Acetylated amino sugars

It will be appropriate to consider first the building units of the carbohydrate residues and it will be immediately apparent that D-mannose and D-galactose with N-acetyl-amino sugars and hexuronic acids play a predominant role. It is proposed, therefore, to discuss briefly some of the more important discoveries connected with the two latter groups and with the carbohydrate sulfates. 

Synthesis of GAGs requires the sequential attachment of an jV-acetyl amino sugar or uronic acid to the protein core molecule (Figure 9.2), each step being catalysed by a... 

The acetylated amino sugars N-acetyl-D-glucosamine and N-acetyl-D-Galactosamine (4) are often encountered as components of glycoproteins. 

Table 4 Results for the aqueous condensation of Ai-acetylated amino sugars with 2,4-pentanedione (1) in the presence of NaHCOa at 90° C...

Acetamidodeoxyhexoses. A further modification of the 4-keto-inter-mediate has been independently shown by Ashwell and by Strominger and associates (Table I, References 20, 21, 22, 23). Transamination reactions with L-glutamate as the amino donor and pyridoxal phosphate as coenzyme led to formation of 3-amino 3,6-dideoxy- and 4-amino 4,6-dideoxyhexoses, respectively. Acetylation with acetyl coenzyme A yields the naturally-occurring N-acetyl amino sugar derivatives. 

Aldonic, uronic, and ascorbic acids, lactones, and N-acetylated amino sugars were separated on sulfonated polystyrene-divinylbenzene, a strong polyanion exchanger (Wheaton and Bauman, 1953). This method is adaptable to neutral carbohydrates without complexation or adsorption, by immersion in strong alkali to ionize the hydroxyl groups (ion chromatography). 

Shown in Fig. 3.3 (C) is the N-NMR spectrum ofHMWDOM isolated from a depth of 15 m near Hawaii. The one major resonance in the spectrum corresponds to amideN (124 ppm >90% ofthe total N). Surface water samples sometimes show a minor resonance near 24 ppm (<10% of the total N), which corresponds to amine N. Amides are commonly present in biological systems within peptide bonds. Other amide containing biochemicals that may be common in oceanic environments include N-acetyl amino sugars such as Af-acetyl glucosamine, iV-acetyl muramic... 

Figure 6 NMR spectra of HMW DOM from surface seawater, (a) C-NMR spectra can be used to quantitatively determine the functional groups, and by inference, the relative importance of different biochemical classes in HMW DOM. The spectra highlight the importance of carbohydrates (100 ppm and 70 ppm), carboxylic acids (175 ppm), and alkyl carbon (10-30 ppm), (b) H-NMR also show the importance of carbohydrates (4 ppm) and alkyl carbon (1 ppm), but additionally show that acetyl groups most likely bound to carbohydrate are an important components. N-NMR show that 80-90% of HMW DON is amide, while 10-20% is free amine. Quantitative analyses for acetate and nitrogen suggest that most amide in surface seawater is bound as A-acetyl amino sugars and protein residues. In the deep ocean HMW DOM however, most amide is nonhydrolyzable, and is of unknown molecular environment.

The C-NMR spectmm of HMW DOM also includes contributions from carboxyl (CO-(OH or NH), 5% of total carbon), and alkyl (CH 14% total carbon) functional groups, which may derive from proteins, lipids, or carbohydrates (deoxy-and methyl sugars). Hydrolysis of HMW DOM followed by extraction with organic solvent yields 4-8% of the total carbon in HMW DOM as acetic acid. Acetyl is easily recognized in the H-NMR spectra of HMW DOM, where it appears as a broad singlet centered at 2 ppm (Figure 6(b)). Free acetic acid and its derivatives are not retained by ultrafiltration, and the acetyl in HMW DOM must be covalently bound to macromolecular material, most likely as an A-acetyl amino sugar. Acetyl contributes up to half the carboxyl carbon in the C-NMR spectrum. 

Several important biochemicals are amides, of which proteins and A -acetyl amino sugars are the most abundant in marine organisms, where proteins contribute up to 80%, and amino sugars up to 10% or the total nitrogen. Molecular-level analyses show both these biochemical classes to be present in HMW DON, but only at low concentrations. For most samples, amino acids account for <15-20% of HMW DON, and aminosugars <1 % of the HMW DON (McCarthy et al., 1996 Kaiser and Benner, 2000 Mannino and Harvey, 2000). Either HMW DON amide is derived from some other component, or the analytical protocols used in molecular-level analyses are not appropriate for measuring these biochemicals in HMW DON. 

The discrepancy between molecular-level and NMR measurements of amide nitrogen can best be resolved by using the two techniques interactively. Proteins and N-acetyl amino sugars both yield amino compounds (free amino acids and amino sugars, respectively) on treatment with acid (Figure 8). Therefore, acid hydrolysis of HMW DON should be accompanied by a change in the N-NMR chemical shift from amide to... 

Figure 8 The effect of mild acid hydrolysis on amides in HMW DOM. Two potentially important classes of biochemicals that likely contribute to HMW DOM are (poly)-N-acetyl amino sugars (top) and proteins (bottom). Mild acid hydrolysis of (poly)-iV-acetyl amino sugars will yield free acetic acid, but will not depolymerize the polysaccharide. The generation of acetic acid will be accompanied by a shift in the N-NMR from amide to amine. In contrast, mild acid hydrolysis of proteins does not yield acetic acid, but may depolymerize the protein macromolecular segments to yield free amino acids. Free amino acids can be quantified by chromatographic techniques and compared to the shift from amide (protein) to amine (free amino acid) in N-NMR.

Figure 9 The effect of mild acid hydrolysis on N-NMR of HMW DOM. Nitrogen in HMW DOM is primarily amide (180 ppm), with smaller amounts of free amine (90 ppm). Treatment of HMW DOM with dilute hydrochloric acid increases the amount of amine and decreases the amount of amide. The decrease in amide equals the amount of acetic acid and amino acids released by hydrolysis of poly-N acetyl amino sugars and proteins. The relative amount of protein and amino sugar can be determined by the ratio of acetic acid to amino acids in the hydrolysis product.

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