Fluorogenic sialic acid glycosides for quantification of sialidase activity upon unnatural substrates

Abstract

Herein we report the synthesis of N-acetyl neuraminic acid derivatives as 4-methylumbelliferyl glycosides and their use in fluorometrically quantifying human and bacterial sialidase activity and substrate specificities. We found that sialidases in the human promyelocytic leukemic cell line HL60 were able to cleave sialic acid substrates with fluorinated C-5 modifications, in some cases to a greater degree than the natural N-acetyl functionality. Human sialidases isoforms were also able to cleave unnatural substrates with bulky and hydrophobic C-5 modifications. In contrast, we found bacterial sialidase isolated from C. perfringens to be less tolerant of sialic acid derivatization at this position, with virtually no cleavage of these glycosides observed. From our results, we conclude that human sialidase activity is a significant factor in sialic acid metabolic glycoengineering efforts utilizing unnatural sialic acid derivatives. Our fluorogenic probes have enabled further understanding of the activities and substrate specificities of human sialidases in a cellular context.

N-Glycolylneuraminic Acid as a Receptor for Influenza A Viruses

“…Because binding to N-glycolyl can, for some viruses, also support influenza infection, and because additional mutations do not affect N-glycolyl specificity, we hypothesized that the neuraminidase of A/Vietnam/1203/04 H5N1 can cleave both N-acetyl and N-glycolyl, therefore maintaining HA-NA balance. To this end, we chemically synthesized N-glycolyl-4-Methylumbelliferyl (Izumi et al, 2001; Zamora et al, 2013) (Scheme S1). We used recombinant tetrameric neuraminidases to analyze the ability of influenza virus NA proteins to cleave N-glycolyl (Bosch et al, 2010).…”

“…2′-(4-Methylumbelliferyl)-α-D-N-glycolylneuraminic acid was synthesized previously (Izumi et al, 2001; Zamora et al, 2013) (Scheme S1). The synthesis used in this paper is partially based on the method used by Zamora et al by protecting the C5 position with a Boc group to selectively deprotect and introduce the glycolyl group.…”

Fluorescently labelled glycans and their applications

“…This approach was extended to more complex 4-methylumbelliferyl fluorogenic glycosides, such as di-, triand oligosaccharides [62][63][64][65][66][67], and challenging monosaccharides such as sialic acids [68][69][70]. 4-Methylumbelliferyl aglycone analogues were also explored to better suit the properties of glycosidases [71][72][73][74].…”

Facile Anomer-oriented Syntheses of 4-Methylumbelliferyl Sialic Acid Glycosides

“…Additionally, the chemistry of 4methylumbelliferyl sialic glycosides has been left unexplored for many years despite existing synthetic routes suffering from poor yields, low stereoselectivity and formation of the undesired glycal side product. [18][19][20][21] To this end, we report herein synthetic strategies to access C-5 derivatised neuraminic acid substrates with a fluorogenic 4-methylumbelliferyl (4-Mu) aglycone functionality in both anomeric configurations as tools to probe sialidase activity and specificity.…”