A method for quantitative analysis of monosaccharides including N-acetylneuraminic acid derived from sialic acid-containing oligosaccharides and glycoproteins is presented. The analysis is based on the combination of chemical and enzymatic methods coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. The present method utilizes a simplified acid hydrolysis procedure consisting of mild hydrolysis (0.1 M TFA) to release sialic acid and strong acid hydrolysis (2.0 N TFA) to produce amino and neutral sugars. Amino sugars released from strong acid hydrolysis of oligosaccharides and glycoproteins were reacetylated and derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) along with neutral sugars in the presence of sodium cyanoborohydride to yield quantitatively the highly stable fluorescent APTS adducts. N-acetylneuraminic acid (Neu5Ac), a major component of most mammalian glycoproteins, was converted in a fast specific reaction by the action of neuraminic acid aldolase (N-acylneuraminate pyruvate-lyase EC 4.1.3.3) to N-acetylmannosamine (ManNAc) and pyruvate. ManNAc was then derivatized with APTS in the same manner as the other monosaccharides. This method was demonstrated for the quantitation of pure Neu5Ac and the species derived from mild acid hydrolysis of 6'-sialyl-N-acetyllactosamine and bovine fetuin glycan. Quantitative recovery of the N-acetylmannosamine was obtained from a known amount of Neu5Ac in a mixture of seven other monosaccharides or from the sialylated oligosaccharides occurring in glycoproteins. The sequence of procedures consists of acid hydrolysis, enzymatic conversion and APTS derivatization which produced quantitative recovery of APTS-monosaccharide adducts. The detection limits for sugars derivatized with APTS and detected by CE-LIF are 100 pmol for Neu5Ac and 50 pmol for the other sugars.
The 2 and E isomers of N-methyl-a-(p-tolyl)-or-phenyl nitrone (7a and 8a) and the Z and E isomers of Nbenzyl-a-(p-tolyl)-a-phenyl nitrone (sa and 10) were prepared. Alkylations of isomeric oxime anions and the reaction of ,V-benzylhydroxylamine (13) with 1,l-dichloro-4'-methyldiphenylmethane (14) were employed in the syntheses. The first-order rates for thermal approach to geometric equilibrium of 7a, Sa, 9a, and 10 were determined in degassed tert-butyl alcohol solutions. Activation parameters for the isomerization of 9a were determined in the same solvent and are AE * = 33.6 i 1.4 kcal/mol; A S * = -4 & 4 eu. The energy barrier to isomerization is substantially larger than would be anticipated from the limited data available from previous studies of geometric isomerizations of nitrones. These results are briefly discussed.
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