A-Acetylmannosamine

This enzyme [EC 4.1.3.3], also known as A-acetylneu-raminate aldolase, will convert A-acetylneuraminate to A-acetylmannosamine and pyruvate. The enzyme will also act on A-glycoloylneuraminate and on O-acetylated sialic acids, other than O -acetylated derivatives. 

This enzyme [EC 4.1.3.20] catalyzes the reaction of N-acylneuraminate 9-phosphate with phosphate to produce A-acetylmannosamine 6-phosphate, phosphoenolpyr-uvate, and water. The protein will act on A-glycoloyl and A-acetyl derivatives. 

Sialic acid aldolase (SA EC 4.1.3.3), also named A-acetylneuraminate pyruvate lyase, has been extensively used by our group in its immobilized form, first for the synthesis of large amounts of A-acety lneuraminic acid [20] and then for many natural and unnatural sialic acids [21], SA catalyzes the reversible aldol reaction of A-acetylmannosamine and pyruvate to give A-acety lneuraminic acid with an optimum pH for activity of 7.5 and an equilibrium constant of 12.7 A/-1 in the synthetic direction (Scheme 3) [10],... 

Immobilized sialyl aldolase (50 mL of gel, 68 U) was added to a mixture of 88% pure A-acetylmannosamine (20 mmol), sodium pyruvate (180 mmol), 1,4-dithiothreitol (0.2 mmol), and sodium azide (20 mg) in 0.05 M potassium phosphate buffer, pH 7 (150 mL). The suspension was gently stirred under nitrogen for 4 d at 37°, the reaction being monitored by t.l.c. in 7 3 propanol - water. The gel was removed by filtration, washed with the buffer, and A-acetylneuraminic acid (2) was isolated by chromatography on Dowex 1 X8 (HCOj-) resin, using a gradient of formic acid as the eluant, in 66% yield. The gel was used in four successive runs. Starting from 17 g of 88% pure A-acetylmannosamine, the procedure allowed the synthesis of 14 g of A-acetylneuraminic acid (2). In the end, the recovered gel retained 80% of its enzymic activity. 

The amino acid sequence deduced from ORF 23 (mnaA) shared homology with bacterial A-acetylglucosamine 2-epimerases that convert A-acetyl-glucosamine into A-acetylmannosamine, a precursor of sialic acids. ORF 9 and ORF 24 (neuB) both produce polypeptides that exhibited 25 and 31%... 

In considering the application of enzyme catalysis to DCC, we were encouraged by the thermodynamic resolution of a dynamic mixture of aldol products by Whitesides and co-workers through the use of a broad-specificity aldolase to lead to reversible formation of carbon-carbon bonds under mild conditions.35 For the current investigation36 we chose a related enzyme, N-acetylneuraminic acid aldolase (NANA aldolase, EC 4.1.3.3), which catalyzes the cleavage of N-acetylneuraminic acid (sialic acid, 27a) to A-acetylmannosamine (ManNAc, 28a), and sodium pyruvate 29 in the presence of excess sodium pyruvate, aldol products 27a-c are generated from... 

A-acetylmannosamine undergoes a chelation-controlled reaction and leads to 90% of the syn-f3-amino alcohol when reacted in a 0.5 M NH4G1 solution. While a dibenzylamino substitutent of a-aminopropionaldehyde is too bulky to enter complexation, a dimethylamino group is not and leads to high levels (99%) of the. sy/z-diastereomer (Table 4).159... 

O-Acetyl-yV-acetylmannosamine 6, prepared from A-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-substimted Neu5Ac derivatives could also be prepared [29-32],... 

Scheme 5 Aldol addition of fluoropyruvate with A-acetylmannosamine or mannose catalyzed by sialic acid aldolase.

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. 

Mammals produce sialic acid by aldolic condensation of phosphoenolpyruvate and Ai-acetylmannosamine 6-phosphate (reaction 12.1). A kinase enzyme catalyses the phosphorylation of A -acetylmannosamine and a phosphatase catalyses the hydrolysis of the phosphate of sialic acid. These phosphorylation and dephosphorylation steps are irreversible, such that the synthesis can be total even with low concentrations of the substrate. A variation of reaction (12.1), observed with the bacterium Neisseria meningitidis, uses non-phosphated /-acetylmannosamine. However, these were not the enzymes used in the preparative synthesis, which used instead a microbial aldolase which catalyses equilibrium (12.2). This enzyme probably plays a catabolic role in these organisms, but it functions in the synthetic sense in the presence of an excess of pyruvate. 

K. J. (2004). Characterization of the metabolic flux and apoptotic effects of 0-hydroxyl- and A-acetylmannosamine (ManNAc) analogs in Jurkat (human T-lymphoma-derived) cells. J. Biol. Chem., 279, 18342-18352. 

Synthesis of iV-acetylneuraminic acid (Neu5Ac) in vivo is catalyzed by Neu5Ac synthetase (EC 4.1.3.19) through the irreversible condensation of phosphoenolpyruvate (PEP) and A/-acetylmannosamine (15) (Scheme 2) [30-32], This enzyme has not yet been isolated and its catalytic activity might be interesting field for exploration. 

Many hexoses were tested in both D- and L-form, resulting in the formation of nonulosonic acid analogues. Table 2 demonstrates the most common enzymatic approach to the synthesis ofNeu5Ac (entry 1) and its analogues (entry 2-12) from modified A-acetylmannosamine derivatives. 

In line with the substrate requirements of FDP aldolase, the specificity of sialic acid aldolase appears to be absolute for pyruvate (the donor), but relaxed for the aldehydic acceptor. As may be seen from Scheme 2.193, a range of mannosamine derivatives have been used to synthesize derivatives of NeuAc [1427-1432]. Substitution at C-2 of A-acetylmannosamine is tolerated, and the enzyme exhibits only a slight preference for defined stereochemistry at other centers. 

The metabolism of A -acetylmannosamine and its relation to the formation of the sialic acids (derivatives of neuraminic acid) are not well understood. The subject has been reviewed by Roseman . 

An extended NeuAc analogue 49 has been prepared by the reaction of 5,6-0-isopropylidene-A-acetylmannosamine with disodium acetone dicarboxylate in the presence of nickel acetate the C-5 and C-6 epimers of 49 were also formed. Deprotection of 49 by hydrogenolysis gave the free acid which decarboxylated readily. 9 Treatment of the Wittig product 50 under Wacker-type conditions led to the glycoside 51 of a 3-ulosonic acid (Scheme 7). ... 

In a further example of indium-mediated additions to aldoses, a-(bromo-methyl)acrylic acid added to A-acetylmannosamine in the presence of indium to give branched derivative 27 and its C-4 epimer. Ozonolysis of 27 afforded N-acetylneuraminic acid. In a study of the oxidation of L-sorbose (5% Pt/AlaOa, O2) to 2-keto-L-gulonic acid it was found that the reaction rate and selectivity was improved in the presence of certain tertiary amines. The synthesis of a carbocyclic analogue of iV-acetyl-neuraminic acid is discussed in Chapter 18. 

The biosynthetic pathway for sialic acid formation in liver was well established nearly thirty years ago, by the elegant work from the groups of Roseman [9-11] and Warren [12] (Figure 1). The enzymatic conversion of UDP-A-acetyl glucosamine to A-acetylmannosamine is the critical step in the overall synthesis of sialic acid because the activity of this epimerase is controlled downstream in the pathway by feedback inhibition by CMP-A -acetylneuraminic acid [13]. Thus, production of ManNAc as a precursor for sialic acid biosynthesis is a closely regulated cellular process. 

Reaction number five, Figure 3, is the first step which is unique and is not shared by other aminosugars in the biosynthetic sequence leading to the sialic acids. This reaction is catalyzed by one enzyme which apparently carries out both an epimerization of the acylamino group at C-2 and hydrolysis of A/ -acetylmannosamine (see Figure 4). 

Another, more classical, control mechanism exists for the feedback regulation of key enzymes for the synthesis of Gm-6-P04 and for N-acetylmannosamine. Each of these is the first enzyme in the metabolic commitment to hexosamine and sialic acid synthesis. Komfeld et al, (1964) showed that UDP-GlcNAc is an efficient feedback inhibitor for L-glutamine-D-fructose-6-phosphate aminotransferase and that CMP-NAN also inhibits UDP-GlcNAc-2-epimerase, which is responsible for the synthesis of A/ -acetylmannosamine. This is an example of the by now familar endproduct inhibition of the first enzyme of a metabolic pathway (see Figure 6). They were also able to demonstrate that in vivo administration of puromycin to rats, which inhibits de novo protein synthesis and also depresses sialic acid and hexosamine utilization, does not lead to an accumulation of UDP-GlcNAc. Furthermore, the turnover of the UDP-hexosamine pool was shown to be slowed down. These data suggest that impairment of the utilization of UDP-hexosamine leads to decreased synthesis of UDP-hexosamines or their precursors (i.e., classical feedback inhibition). 

In 1969, Schoop et aL found that pig submaxillary slices could synthesize NGN from acetate, N-acetylmannosamine, or N-acetylneu-raminic acid, all labeled in the acetate moiety. Schauer (1970a 1970Z ) demonstrated an enzyme system in high-speed supernatants of hog submaxillary gland extracts capable of oxidizing N-acetylneuraminic acid or A -acetylmannosamine to A/ -glycolylneuraminic acid. The enzyme is called N-acetylneuraminaterOg-oxidoreductase and requires O2 and ascorbate or NADPH for activity. It appeared that free N-acetylneuraminic acid is the substrate, but it is possible that CMP-NAN or the 9-phosphate ester is the substrate. 

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