N-acetylmannosamine

Figure 6. Synthesis of 9-0-acetyl-N-acetylneuraminic acid. The aldol acceptor was prepared from N-acetylmannosamine and isopropenyl acetate in DMF catalyzed by protease N obtained from Amano. The aldol condensation was carried out by using N-acetylneuraminic acid aldolase as catalyst.

Fig. 4.3.2 Human sialic acid metabolism and genetic defects. -6P -6-Phosphate, -9P -9-phos-phate, CMP cytidine 5 -monophosphate, CTP cytidine 5 -triphosphate, UDP-GlcNAc uridine diphosphate-N-acetyl-D-glucosamine, ManNAc N-acetylmannosamine, NeuAc N-acetylneur-aminic acid, OGS oligosaccharides, SASD sialic acid storage disease...

O-Acetyl-N-acetylmannosamine 6, prepared from Af-acetylmannosamine either by chemical acetylation [28] or by transesterification catalyzed by subtilisin [31], led to 9-0-acetyl Neu5Ac 7 [29], a receptor of influenza C virus occurring on human erythrocytes. Several other 9-O-substituted Neu5Ac derivatives could also be prepared [29-32]. 

In mammals the epimerase (Eq. 20-7, step a) probably utilizes a similar chemical mechanism but eliminates UDP and replaces it with HO" to give free N-acetylmannosamine, which is then phosphor-ylated on the 6-hydroxyl (Eq. 20-7, step b). ManNAc may also be formed from free GlcNAc by another 2-epimerase (step a").47C/d... 

The aldol condensation of the hexosamine and pyruvic acid was shown to have been preceded by a 2-epimerization of N-acetylglucosa-mine to N-acetylmannosamine when Roseman and Comb (25) identified... 

Radiolabeling of Brain Glycolipids. Thy-1 glycolipid was labeled biosynthetically using a previously described method (7). Five to seven day mice of either AKR/J (H-2, Thy-1.1) or ICR Swiss (Thy-1.2) strain mice were used for each preparation. Each mouse pup was injected intracranially with 8 pi of sterile saline solution containing 5 pci [1- 4C]-N-acetylmannosamine (ManNAc) (54.5 mCi/mmol, New England Nuclear, Boston MA). This solution was injected into both sides of the head at a point 1-2 mm anter-... 

N-acetylmannosamine. Cells were cultured at 37°C in flasks at a concentration of 5 x 10 cells/ml. The cells reached a concentration of 2 x 106 cells/ml after about 48 hours and were washed once with phosphate buffered saline (PBS) and the pellet collected by centrifugation was extracted as described below. 

This problem was approached by incorporating radioactive percur-sors into the glycolipids of both brain and lymphoma cells of the Thy-1.2 and 1.1 types. We have used C-palmitate as a per-cursor of ceramide, and - C-N-acetylmannosamine as a percursor of sialic acid (7). Glycolipids were isolated and the radioactive gangliosides were resolved by two-dimensional thin layer chromatography in two different solvent systems followed by autoradiography. 

N-acetylglucosamine (see Chapter 9) is a component of glycoproteins, connective tissue proteoglycans, and complex lipids. It may be synthesized in the human organism from fructose-6-phosphate, as indicated in Figure 18.17. N-acetylglucosamine is also a precursor of N-acetylmannosamine, which along with pyruvic acid participates in the biosynthesis of sialic acid. 

In gangliosides, the most complex sphingolipids, an oligosaccharide chain attached to the ceramide contains at least one acidic sugar. The acidic sugar is H-acetylneuraminate or -glycolylneuraminate. These acidic sugars are called sialic acids. Their nine-carbon backbones are synthesized from phosphoenolpyruvate (a three-carbon unit) and N-acetylmannosamine 6-phosphate (a six-carbon unit). 

Neu5Ac aldolase has also been used for the synthesis of 3-deoxy-D-mfl7j jo-octulosonic acid (KDO, 17) [16,17,20]. Condensation of o-arabinose (18) with pyruvate gave a mixture of KDO and 4-epi-KDO (19). Wong and coworkers have since reported the isolation of a KDO aldolase which produces KDO with complete stereospecificity at C-4 and also accepts a wide variety of carbohydrate substrates [51]. N-Acetylneuraminate synthase, found in Neisseria meningitidis, has been used to catalyse the condensation of 6-azido-6-deoxy-N-acetylmannosamine with phosphoenolpyruvate to give 9-azido-9-deoxy-Neu5Ac [52]. 

Natural sialic acids (Schauer 1982 1991) are derivatives of 5-amino-3,5-dideoxy- D-glycero-D-galacto-nonu osonic acid 12.1. This awkward name has been replaced by neuraminic acid . The most common derivative is N-acetyl-neuraminic acid 12.2 whose configuration is easy to memorize because, in the Fischer representation, 12.3, it is presented as an aldolic condensation product of N-acetylmannosamine (2-acetamido-2-deoxy-D-mannose) and pyruvic acid. When the expression sialic acid is used without any other precision, it is in reference to derivative 12.2. It exists in the free state or glycosidated in the d-conformation, which allows an equatorial disposition of the three-carbon side chain. Structure 12.2 represents the stable /3-anomer of the free sugar with an axial anomeric hydroxyl group and all-equatorial non-anomeric substituents. An X-ray spectrum of this crystallized /3- anomer confirms this conformation and reveals, moreover, that the side chain has the zig-zag conformation with two... 

Sialic acid aldolase catalyzes the condensation of pyruvate and N-acetylmannosamine to form sialic acid, an acidic sugar involved in a number of biochemical recognition processes. Like other aldolases, sialic acid aldolase accepts a number of aldoses as substrates (12,13). Mannose, 2-deoxyglucose, and many 6-substituted or 6-modified mannose or N-acetylmannosamine, for example, are good substrates for the enzyme. We have prepared 9-deoxy-9-fluorosialic acid and a 7,9-difluoroderivative of sialic acid using sialic acid aldolase as catalyst (Figure 4). In an attempt to prepare 3-deoxy-3-fluorosialic acid, it was found that 3-fluoropyruvate is not a substrate for the enzyme. 

Figure 7.20. The glycal mechanism for the conversion of UDP-N-acetylglucosamine (UDP-GIcNAc) to UDP-N-acetylmannosamine (UDP-ManNAc) catalyzed by UDP-CIcNAc epimerase [109],...

N-Acetylneuraminic acid aldolase catalyzes the cleavage of N-acetylneuraminic acid (Neu5Ac) to N-acetylmannosamine (ManNAc) and pyruvate (Pyr). The reverse reaction can be employed to synthesize N-acetylneuraminic acid, which plays an important physiological role as a terminal sugar residue of glycosylated proteins 1481 (Eq. (3)). 

CMP-N-acetylneuraminic acid (CMP-NeuAc). CMP-N-acetylneuraminic acid has been prepared enzymatically on small scales (> 0.5 mmol) from CTP and NeuAc, under catalysis by CMP-NeuAc synthetase (EC 2.7.7.43) [131l An improvement in this procedure, involving in situ production of CTP from CMP under adenylate kinase and pyruvate kinase catalysis, is suitable for multigram-scale synthesis11321. Adenylate kinase catalyzes the equilibration of CTP and CMP to CDP, which is subsequently phosphorylated by pyruvate kinase to provide CTP. A one-pot synthesis of CMP-NeuAc based on this procedure involves the in situ synthesis of NeuAc from N-acetylmannosamine and pyruvate, catalyzed by sialic acid aldolase (Fig. 11.3-12)[10S1. Chemical syntheses of CMP-NeuAc have also been reported11421. 

NeuAc aldolase, or sialic acid aldolase, catalyzes the reversible condensation of pyruvate with N-acetylmannosamine (ManNAc) to form NeuAc (sialic acid N-acetyl-5-amino-3,5-dideoxy-D-g ycero-D-ga acto-2-nonulopyranosonic acid) (Fig. 14.1-... 

The synthesis of NeuAc in vivo is accomplished using NeuAc synthetase11111. This aldolase catalyzes the irreversible condensation of PEP and N-acetylmannosamine. Although this enzyme has not yet been isolated and characterized, it may prove synthetically useful in the future. 

N-Acetylglycosaminyl transferase 924 N-Acetylmannosamine 1047 N-Acetylmuramic acid 938 N-Acetylneuramic acid 811, 1047 N-Acetylneuraminic acid 772 Acetylphosphate 1075 Acetylcysteine... 

N-Acetylneuraminic acid aldolase catalyzes the reversible aldol condensation of pyruvate (23) and N-acetylmannosamine (22 ManNAc) to form N-acetylneuraminic acid (24 NeuAc N-acetyl-5-amino-3,5-dideoxy-D-glycerogalacto-2-nonulopyronic acid Scheme 6).80-83 In vivo the enzyme has a catabolic function and the equilibrium for this reaction is near unity the presence of excess pyruvate can shift this equilibrium. NeuAc and other derivatives of neuraminic acid are termed sialic acids. These compounds are found at the termini of mammalian glycoconjugates and play an important role in cellular recognition.84-89 The production of analogs of NeuAc is a point of great interest to synthetic and medicinal chemists. The enzymatic approach has not been fully explored but it may be a practical alternative to the chemical synthesis of certain sialic acids.89... 

The incorporation of new functional groups can also be accomplished using the metabolic machinery for posttranslational protein modifications. These methods rely on the ability of some modification enzymes to process and install analogs of their natural substrates containing reactive handles of interest. In an early demonstration of this technique, it was shown that derivatives of N-acetylmannosamine 40a bearing ketones 40b) [62] or azides 40c [63] in the acyl moiety are tolerated by enzymatic pathways that produce sialic acid. By feeding these unnatural building blocks to cell cultures,... 

The use of this reaction in the biological context was first demonstrated for the chemospecific labeling of Jurkat cell surfaces [63]. Metabolic engineering with N-acetylmannosamine derivative 40c was used to incorporate azides into sialic acid groups on cell surfaces. The cells were then incubated with biotinylated phosphine 49, and the extent of the reaction was quantified by flow cytometry after treatment with fluorescent avidin. Importantly, neither the azide nor the phosphine displayed any reactivity with the cell-surface groups in the absence of its reactive partner. In addition, the cells showed unchanged growth rates after modification. 

---