Migration of O-acetyl esters within polyhydroxy groups on the linear C7-C8-C9 side chain of sialic acids(Sias) such as N-acetylneuraminic acid (Neu5Ac) under physiological conditions is well recognized. However, migration in mono-O-acetyl-Neu5Ac is assumed to be unidirectional from C7 (in Neu5,7Ac2) to C9 (the primary hydroxyl group, giving Neu5,9Ac2). Surprising resistance to mild periodate oxidation of glycosidically-bound Neu5,9Ac2 (which should cleave C-C bond between C7- and C8 hydroxyls) was assumed to be due to cis-trans conformation and/or internal hydrogen bonds. Indeed, early literature refers to a compound eventually found to be Neu5,9Ac2 as “8-O-acetyl-sialic acid”, but the latter is now considered only a possible transient intermediate. Here we show evidence suggesting reverse migration of 9-O-acetyl groups to the 8-O-position and possibly the 7-O-position of Neu5Ac, using an improved method of low temperature 1,2-Diamino-4,5-methylenedioxybenzene dihydrochloride (DMB) derivatization in 1.4 M of acetic acid instead of 0.02 M of trifluoroacetic acid, to avoid pH changes induced by sample buffer. In free Neu5,9Ac2, 20% of 9-O-acetyl group migrated to 8-O at pH ranges &lt;5.0 after 2 hour-preincubation at 37○C. No significant migration occurred during the low temperature DMB derivatization. A smaller amount of 9-O-acetyl-group (~6%) in a Neu5,9Ac2–containing sialoside migrated to the C8-OH with a pH increase from 5.0 to 8.0. An ongoing interconversion between Neu5,9Ac2 and Neu5,8Ac2 was further suggested by resistance to mild periodate oxidation and the resulting product loss detected on HPLC. Furthermore, the synthetic analog 9-N-acetyl-Neu5Ac, in which the N-acetyl group does not migrate was much more sensitive to periodate. Notably, a sialoglycan microarray study on esterase-inactivated HE proteins of porcine torovirus and bovine coronavirus (which preferably bind 9-O-acetyl Sias), showed much stronger binding intensity to sialosides containing 9-N-acetyl-Neu5Ac than those containing Neu5,9Ac2. Taken together, data suggest that O-acetyl group migration in Sias may be bidirectional between 8- and 9-positions. Dynamic O-acetyl group shifting should impact on biological recognition involving Sias, such as viral infections, and may also be finely regulated by pH changes.

Preliminary evidence for reversible O-acetyl group migration at 9- and 8-carbon position in sialic acids

2019 Fall National Meeting

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.