Sialic acids purification

Kean, E. L. and Roseman, S. The sialic acids Purification and properties of cytidine 5 -monophosphosialic acid synthetase. J. Biol Chem., 1966, 241, 5643-5650. 

Higa, H. H., Manzi, A., and Varki, A., 1989b, O-Acetylation and de-O-acetylation of sialic acids. Purification, characterization, and properties of a glycosylated rat liver esterase specific for 9-0-acetylated sialic acids, J. Biol. Chem. 264 19435-19442. 

Exact analysis of sialic acid is required in biologieal experiments where the biological role of sialic acid is frequently studied with the aid of sialidases, and the amount of sialic acids released is determined. This is also important for periodate oxidation studies on biological systems, where modification of sialic acids by periodate is only assumed, but chemical analysis of this effect by isolation and analysis of the modified sialic acids is seldom performed. These uncertainties in determinations of sialic acid can be overcome by the purification procedures already described. Furthermore, it must be stressed that unequivocal determination of the structure of a sialic acid, especially... 

Although most of the sialidases have been purified by classical methods of enzyme isolation, affinity chromatography is now coming more and more into use, as is indicated by the foregoing examples of purification of sialidases. The adsorbent first used for chromatography of sialidases was sialic acid bound to the surface of intact erythrocytes ... 

Several alternative methods for the determination of sialic acid in body fluids and tissues have been described. Most of these methods make use of the classic periodate-TBA assay in combination with purification using HPLC [13]. Another method makes use of fluorometric HPLC of sialic acids after derivatization with a fluorogenic compound [9]. The most promising new method for the determination of free sialic acid in urine (and probably also other body fluids and tissues) is the HPLC-tandem mass spectrometry method [19]. This method is rapid, accurate, and sensitive, and is more robust than earlier methods. The only disadvantage is the expensive equipment that is required, which makes it only economical for specialized metabolic laboratories. Since this equipment is used for many different metabolic assays, the investment is certainly warranted, and nowadays almost essential for any metabolic laboratory. 

Sialic acid was partially-purified from lipid extracts after hydrolysis in 0.05NHC1 (total sialic acid) or without hydrolysis (free sialic acid) as described in Methods. The amounts of gly-cosidically-bound sialic acid (total minus free) and free sialic acid were determined by the TBA assay. The hydrolysed samples were also analyzed using gas liquid chromatography by the method of Yu and Ledeen (15) and shown to possess essentially identical amounts of total sialic acid. However, the crude lipid extracts were too impure to make GLC the method of choice in the absence of extensive purification of the glycolipids. 

Melkerson-Watson LJ, Sweeley CC. Purification to apparent homogeneity by immunoaffinity chromatography and partial characterization of the GM3 gangUoside-forming enzyme, CMP-sialic acid lactosylceramide alpha 2,3-sialyltransferase (SAT-1), from rat liver Golgi. J. Biol. Chem. 1991 266 4448-4457. 

The usual procedure to remove the protecting silyl group (tetrabutylammonium fluoride in tetrahydrofuran) has caused acetyl rearrangements rather than to give a sialic acid derivative with a free C-9 primary hydroxyl function. Compound 5c, in which the free hydroxyl appears to be the secondary one at C-7, has been obtained as the sole reaction product in a 85% jdeld, after chromatographic purification. The overall yield of 5c based on 3a has been 83% [30]. 

Supports prepared with sialic acid have also been used in the purification of insulin, as this sugar and N-acetylglucosamine have been found to take part in the interaction between insulin and its glycosylated receptor. The affinity between insulin and sialic acid-bearing supports has been found rather strong i a ... 

H. Lakhiari, J. Jozefonvicz, and D. Muller, Separation and purification of insulins on coated silica support functionalized with sialic acid by affinity chromatography, J. Liquid Chromatogr. Related Technol. 19 2423 (1996). 

Halcomb and Chappell developed a route to CMP-NeuAc 88 that promises to be general for the synthesis of virtually any derivative thereof [44,45]. The route (Scheme 36) utilizes a condensation of sialic acid derivative 99 with the phosphoramidite 112 to afford the phosphite 113 in 62% yield. Oxidation of the phosphite provided the phosphotriester 114 [46], which was taken directly to the next transformation without purification (owing to its instability to chromatography). Deal-lylation of the phosphate gave compound 115 (61% for two steps), which was stable to silica gel chromatography. Compound 115 was deacylated with methoxide, and its methyl ester was subsequently saponified with NaOH to provide CMP-NeuAc 88. The derivatives shown in Scheme 37 were synthesized according to this protocol and were investigated as substrates for sialyltransferases (see below). 

---