Sialic acids enzymatic method

Van Lenten, L., and Ashwell, G. (1971) Studies on the chemical and enzymatic modification of glycoproteins. A general method for the tritiation of sialic acid-containing glycoproteins. J. Biol. Chem. 46, 1889-1894. 

Initially, the separation of sialic acids was mainly carried out by cellulose chromatography at low temperature [6,237]. However, nowadays HPLC fractionations using different column materials, elution protocols and detection techniques have replaced this approach [6,11,268]. The application of HPLC has also introduced a rapid method for tentative assignments of sialic acids in complex mixtures, based on elution times of known standards, being more reliable when more than one HPLC procedure is followed. Moreover, a rapid method for quantification of released sialic acids has become available. Due to the relatively short HPLC runs, also fast transitions between members of the sialic acid family due to migration of substituents, introduction of substituents, cleavage of substituents, or other (enzymatic) modifications can easily be monitored. 

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,... 

Notes and discussion. A major drawback of enzymatic sialylation with sialyl-transferases is the strict acceptor specificity of these enzymes. This synthesis addresses this limitation by showing a widely applicable transfer of sialic acid (NeuAc) from a donor substrate of the sequence NeuAca-(2 —3)Gal-ORi to virtually any galactose acceptor (Gal-OR2). This method uses the less substrate specific glycosidase from Trypanosoma cruzi. The problem with this method is that the product is made at the expense of another sialoside, used as the donor substrate, and as NeuAc transfer is a reversible process, it is difficult to drive the equilibrium in favour of the desired sialoside. For this reason the sialoside donor substrate is regenerated in situ by an a-(2 3)-sialytransferase enzyme, thus enhancing the production of the desired product. The specificity of the sialyltransferase ensures that only the galactose byproduct, formed from the sialyl donor, is re-sialyated as the Gal-OR2 acceptor substrate is a poor substrate. Due to the broad specificity of the trans-sialidase, many a-NeuAc-(2 3)-Gal-OR sequences can be synthesised by... 

Neutral monosaccharides, uronic acids, hexosa-mines, and sialic (neurominic acids) are identified and determined by specific colorimetric reactions. The principle behind the techniques rests on the condensation of the degraded products of the neutral monosaccharides (hexose, pentose and methyl pentose) by sulfuric acid with anthrone, cysteine hydrochloride, orcinol, and phenol reagents. Uronic acids may be determined by colorimetric and manometric procedures. While sialic acids are determined after chemical/enzymatic hydrolysis, gravimetric and Van-Soest detergent based methods are used to determine cellulose, hemicellulose, and fiber. 

In the chemo-enzymatic synthesis of LLG-3 performed by Withers et al. [34], the glycoside of 8-0-Me-Neu5Ac with glycolic acid 59 was synthesized from NeuSAc derivative 58 by a chemical method because of the absence of a sialyltransferase capable of sialyltransfer to the hydroxyl group of the glycosyl amide in the penultimate sialic acid residue and an 8-O-methyltransferase for NeuAc (Scheme 11.10). 

The monosaccharide sialic acid remains relatively costly for preparative-scale synthesis. Although it has been demonstrated that NeuAc can be generated in situ by reaction of ManNAc with pyruvate, catalyzed by NeuAc aldolase [27], ManNAc is also relatively expensive and difficult to prepare. Therefore, a method for the generation of NeuAc from the inexpensive monosaccharide GlcNAc in a two-enzyme system might also prove useful in regeneration schemes. It has been shown that GlcNAc can be converted to ManNAc chemically [28], or enzymatically by the... 

---