Activation and transfer of novel synthetic 9-substituted sialic acids.
In this report several NeuAc analogues differently modified at position C-9 were tested as substrates for CMP sialic acid synthase from bovine brain: the hydroxy group at C-9 was replaced by an amino, acetamido, benzamido, hexanoylamido and azido group. The synthase was partially purified by chromatography on CDP-hexanolamine--Sepharose. CMP-glycosides synthesized were measured by analytical HPLC at 275 nm. Each NeuAc analogue was activated to the respective CMP-glycoside: Km-values varied from 0.8 mM to 4.6 mM, the Km for NeuAc was 1.4 mM. Thus affinity of the enzyme was influenced only moderately by chemical modification at C-9. CMP-glycosides were synthesized on a preparative scale with good yield and characterized by analytical HPLC. In addition, 500-MHz 1H-NMR data of CMP-9-amino-NeuAc and CMP-9-acetamido-NeuAc were obtained. Each CMP-activated NeuAc analogue was a suitable donor substrate for Gal beta 1-4GlcNAc alpha 2,6-sialyltransferase from rat liver. Transfer was determined by the thiobarbituric acid method and by analytical HPLC at 200 nm. The results demonstrate that synthetic, not naturally occurring, non-labelled NeuAc analogues can be incorporated into glycoprotein with high yield.
Gross
H J
HJ
Institut für Biochemie II der Universität Heidelberg, Federal Republic of Germany.
Bünsch
A
A
Paulson
J C
JC
Brossmer
R
R
D000818:Animals; D001212:Asialoglycoproteins / Q000378:metabolism; D001711:Biotransformation; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D006027:Glycosides / Q000096:biosynthesis; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D019158:N-Acetylneuraminic Acid; D051381:Rats; D012794:Sialic Acids / Q000378:metabolism*; D012799:Sialyltransferases / Q000378:metabolism; D013379:Substrate Specificity
D001212:Asialoglycoproteins; D006027:Glycosides; D012794:Sialic Acids; D012799:Sialyltransferases; D019158:N-Acetylneuraminic Acid