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Authors: Japan Bioindustry Association, Advanced Industrial Science and Technology, Japan
- Deacetylation of glycopeptide with an esterase prevents base-induced side reactions, b-elimination, and epimerization
- An esterase from B. subtilis was utilized for Deacetylation of sugar hydroxyl groups on glycopeptide synthesis
- Deacetylation with an esterase and glycosylations with glycosyltransferases were accomplished in one-pot
Glycopeptides are fragments of glycoproteins and are important in evaluating the biological roles of carbohydrates in glycoproteins. Fmoc solid-phase peptide synthesis using acetyl-protected glycosylated amino acids is a common strategy for the preparation of glycopeptides, but this approach normally requires chemical de-O-acetylation with a base that β-eliminates sugar residues and epimerizes the peptide backbone. Here we demonstrate a facile new chemoenzymatic synthetic strategy for glycopeptides, using an esterase for the de-O-acetylation of sugar residues and glycosyltransferases for successive sugar elongations at neutral pH.
ESI-MS spectra were collected using an Advion expression Compact Mass Spectrometer (CMS) in negative ion mode.
There are many separation and detection methods that are available for the determination of saccharides. These include GC-MS with derivatization, HPLC-UV with derivatization, HPAEC-PAD (High-performance anion-exchange chromatography coupled with pulsed amperometric detection), and LC-MS. The availability of a sensitive and accurate method is still a challenge. For an LC-MS method, given the high polarity, hydrophilicity and low proton affinity of these compounds, it can be difficult to ionize them directly by electrospray by ESI or APCI. The chemical diversity and numerous isomers of carbohydrates further complicates their analysis with LC-MS.
In this Application Note, post-column chloride attachment is used for the analysis of samples containing carbohydrates using the Advion expression® Compact Mass spectrometer (CMS). The 35Cl and 37Cl isotopes also provide confirmation for the formation of the chloride adducts in negative ion APCI mode. Four carbohydrates (Fructose, Glucose, Sucrose and Lactose) are used for method verification.
Q: WHAT IS THE FOCUS OF YOUR LAB’S RESEARCH?
A: The research in the Biosyn group is focused on the design, synthesis and function of the four major types of biomolecules: nucleic acids, carbohydrates, peptides and lipids and hybrid structures thereof. These biomolecules and their derivatives are used in drug discovery and chemical biology, to develop synthetic methodology or as an inspiration for mimetic design.
Q: WHAT WAS YOUR PREVIOUS EXPERIENCE?
A: I started as organic chemist on Pt-adducts on self-made DNA fragments under supervision of Professor Dr. J. Reedijk. Following that, I switched to synthesis of bio-organic molecules on solid support under supervision of Professor Dr. J.van Boom. Currently I am working with sophisticated analytical equipment including many (prep) LCMS systems under supervision of Professor Dr. H. Overkleeft. With this equipment we show our facility to many other workgroups within and outside the University of Leiden.
Q: WHY DID YOU INCORPORATE THE EXPRESSION CMS INTO YOUR LABORATORY?
A: It’s easy, walk-up use with straightforward swapping of ESI and APCI sources make it a reliable, productive tool for determining synthetic success.
Q: HOW DID THE EXPRESSION CMS HELP RESOLVE YOUR CHALLENGES?
A: I incorporated the expression CMS as TLC/MS system because of its simplicity, low cost and the brilliant combination with the Plate Express (as a TLC interface).
Q: TO WHOM WOULD YOU RECOMMEND THE EXPRESSION CMS?
A: I recommend the expression CMS to people who want to use a robust system with simple handling (like organic chemists).
Fully characterize oligosaccharides that are missed by LC/MS/MS.
Download this product note to find out how.
Zamfir, A.;Vakhrushev, S.; Sterling , S.; Niebel, H.J.; Allen, M.; Peter-Katalinic, J.
Anal. Chem. 2004;76:2046-2054