Large scale preparation of high mannose and paucimannose N-glycans from soybean proteins by oxidative release of natural glycans (ORNG)

Abstract

Despite the important advances in chemical and chemoenzymatic synthesis of glycans, access to large quantities of complex natural glycans remains a major impediment to progress in Glycoscience. Here we report a large-scale preparation of N-glycans from a kilogram of commercial soy proteins using oxidative release of natural glycans (ORNG). The high mannose and paucimannose N-glycans were labeled with a fluorescent tag and purified by size exclusion and multidimensional preparative HPLC. Side products are identified and potential mechanisms for the oxidative release of natural N-glycans from glycoproteins are proposed. This study demonstrates the potential for using the ORNG approach as a complementary route to synthetic approaches for the preparation of multi-milligram quantities of biomedically relevant complex glycans.

Characterization and Analysis of Food-Sourced Carbohydrates

“…There have been a limited number of examples in which a more significant amount of starting material is used which allowed the production of sufficient amount carbohydrates for nuclear magnetic resonance (NMR) analysis. Isolation of high mannose N-glycans from soy proteins and egg yolks by a preparative scale multidimensional HPLC method has been reported [41,42]. However, even after multidimensional HPLC and with the use of analytical columns, some fractions are still mixtures of isomers that are very difficult to separate.…”

Preparation of Complex Glycans From Natural Sources for Functional Study

“…The ORNG approach is fast, easy to operate, and can be applied to multi-kilograms of natural materials to produce gram-scale natural complex glycans. In our most recent study, the ORNG approach was demonstrated as a complementary route for the preparation of multi-milligram quantities of purified high-mannose N-glycans (Zhu et al, 2018a ).…”

“…With gram-scale glycans from a natural source are available because of the ORNG technique, development of preparative-scale purification becomes practical and provides an effective route to address the lack of glycans for functional study. We have reported isolation of high mannose N-glycans from soy proteins and egg yolks by a preparative scale multidimensional HPLC method (Zhu et al, 2018a , b ). However, even after multidimensional HPLC, some fractions are still mixtures of isomers that are very difficult to separate even on analytical columns.…”

O-Benzylhydroxylamine (BHA) as a Cleavable Tag for Isolation and Purification of Reducing Glycans

“…Debenzylation of 2 mg of glycan-BHA conjugates was catalyzed by 2 mg of 10% Pd/C and 50 mg of ammonium formate in 0.2 mL of water at room temperature for 12 h. After the reaction was complete, the residue was submitted to a Sephadex G25 column to remove excess BHA tags. 19…”

“…Gram-scale glycopeptides and glycans can be isolated from a large amount of egg yolk. 19 As a more general approach, we have developed the oxidative release of natural glycans (ORNG), which uses household bleach to directly release gram-scale glycans from kilogram-scale natural materials such as animal and plant tissues. 20 Another challenge is the separation of individual glycans from a complex mixture that occurs in nature by chromatography.…”