N-Mannosamine

The second pathway for the formation of 3-deoxyulosonic acids is, at present, represented only by the condensation of pyruvate with iV -acetyl-n-mannosamine to form a nonulosaminic acid (iV-acetylneuraminic acid). - This reaction is readily reversible, but the point of equilibrium is 90 per cent toward degradation and 10 per cent toward synthesis of the iV-acetyl-neuraminic acid. ... 

O-Prolected sugar thioformamides have also been prepared in 44-87% yields by tri-n-butyltin hydride reduction of isothiocyanate precursors in ether.156 For the preparation of 5-A-thioacylneuraminic acids (84), a chemoenzymatic route based on the M-acetylneuraminate pyruvate lyase-mediated condensation of the corresponding A -thioacyl-n-mannosamine derivatives (83) and sodium pyruvate has been reported.189 The enzyme-catalyzed aldol reaction was performed at pH 6.8 and afforded the desired compound in 55% yields (Scheme 24). 

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]. 

We propose that the first step in NDP-kasugamine biosynthesis is 2-epimeriza-tion of the postulated UDP-A -acetyl-D-glucosamine precursor, which is suggested by the similarity of the KasQ protein with known UDP-(Af-acetyl-)D-glucosamine 2-epimerases and catalyzes the conversion to UDP-N-acetyl-D-mannosamine. To... 

D-Erythrose-4-phosphate = D-Arabinose-5-phosphate = N -Acetyl-D-mannosamin-6-phosphate... 

N-Acetvlneuraminic Acid Aldolase. A new procedure has also been developed for the synthesis of 9-0-acetyl-N-acetylneuraminic acid using the aldolase catalyzed reaction methodology. This compound is an unusual sialic acid found in a number of tumor cells and influenza virus C glycoproteins (4 ). The aldol acceptor, 6-0-acetyl-D-mannosamine was prepared in 70% isolated yield from isopropenyl acetate and N-acetyl-D-mannosamine catalyzed by protease N from Bacillus subtilis (from Amano). The 6-0-acetyl hexose was previously prepared by a complicated chemical procedure (42.) The target molecule was obtained in 90% yield via the condensation of the 6-0-acetyl sugar and pyruvate catalyzed by NANA aldolase (Figure 6). With similar procedures applied to KDO, 2-deoxy-NANA and 2-deoxy-2-fluoro-NANA were prepared from NANA. 

N-acetyl D-glucosamine A/-acetyl D-mannosamine N-acetyl D-galactosamine... 

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... 

A-Acetyl neuraminic acid aldolase [from Clostridium perfringens, A-acetylneuraminic acid pyruvate lyase] [9027-60-5] [EC 4.1.3.3]. Purified by extraction with H20, protamine pptn, (NH4)2S04 pptn, Me2CO pptn, acid treatment at pH 5.7 and pptn at pH 4.5. The equilibrium constant for pyruvate + n-acetyl-D-mannosamine ++ /V-acetylneuraminidate at 37° is 0.64. The Km for A-acetylneuraminic acid is 3.9mM in phosphate at pH 7.2 and 37°. [Comb and Roseman Methods in Enzymology 5 391 1962). The enzyme from Hogg kidney (cortex) has been purified 1700 fold by extraction with H20, protamine sulphate pptn, (NH4)2S04 pptn, heat treatment between 60-80°, a second (NH4)2S04 pptn and starch gel electrophoresis. The Km for A-acetylneuraminic acid is 1.5mM. [Brunetti et al. JBC 237 2447 1962). 

The sialic acid aldolase-catalyzed condensation of D-mannose 8 and pyruvate led, in an excellent yield, to the synthesis of KDN 9 [33], a natural deaminated neuraminic acid first isolated from rainbow trout eggs [34] and then discovered in other species. The discovery that sialic acid aldolase accepts as substrates D-mannose substituted on the 2-position, even by bulky substituents such as phenyl, azido, or bromine, opened the route to novel unnatural sialic acid derivatives [35-39]. Pentoses also are substrates. N-Substituted neuraminic acids could be prepared either directly from the corresponding Af-substituted mannosamine, such as N-thioacyl derivatives [40], or after reduction and acylation of 5-azido-KDN [41]. Recently, AT-carbobenzyloxy-D-mannosamine was converted, in a good yield, into the N-carbobenzyloxy-neurarninic acid, further used as a precursor of a derivative of castanospermine [42]. 

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... 

Scheme 12.—Chemical Synthesis of the Furanose Derivative of N-Acetyl-D-mannosamine.

When HeLa cells were.cultured in medium supplemented with 5 mM sodium butyrate, their content of GM3 increased (Fig.2a). Increases varied from 3.5 to 5-fold depending on the experiment (4,8,12,13). When the butyrate was removed and the cells were cultured in normal medium for 24 h, the GM3 levels returned to those found in untreated cells (Fig. 2a). Similar results were observed when N-[acetyl-3H]-D-mannosamine, a precursor of sialic acid, was also included in the culture medium. In the butyrate-treated cells, radioactivity associated with GM3 increased 6.5-fold and 24 h after butyrate was removed, the amount of labeling returned to control values (Fig. 2b). We also were able to label the GM3 by means of a cell surface labeling technique. Control and butyrate-treated cells were exposed to 10 mM sodium periodate and the oxidized sialyl residues were reduced with NaBSfy. There was 5.5-fold more 3h associated with the GM3 recovered from the butyrate-... 

HeLa cells were cultured in medium containing N[acetyl-3H]-D-mannosamine (50 pCit mL) for 24 hr with (solid bars) and without (open bars) 5 mM sodium butyrate. In addition, butyrate-treated cells were cultured an additional 24 hr in fresh medium (without label and butyrate) (hatched bars). The cells were harvested and analyzed for GM3 content and radioactivity. (Data from Refs. 8, Vi.)... 

A HeLa cells cultured in medium containing 5 mM sodium butyrate and 50 ixCi/mL N-[acetyl-sH]-D-mannosamine after 24 hr, one set of cells harvested and medium replaced with fresh medium with and without 0.5 pg/mL cycloheximide on the other two sets, which were harvested after another 19 hr cells then analyzed for radioactive GM3. B Essentially same as A except the cells were cultured in unlabeled medium and assayed for sialyltransferase activity. C same as B except the cells were assayed for GM3-... 

---