ManNAc

N-Acetylneuraminic acid aldolase (or sialic acid aldolase, NeuA EC 4.1.3.3) catalyzes the reversible addition of pyruvate (2) to N-acetyl-D-mannosamine (ManNAc (1)) in the degradation of the parent sialic acid (3) (Figure 10.4). The NeuA lyases found in both bacteria and animals are type I enzymes that form a Schiff base/enamine intermediate with pyruvate and promote a si-face attack to the aldehyde carbonyl group with formation of a (4S) configured stereocenter. The enzyme is commercially available and it has a broad pH optimum around 7.5 and useful stability in solution at ambient temperature [36]. 

Scheme 2.7 Aldol reaction of ManNAc analogues and sodium pyruvate to produce sialic acid, catalyzed by A-acetylneuraminic acid (NANA) aldolase.

Synthetic studies for sialic acid and its modifications have extensively used the catabolic enzyme N-acetylneuraminic acid aldolase (NeuA E.C. 4.1.3.3), which catalyzes the reversible addition of pyruvate (70) to N-acetyl-D-mannosamine (ManNAc, 11) to form the parent sialic acid N-acetylneuraminic acid (NeuSNAc, 12 Scheme 2.2.5.23) [1, 2, 45]. In contrast, the N-acetylneuraminic acid synthase (NeuS E.C. 4.1.3.19) has practically been ignored, although it holds considerable synthetic potential in that the enzyme utilizes phosphoenolpyruvate (PEP, 71) as a preformed enol nucleophile from which release of inorganic phosphate during... 

The occurrence of phosphoric ester groups at 0-9 is long established, as Neu5Ac9P was recognized as the condensation product of enolpyruvate phosphate (PEP) and ManNAc 6-phosphate in the biosynthetic pathway of sialic acids (see Section V,l). 

The pathway of the biosynthesis of Neu5Ac demonstrates the origin of sialic acids from the cellular hexose and hexosamine pools. These sugars are, therefore, suitable components for the study of the biosynthesis of sialic acid. However, only ManNAc has been shown to be a relatively specific precursor of sialic acids, as may be seen from the distribution of radioactivity between the individual monosaccharides of glycoconjugates after incubation. Injections of radioactive ManNAc into animals, or incubation of surviving tissue slices or individual cells with this compound, give incorporation of label mainly into the sialic acids.226 227... 

That secretion of 2-acetamidoglucal, which is known to be an intermediate in the complex, 2-epimerase reaction, but cannot be isolated under normal conditions, may point to another defect in this enzyme that may be independent of the presence or absence of a feedback-inhibition receptor site for CMP-Neu5Ac has been discussed.233 An excess of 2-acetamidoglucal may be converted spontaneously into GlcNAc and this, enzymically, into ManNAc both compounds are secreted in the urine of the sialuria patient. As a consequence of these reactions, the cellular concentration of ManNAc may increase to a level leading to the synthesis of additional Neu5Ac from this compound by the action of acylneuraminate pyruvate-lyase. All of these reactions, and known or theoretical interactions, were summarized in Fig. 3 of Ref. 233. 

Nonphysiological compounds have also been described as influencing the overall metabolism of sialic acid. Administration of ethanol (2 g/kg) to rats significantly decreases the sialic acid content of brain tissue.246 Convulsions induced by pentylenetetrazole (6,7,8,9-tetrahy-dro-5/f-tetrazoloazepine) are accompanied by a diminution in the rate of biosynthesis of polysialogangliosicles GT, and GQn, in rat brain.227 Such ManNAc analogs as 2-acetamido-l,3,4,6-tetra-0-acetyl-2-deoxy-D-mannopyranose or the 2-(trifluoroacetamido) derivative lead to a marked lowering of the incorporation of radioactivity from labelled ManNAc into glycoconjugate sialic acids of murine, erythroleukemia (Friend) cells.247... 

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...

The enzymatic preparation of the activated sugar nucleotide may also involve a cofactor regeneration system. An example of this is an economic one-pot procedure, in which N-acetylneuraminic acid (NeuAc) is generated in situ from IV-acetylmannosamine (ManNac) and pyruvate with sialic acid aldolase and then converted irreversibly to CMP-NeuAc ([14], see also Sec. III). 

In this one-pot procedure NeuAc 16 is generated from ManNAc 15 and pyruvic acid in situ with sialic acid aldolase and then converted irreversibly to CMP-NeuAc 17. CMP is converted to CDP with myokinase and ATP. The released ADP is converted to ATP with pyruvate kinase and PEP. CDP is then converted to CTP also with pyruvate kinase and phosphoenolpyruvate (PEP). The formed CTP reacts with NeuAc catalyzed by NeuAc synthetase to give 17. 

To HEPES buffer (100 mL, 200 mM, pH 7.5) were added ManNAc 15 (1.44 g, 6 mmol), PEP sodium salt (1.88 g, 8 mmol), pyruvic acid sodium salt (1.32 g, 12 mmol), CMP (0.64 g, 2 mmol), ATP (11 mg, 0.02 mmol), pyruvate kinase (300 U), myokinase (750 U), inorganic pyrophosphatase (3 U), /V-acctylneuraminic acid aldolase (100 U), and CMP-sialic acid synthetase (1.6 U). The reaction mixture was stirred at room temperature for 2 days under argon, until CMP was consumed. The reaction mixture was concentrated by lyophilization and directly applied to a Bio-Gel P-2 column (200-400 mesh, 3 x 90 cm), and eluted with water at a flow rate of 9 mL/h at 4°C. The CMP-NeuAc fractions were pooled, applied to Dowex-1 (formate form), and eluted with an ammonium bicarbonate gradient (0.1-0.5 M). The CMP-NeuAc fractions free of the nucleotides were pooled and lyophilized. Excess ammonium bicarbonate was removed by addition of Dowex 50W-X8 (H+ form) to the stirred solution of the residual powder until pH 7.5. The resin was filtered off and the filtrate was lyophilized to yield the ammonium salt of CMP-NeuAc 17 (1.28 g, 88%). 

UDP-GlcNAc can be converted to UDP-N-acetyl-mannosamine (UDP-ManNAc) with concurrent elimination of UDP (Eq. 20-7). , 7b This unusual epimeri-zation occurs without creation of an adjacent carbonyl group that would activate the 2-H for removal as a proton. As indicated by the small arrows in Eq. 20-7, step a, the UDP is evidently eliminated. In a bacterial enzyme it remains in the E-S complex and is returned after a conformational change involving the acetamido group. This allows the transient C1-C2 double bond to be protonated from the opposite side (Eq. 20-7, step a).47 In bacteria the UDP-ManNAc may be dehydrogenated to UDP-N-acetylmannos-aminuronic acid (ManNAcA). Both ManNAc and ManNAcA are components of bacterial capsules.47... 

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 six-carbon chain of ManNAc 6-P can be extended by three carbon atoms using an aldol-type condensation with a three-carbon fragment from PEP (Eq. 20-7, step c) to give N-acetylneuraminic acid (sialic acid).48 Tire nine-carbon chain of this molecule can cyclize to form a pair of anomers with 6-membered rings as shown in Eq. 20-7. In a similar manner, arabi-nose 5-P is converted to the 8-carbon 3-deoxy-D-manno-octulosonic acid (KDO) (Fig. 4-15), a component of the lipopolysaccharide of gram-negative bacteria (Fig. 8-30), and D-Erythrose 4-P is converted to 3-deoxy-D-arafrmo-heptulosonate 7-P, the first metabolite in the shikimate pathway of aromatic synthesis (Fig. 25-1).48a The arabinose-P used for KDO synthesis is formed by isomerization of D-ribulose 5-P from the pentose phosphate pathway, and erythrose 4-P arises from the same pathway. 

It seems possible that a block mechanism also operates in the assembly of the chain of polysaccharide 42, present in the cell wall of Bacillus cereus.124 In this case, formation of the polymer from a-GlcNAc-pp-Bpr, UDP-ManNAc, UDP-GalNAc, and UDP-Glc was observed.74... 

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