Enzymatic Cascades for Tailored 13C6 and 15N Enriched Human Milk Oligosaccharides

Molecules

Raphael Heinzler¹  Matthias Franzreb¹  Thomas Fischöder²  Lothar Elling²  Udo Reichl³  Erdmann Rapp³  Samanta Cajic³  Valerian Grote³ 

[1] Institute of Functional Interfaces, Karlsruhe Institute of Technology, 76131 Karlsruhe, Germany;Laboratory for Biomaterials, Institute for Biotechnology and Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University, Pauwelsstraße 20, 52074 Aachen, Germany;Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstraße 1, 39106 Magdeburg, Germany;

关键词: glycobiology;    biocatalysis;    human milk oligosaccharides;    isotopic labeling;    quantification: nucleotide sugars;    lacto-n-biose type;    lacto-n-neo type;    glycosyltransferases;    capillary gel electrophoresis;    maldi-ms;   

DOI  :  10.3390/molecules24193482

来源: DOAJ

【 摘 要 】

Several health benefits, associated with human milk oligosaccharides (HMOS), have been revealed in the last decades. Further progress, however, requires not only the establishment of a simple “routine” method for absolute quantification of complex HMOS mixtures but also the development of novel synthesis strategies to improve access to tailored HMOS. Here, we introduce a combination of salvage-like nucleotide sugar-producing enzyme cascades with Leloir-glycosyltransferases in a sequential pattern for the convenient tailoring of stable isotope-labeled HMOS. We demonstrate the assembly of [¹³C₆]galactose into lacto-N- and lacto-N-neo-type HMOS structures up to octaoses. Further, we present the enzymatic production of UDP-[¹⁵N]GlcNAc and its application for the enzymatic synthesis of [¹³C₆/¹⁵N]lacto-N-neo-tetraose for the first time. An exemplary application was selected—analysis of tetraose in complex biological mixtures—to show the potential of tailored stable isotope reference standards for the mass spectrometry-based quantification, using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) as a fast and straightforward method for absolute quantification of HMOS. Together with the newly available well-defined tailored isotopic HMOS, this can make a crucial contribution to prospective research aiming for a more profound understanding of HMOS structure-function relations.

【 授权许可】

Unknown