On‐Chip Synthesis and Label‐Free Assays of Oligosaccharide Arrays

Abstract

Oligosaccharides, like proteins and DNA, are ubiquitous biopolymers that mediate essential functions in organisms. [1] Yet, an understanding of the many roles that carbohydrates play is still at an early stage and essentially absent when compared to our knowledge of the functions of proteins and nucleic acids. [2] This contrast reflects the lack of convenient and flexible tools for the synthesis and biochemical analysis of oligosaccharides and their conjugates. The development of biochips-glass slides patterned with an array of hundreds to thousands of biomolecules-has been particularly important to studies in genomics and proteomics, and first became practical with the on-chip synthesis of oligonucleotides and peptides. [3,4] Herein we present a strategy for the on-chip synthesis of oligosaccharide arrays and we demonstrate that combining these arrays with mass spectrometry permits us to perform label-free assays of glycosyltransferase activities. This method represents a significant addition to the molecular strategies now used to discover and investigate the biological functions of oligosaccharides.Since our report of a carbohydrate array five years ago, [5] several examples demonstrating the preparation and application of oligosaccaharide arrays have been reported. [6] Wong, Paulson, and co-workers prepared 200 amino-substituted oligosaccharides and immobilized these molecules on N-hydroxysuccinimidate-functionalized glass slides for studies of the binding of lectins and viruses. [7] Gildersleeve and coworkers prepared arrays by printing conjugates of carbohydrates and bovine serum albumin and glycoproteins onto epoxide-functionalized glass slides and used these arrays to profile multiple lectins.[8] Seeberger and co-workers used solid phase methods to prepare seven sulfhydryl-terminated oligosaccharides and immobilized these reagents onto maleimidefunctionalized glass slides for assaying the interactions between proteins and carbohydrate epitopes. [9] In collaboration with the group of Seeberger we prepared thiol-modified high mannose oligosaccaharides, which were immobilized on self-assembled monolayers, for assays involving lectin binding.[10] The effort required to synthesize the carbohydrate reagents in this and related work is substantial, and limits the size of arrays. Additionally, the need for labeling strategies can make it difficult to perform assays and, further, is not well-suited to the identification of unanticipated activities.Our approach takes advantage of the combination of monolayers with matrix assisted laser desorption-ionization mass spectrometry (in a technique referred to as self-assembled monolayers for matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (SAMDI-TOF MS)) to directly observe the synthetic intermediates at the surface and therefore to optimize the reactions used in the synthesis of the oligosaccharides. [11,12] This method is also useful in performing assays of enzymes that modify the immobilized oligosaccharides and has been applied to nanopart...

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2007–2008

“…Aminated sugars were attached by succinimide ester chemistry whereas underivatized glycans were attached using hydrazide chemistry. Ban and Mrksich (2008) have prepared a 6 Â 4 array containing the disaccharides Gal-Gal, Glc-Gal, Gal-Glc, Glc-Glc, Gal-GlcNAc, and Glc-GlcNAc in all four linkages (b1 ! 2, b1 !…”

Oligosaccharide Synthesis on Solid, Soluble Polymer, and Tag Supports

“…Although oligosaccharide synthesis on solid, soluble polymer, and tag supports has been dramatically developed as described, a high level of technical expertise is still required for the oligosaccharide synthesis. As the direct construction and assay techniques of the oligosaccharides library on the array, 110 as well as the utilization of the new surface platform for the iterative glycan synthesis, 111 have rapidly been emerging, further improvements in the oligosaccharide synthesis on the solid supports is expected to establish the general and efficient methods, which will speed up the elucidation of the biological functions of oligosaccharides as well as clinical applications of oligosaccharide-based drugs. …”

Natural glycan microarrays

“…Many different linker and spacer designs available; in situ synthesis [101,102] Derivatization and fractionation approach restricts the options for linker design and immobilization chemistry Glycan structural assignment 1 H NMR and 13 C NMR can ascertain anomeric purity and structural integrity [36]; SAMDI-TOF MS [102] MS; characterization by chromatographic, electrophoretic, and mass spectrometric means [34] GPI: Glycosylphosphatidylinositol; HPTLC: High-performance thin-layer chromatography; MS: Mass spectrometry; NGL: Neoglycolipid; NMR: Nuclear magnetic resonance; SAMDI-TOF: Self-assembled monolayer desorption ionization-time-of-flight. Natural glycan microarrays be generated using 6-biotinyl-aminocaproyl hydrazide (BACH [ Figure 2C]), resulting in a closed-ring innermost monosaccharide structure [60,61].…”