Enzymatic modular synthesis of deuterium-labeled human milk oligosaccharides and application in sequence assignment...

Human milk oligosaccharides (HMOs) are crucial nutritional and bioactive components of human milk, providing multiple health benefits to neonates. Nevertheless, their structural heterogeneity and complexity present a major difficulty for their separation, sequence assignment and quantitation, hindering their further detailed structure-function studies. In response to this challenge, we established a robust enzymatic synthesis platform for the rapid and scalable production of deuterium-labeled linear and branched oligosaccharides representing the major structures present in huma milk, including variously fucosylated and sialylated sequences. These synthesized heavy isotope-labeled oligosaccharides were employed in mass spectrometry (MS)-based methodologies for sequence determination by establishing MS fragmentation pattern and for absolute quantitation as internal standards. The site-specific GlcNAc-D₃ labeling is particularly important for assignment of asymmetric β1,3/6-branching patterns, type 1 and type 2 backbone chains and fucosylated blood group H(O) and Lewis a and x epitopes by tandem MS. The library of 18 well-defined D₃-labeled HMOs provides reliable internal standards for absolute rather than relative quantitation of oligosaccharides in complex mammalian secretions. These advantages have significant potential values in the studies of HMO structure-function relationships and their quality control in specialized formulations, such as functional foods for gut health and infant formulas.