Abstract
A mild methylation method is detailed in this work. It uses 1N NaOH to deprotonate primary and secondary hydroxyl groups for short time. Excess 1N NH4OH (1.00 ml, pH 11.4) is added, followed by NaHCO3 to make the carbonate esters by nucleophilic substitution of the deprotonated primary and secondary OH groups. The reaction conditions include; ambient temperature and for an appropriate time. Then NaBH4 is added and the reaction conditions are; standing at ambient temperature for at least 4 hours. This converts the carbonate esters to the methyl ethers. Work-up includes transferring the methylated switchgrass leaf section to a vial containing 1N HCl (1.00 ml). The leaf needs to be submerged in the acid. The reaction conditions include; heating the reaction vessel at 72°C, for 4 days. The contents of the vial are reduced in volume to syrup with a stream of air. The mixture dissolves completely in methanol. Mass spectrometry is done on a methanol solution in the negative ion ESI mode. The mass spectral interpretation reveals the presence of methylated glucan amimo acids and glucan dipeptides. The molecules identified were per-O-methylated glucans linked to either serine or asparagine through a di-(hydrido) di-phospho di-hydratephe moeity. It is possible that one ion is derived from a phosphorylated tyrosine linked to either the serine or asparagine linked to an originally di-phospho cellulo-glucan.
Highlights
Methylation methods have been used to determine the position of substitution for oligosaccharides and polysaccharides. [1, 2] the conditions of most methods require the use of strong base, to ionize methylatable hydroxyl groups on secondary and primary alcohols
[4] This is done by making the carbonate esters and treating the newly formed carbonate ester with NaBH4 in NH4OH
[6] These reaction conditions allow us to isolate glycan-protein linkages for cellulose and their submission to mass spectral analysis and interpretation. [7,8,9,10] We show support for the phosphorylation of tyrosine linked to serine or asparagine to cellulo-glucan. [10,11,12,13] There are three bovine glycan-peptide linkages that we have isolated via this method. [14,15,16]
Summary
Methylation methods have been used to determine the position of substitution for oligosaccharides and polysaccharides. [1, 2] the conditions of most methods require the use of strong base, to ionize methylatable hydroxyl groups on secondary and primary alcohols. Methylation methods have been used to determine the position of substitution for oligosaccharides and polysaccharides. [1, 2] the conditions of most methods require the use of strong base, to ionize methylatable hydroxyl groups on secondary and primary alcohols. Strong base will cause degradation of carbohydrates. The method proposed here uses the reduction of carbonate esters to ethers. [5] The reduction of sulfate and phosphate esters is accomplished via this method, as well. [6] These reaction conditions allow us to isolate glycan-protein linkages for cellulose and their submission to mass spectral analysis and interpretation. [10,11,12,13] There are three bovine glycan-peptide linkages that we have isolated via this method. The method proposed here uses the reduction of carbonate esters to ethers. [4] This is done by making the carbonate esters and treating the newly formed carbonate ester with NaBH4 in NH4OH. [5] The reduction of sulfate and phosphate esters is accomplished via this method, as well. [6] These reaction conditions allow us to isolate glycan-protein linkages for cellulose and their submission to mass spectral analysis and interpretation. [7,8,9,10] We show support for the phosphorylation of tyrosine linked to serine or asparagine to cellulo-glucan. [10,11,12,13] There are three bovine glycan-peptide linkages that we have isolated via this method. [14,15,16]
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