ManNAc- 6-phosphate

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). 

Higher organisms and some bacteria utilize PEP-dependent N-acetylneuraminic acid synthase (NeuS EC 2.5.1.56) for the generation of sialic acids and related compounds (Scheme 17.3) [17, 21]. In mammaHan cells, NeuSAc is synthesized from the phosphorylated ManNAc-6-phosphate precursor by the PEP-dependent N-acetylneuraminic acid 9-phosphate synthase (Neu9PS EC 2.5.1.57). By simultaneous release of inorganic phosphate from the enol ester upon C-C bond formation, the additions are essentially irreversible. 

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... 

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 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. 

In contrast to other monosaccharides, activated sialic acid donors are biosynthesized from A -acetylmannosamine (ManNAc) or directly from sialic acids (Sia), including A-acetylneuraminic acid (NeuAc), via a more complex pathway (21). ManNAc is phosphorylated at the at the 6-hydroxyl group and condensed with phosphoenolpyruvate to give A -acetylneuraminic acid-9-phosphate (NeuAc-9-P). Phosphate ester hydrolysis is followed by direct condensation with CTP to give CMP-NeuAc (Figure 3). Sialic acids can intercept this pathway directly via enzymatic reaction with CTP. 

In addition to using MOE to improve the presentation of the normal sugars on glycoproteins, such as the human Neu5Ac form of sialic acid derived from ManNAc feeding, this methodology can be extended to control the type of sialic acid added to recombinant therapeutics. For example, Viswanathan and co-workers showed that mannose-6-phosphate, used in place of ManNAc, allowed their glycoengineered insect cells to produce the 2-keto-... 

N-acetylneuraminate-9-phosphate synthase 4.1.3.20 ManNAc-6-P, PEP 7 — cytosol Watson etal. 1966... 

The anabolic route to NeuSAc proceeds via a N-acetyl-D-neuraminyl-9-phosphate (Neu5Ac9P) synthase which catalyzes Neu5Ac9P formation from ManNAc-6-P and PEP. The enzyme has been found in rat liver and bovine submandibular glands (Roseman et al. 1961, Warren and Felsenfeld 1961 a,b, 1962). 

Other modes of NeuSAc formation include the action of NeuSAc pyruvate-lyase in animals and bacteria, but the equilibrium of this reaction lies in the catabolic direction, and no activity was found in tissues secreting sialic acid-containing polymers (Brunetti et al. 1962). The catabolic function of the lyase is discussed below (section IV. 4). Furthermore, ManNAc can be condensed with PEP to give NeuSAc and phosphate. This reaction has been found in Neisseria meningitidis (Blacklow and Warren 1962, Brossmer et al. 1980) but never in animal tissue. 

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