Reductive Alkaline Release of N‐Glycans Generates a Variety of Unexpected, Useful Products

Abstract

Release of O-glycans by reductive β-elimination has become routine in many glyco-analytical laboratories and concomitant release of N-glycans has repeatedly been observed. Revisiting this somewhat forgotten mode of N-glycan release revealed that all kinds of N-glycans including oligomannosidic and complex-type N-glycans from plants with 3-linked fucose and from mammals with or without 6-linked fucose and with sialic acid could be recovered. However, the mass spectra of the obtained products revealed very surprising facts. Even after 16 h incubation in 1 M sodium borohydride, a large part of the glycans occurred in reducing form. Moreover, about one third emerged in the form of the stable amino-functionalized 1-amino-1-deoxy-glycitol. When avoiding acidic conditions, considerable amounts of glycosylamine were observed. In addition, a compound with a reduced asparagine and de-N-acetylation products, in particular of sialylated glycans, was seen. The relative yields of the products reducing glycosylamine, reducing N-glycan, 1-amino-1-deoxy-glycitol or glycitol could be controlled by the release conditions, foremost by temperature and borohydride concentration. Thus, chemical release of N-glycans constitutes a cost-saving alternative to enzymatic hydrolysis for the preparation of precursors for the production of reference compounds for various formats of N-glycan analysis. Moreover, it allows to obtain a stable amino-functionalized glycan derivative, which can be employed to construct glycan arrays or affinity matrices.

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2017–2018

“…Although β‐elimination is the preferred method for release of O ‐glycans, it is rarely used for N ‐glycans. However, the reaction has been revisited with some surprising results (Figl & Altmann, 2018). Thus, even after 16 h incubation in 1 M sodium borohydride, a large part of the glycans were released in reducing form, unlike the reduced form that is the normal product of β‐elimination.…”

Simple Routes to Stable Isotope-Coded Native Glycans

“…Yet other approaches employ ammonia to overcome alkaline peeling . Against intuition, reducing N -glycans can also be obtained by reductive alkaline release, whichdepending on the reaction conditionsalso provides species with an amine on C1 of the linking GlcNAc . It seems, however, that for small- to medium-scale glycan preparations, the application of PNGase F is preferred due to its high specificity and avoidance of side products.…”

“… 41 Against intuition, reducing N -glycans can also be obtained by reductive alkaline release, which—depending on the reaction conditions—also provides species with an amine on C1 of the linking GlcNAc. 42 It seems, however, that for small- to medium-scale glycan preparations, the application of PNGase F is preferred due to its high specificity and avoidance of side products. While reducing glycans are required for subsequent labeling reactions, underivatized reduced glycans are usually employed for PGC chromatography to simplify the analysis of isomeric structures.…”