Versatile fluorescent derivatization of glycans for glycomic analysis

Area of Science:

• Glycomics and glycobiology
• Carbohydrate chemistry
• Bioconjugation techniques

Background:

• Functional glycomics integrates glycan structure and carbohydrate-binding protein (CBP) recognition.
• Glycan array technology is crucial for exploring these interactions.
• Current methods for preparing glycans for arrays are complex, especially for generating amine derivatives.

Purpose of the Study:

• To develop a straightforward method for derivatizing glycans with a primary amine for conjugation.
• To create fluorescently labeled glycans (glycan-DAP conjugates or GDAPs) for broader applications.
• To enable the efficient generation of new glycan arrays from natural glycans.

Main Methods:

• Derivatization of various glycans (milk sugars, N-glycans, glycosaminoglycans, chitin-derived glycans) using 2,6-diaminopyridine (DAP).
• Verification of glycan-DAP conjugates (GDAPs) using High-Performance Liquid Chromatography (HPLC) and mass spectrometry.
• Covalent conjugation of GDAPs to surfaces like N-hydroxysuccinimide (NHS)-activated glass slides, maleimide-activated protein, carboxylated microspheres, and NHS-biotin.

Main Results:

• A versatile method was established to convert diverse glycans into GDAPs containing a primary amine.
• HPLC and mass spectrometry confirmed successful derivatization.
• Conjugated GDAPs demonstrated effective recognition by relevant CBPs, validating their utility.
• Quantifiable fluorescent derivatives were successfully generated on multiple platforms.

Conclusions:

• GDAP derivatives offer a versatile tool for biologists.
• This method facilitates the quantification and covalent capture of small amounts of glycans.
• It supports the exploration of glycan structures and functions, including the creation of novel glycan arrays.