Epidoramectin Monosaccharide

Saccharides form one of the major constituents of biological macromolecules in living organisms. Many biological processes including protein folding, stability, immune response and receptor activation are regulated by glycosylation. In this work, we optimized a capillary electrophoresis method with capacitively coupled contactless conductivity detection for the separation of eight monosaccharides commonly found in glycoproteins, namely D-glucose, D-galactose, D-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-fucose, N-acetylneuraminic acid, and D-xylose. A highly alkaline solution of 50 mM sodium hydroxide, 22.5 mM disodium phosphate, and 0.2 mM CTAB (pH 12.4) was used as a background electrolyte in a 10 µm id capillary. To achieve baseline separation of all analytes, a counter-directional pressure of -270 kPa was applied during the separation. The limits of detection of our method were below 7 µg/ml (i.e., 1.5 pg or 1 mg/g protein) and the limits of quantification were below 22 µg/ml (i.e., 5 pg or 3 mg/g protein). As a proof of concept of our methodology, we performed an analysis of monosaccharides released from fetuin glycoprotein by acid hydrolysis. The results show that, when combined with an appropriate pre-concentration technique, the developed method can be used as a monosaccharide profiling tool in glycoproteomics and complement the routinely used LC-MS/MS analysis.

Monosaccharides play important roles in living organisms. They are present in essential glycoproteins, nucleic acids, and glycolipids as well as cell walls and bioactive natural product glycosides and polysaccharides. Monosaccharides are optically active, and as a routine, scientists make sure that their absolute configurations are determined when new natural glycosides are isolated. Many determination methods for the absolute configuration of monosaccharides have been reported, and thus far, taking advantage of their optical rotation differences is the most used and efficient method to distinguish enantiomers. This method, however, is not very convenient, because it requires a milligram amount of each pure sample and the availability of a polarimeter. Identification methods dealing with comparison of the retention times of the d- and l-diastereomeric monosaccharide derivatives by GC, TLC Rf values, HPLC, or UPLC have been also reported. Although effective, these methods still require sample preparation and a few milligrams of the test compounds. A new method with simple sample preparation to distinguish enantiomers of monosaccharides by analyzing the 1H NMR spectra of their diastereomeric derivatives has been developed. The monosaccharide components of a commercially available saponin-rich Panax ginseng and monoglycosides have been successfully identified using this procedure.

Saccharides play critical roles in many forms of cellular activities. Saccharide structures are however complicated and similar, setting a technical hurdle for direct identification. Nanopores, which are emerging single molecule tools sensitive to minor structural differences between analytes, can be engineered to identity saccharides. A hetero-octameric Mycobacterium smegmatis porin A nanopore containing a phenylboronic acid was prepared, and was able to clearly identify nine monosaccharide types, including D-fructose, D-galactose, D-mannose, D-glucose, L-sorbose, D-ribose, D-xylose, L-rhamnose and N-acetyl-D-galactosamine. Minor structural differences between saccharide epimers can also be distinguished. To assist automatic event classification, a machine learning algorithm was developed, with which a general accuracy score of 0.96 was achieved. This sensing strategy is generally suitable for other saccharide types and may bring new insights to nanopore saccharide sequencing.