Novel Reversible Fluorescent Glycan Linker for Functional Glycomics

Abstract

To aid in generating complex and diverse natural glycan libraries for functional glycomics, more efficient and reliable methods are needed to derivatize glycans. Here we present our development of a reversible, cleavable bifunctional linker 3-(methoxyamino)propylamine (MAPA). As the fluorenylmethyloxycarbonate (Fmoc) version (F-MAPA), it is highly fluorescent and efficiently derivatizes free reducing glycans to generate closed-ring derivatives that preserve the structural integrity of glycans. A library of glycans were derivatized and used to generate a covalent glycan microarray using Nhydroxysuccinimide derivatization. The array was successfully interrogated by a variety of lectins and antibodies, demonstrating the importance of closed-ring chemistry. The glycan derivatization was also performed at large scale using milligram quantities of glycans and excess F-MAPA, and the reaction system was successfully recycled up to five times, without an apparent decrease in conjugation efficiency. The MAPA-glycan is also easy to link to protein to generate neoglycoproteins with equivalent glycan densities. Importantly, the MAPA linker can be reversibly cleaved to regenerate free reducing glycans for detailed structural analysis (catch-and-release), often critical for functional studies of undefined glycans from natural sources. The high conjugation efficiency, bright fluorescence, and reversible cleavage of the linker enable access to natural glycans for functional glycomics.

Differential recognition of oligomannose isomers by glycan-binding proteins involved in innate and adaptive immunity

“…S1). Whereas AEAB labeling is through glycan reduction, F-MAPA derivatization retains the integrity of their reducing terminal glycan ( 39 ). Probes prepared with oligomannose library 1 (#1 to 84) comprised oligomannose array set 1, whereas probes prepared with glycans in oligomannose library 2 (#1 to 100) comprised oligomannose array set 2.…”

Glycan Microarrays as Chemical Tools for Identifying Glycan Recognition by Immune Proteins

“…The F-MAPA can also be cleaved upon treatment of N-chlorosuccinimide (NCS) to regenerate free reducing glycans. This figure is reprinted (adapted) with permission fromWei et al (2019) Copyright 2019 American Chemical Society.…”

Preparation of Complex Glycans From Natural Sources for Functional Study

“…One inherent problem with commonly used reductive amination is breaking the reducing end ring structure, affecting the glycan structural integrity. To address this drawback, new methods and linkers have been reported, such as 2-amino-methyl-N,O-hydroxyethyl (AMNO) (Bohorov et al, 2006 ) and N-Fmoc-3-(methoxyamino)propylamine (F-MAPA) (Wei et al, 2019 ). We also developed a procedure to prepare HMO-AEAB conjugates with an intact reducing end ring structure (Yu et al, 2012 ).…”

“…It can also serve as an affinity tag due to the hydrophobicity. The amino group can be easily regenerated for solid-phase immobilization in microarray printing (Kamoda et al, 2005 ; Yamada et al, 2013 ; Wei et al, 2019 ). We have successfully installed an Fmoc tag on the released glycan from natural O-glycanconjugates and glycosyphingolipids in our ORNG method (Song et al, 2016 ).…”