction of the stable isotope is through a glycosylation modification of the coumarin ring by uniformly ¹³C-labeled glucose.In this paper,several kinds of glycosyl-modified fluorescent probes containing hydroxylamine functional groups were synthesized by two different methods:glycosidic bond formation and click chemistry.The synthesized products were characterized by NMR spectroscopy,mass spectrometry and fluorescence spectroscopy.Maltoheptaose was used to test the role of the fluorescent probes we synthesized in liquid chromatography and mass spectrometry based on precolumn fluorescence derivatization.The results showed that the synthesized probes can efficiently derivatize maltoheptaose.The derivatives can be well separted and analyzed by liquid chromatography with fluorescence detector.Mass spectrometry combined with ¹³C stable isotope labeling has also achieved the expected results,which is expected to be used in more complex structural analysis of carbohydrates.The major achievements completed are listed below.1.Two different glycosylation methods were used to introduce sugar moiety into small molecule fluorescent probe containing hydroxylamine functional groups.Most of the synthetic work in the designed route was completed,however,the removal of the protective groups on the hydroxylamine in the final step failed.Further exploration on the final step is needed.2.Click chemistry was used to synthesize small-molecular fluorescent probes containing short or long fatty chains.The probes containing long fatty chains were simultaneously labeled with ¹³C isotope.These probes were used for the structural analysis of maltoheptaose based on liquid chromatography and mass spectrometry.3.The glycosyl moiety in the fluorescent probe is further modified.We attempted to protect the hydroxyl group in the sugar moeity by the formation of methyl ether and benzyl ether,respectively.We successfully synthesized methyl ether-protected probes for liquid chromatography and mass spectrometry analysis of maltoheptaose.