Abstract
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.
Highlights
It has become increasingly apparent that human milk oligosaccharides (HMOS) consist of a cocktail of complex glycans and play important biological roles well beyond simple nutrition
Regarding the repetitive batch synthesis strategy, a centrifugal concentrator was utilized to carry out the enzymatic synthesis and for the subsequent product separation of the respective batch
We strived to establish an approach to the enzymatic synthesis of stable isotope-labeled HMOS that can be employed as internal standards for quantitative analysis
Summary
It has become increasingly apparent that human milk oligosaccharides (HMOS) consist of a cocktail of complex glycans and play important biological roles well beyond simple nutrition. HMOS are present in large quantities and are the third most abundant group of compounds in human milk, directly after lactose and lipids [3,4,5]. They display a unique and extremely high structural diversity [2,3,6,7], with approximately 150 elucidated structures so far [8]. It has been found that biological functions depend on both, the specific structure and the quantity of the HMOS present [8,9] Striking is their remarkable variability in composition and concentration of milk oligosaccharides (milk OS) during lactation depending on the donor phenotype [10]. A rising amount of HMOS is authorized as a nutrition supplement, which promotes an urgent future request for a rapid and robust “routine” quantification method for suitable quality control [11,12,13]
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