Quantitative Standards of 4‐O‐Acetyl‐ and 9‐O‐Acetyl‐N‐Acetylneuraminic Acid for the Analysis of Plasma and Serum

Abstract

N‐Acetylneuraminic acid (sialic acid, Neu5Ac) is one of a large, diverse family of nine‐carbon monosaccharides that play roles in many biological functions such as immune response. Neu5Ac has previously been identified as a potential biomarker for the presence and pathogenesis of cardiovascular disease (CVD), diabetes and cancer. More recent research has highlighted acetylated sialic acid derivatives, specifically Neu5,9Ac2, as biomarkers for oral and breast cancers, but advances in analysis have been hampered due to a lack of commercially available quantitative standards. We report here the synthesis of 9‐O‐ and 4‐O‐acetylated sialic acids (Neu5,9Ac2 and Neu4,5Ac2) with optimisation of previously reported synthetic routes. Neu5,9Ac2 was synthesised in 1 step in 68 % yield. Neu4,5Ac2 was synthesised in 4 steps in 39 % overall yield. Synthesis was followed by analysis of these standards via quantitative NMR (qNMR) spectroscopy. Their utilisation for the identification and quantification of specific acetylated sialic acid derivatives in biological samples is also demonstrated.

Elevated concentrations of Neu5Ac and Neu5,9Ac2 in human plasma: potential biomarkers of cardiovascular disease

“…Analysis of sialic acids was carried out using the DMB method [ 22 , 24 , 28 ]. Two samples were required per analysis, one to quantify Neu5Ac and one to quantify Neu5,9Ac 2 .…”

“…One nmol standards of Neu5Ac and Neu5,9Ac 2 [ 22 ] were also labelled using DMB (Scheme 1 ). 20 μL of labelling solution (3.5 mg DMB dye, 2.2 mL mercaptoethanol (1.4 M), 20 mg sodium dithionite) was added to each standard.…”

“…Quantitative analysis of these sialic acid derivatives as biomarkers for CVD requires a sensitive and specific analytical technique with a sufficiently low limit of detection. This is because Neu5Ac is present in sufficiently large quantities (average 45.49 mg/100 mL in healthy controls), with Neu5,9Ac 2 being present in quantities 100–200 times less (average 0.29 mg/100 mL in healthy controls) [ 22 ]. Ideally, the assay of choice will also analyse total sialic acid and not just sialic acids present on N -glycans.…”

“…Neu5Ac is commercially available in sufficiently large quantities but Neu5,9Ac 2 is not, and therefore must be chemically synthesised. For the accurate quantitation of Neu5,9Ac 2 in plasma samples, we have previously synthesised Neu5,9Ac 2 and analysed the standard using quantitative nuclear magnetic resonance (QNMR) techniques [ 22 ].…”