Chemoenzymatic Synthesis of Asymmetrically Branched Human Milk Oligosaccharide Lacto-N-Hexaose

Abstract

We herein reported the first chemoenzymatic synthesis of lacto-N-hexaose (LNH) by combining chemical carbohydrate synthesis with a selectively enzymatic glycosylation strategy. A tetrasaccharide core structure GlcNH2β1→3 (GlcNAcβ1→6) Galβ1→4Glc, a key precursor for subsequent enzymatic glycan extension toward asymmetrically branched human milk oligosaccharides, was synthesized in this work. When the order of galactosyltransferase-catalyzed reactions was appropriately arranged, the β1,4-galactosyl and β1,3-galactosyl moieties could be sequentially assembled on the C6-arm and C3-arm of the tetrasaccharide, respectively, to achieve an efficient LNH synthesis. Lacto-N-neotetraose (LNnH), another common human milk oligosaccharide, was also synthesized en route to the target LNH.

Engineered plants provide a photosynthetic platform for the production of diverse human milk oligosaccharides

“… 25 ), Hp0826 (ref. 26 )), one ß-1,3-galactosyltransferase ( Cvß3GalT 27 ) and one ß-1,3- N -acetylglucosaminyltransferease ( NmLgtA 28 ) resulted in the production of lactose and various neutral HMOs with degrees of polymerization ranging from three to seven. Notably, N. benthamiana transiently expressing this pathway produced the tetrasaccharides LNT ( m / z 708.2559 ) and LNnT ( m / z 708.2559), which represent principal type I and type II HMOs in human milk, respectively 29 (Fig.…”

Plant-based production of diverse human milk oligosaccharides

“…Neutral HMOs function as the core scaffolds of other more complex HMOs (i.e., fucosylated and acidic); thus, we first targeted the type I and type II neutral HMO core structures, lacto- N -tetraose (LNT) and lacto- N -neotetraose (LNnT). Expression of a neutral HMO biosynthetic pathway using two ß-1,4-galactosyltransferases ( GalTPM1141 24 , Hp0826 25 ) , one ß-1,3-galactosyltransferase ( Cvß3GalT 26 ), and one ß-1,3- N -acetylglucosaminyltransferease ( NmLgtA 27 ) resulted in the production of lactose and various neutral HMOs with degrees of polymerization ranging from three to seven. Notably, N. benthamiana transiently expressing this pathway produced the tetrasaccharides LNT ( m/z 708.2559 ) and LNnT ( m/z 708.2559), which represent major type I and type II HMOs in human milk, respectively 28 (Fig.…”

Expression and Purification of Cp3GT: Structural Analysis and Modeling of a Key Plant Flavonol-3-O Glucosyltransferase from Citrus paradisi

“…Many GTs exhibit exceptional regio-, stereo-, and substrate specificities, making them invaluable tools for the forming of specific glycosidic bonds [16][17][18][19]. Through rational mutagenesis and directed evolution strategies, GTs have been engineered to facilitate the efficient synthesis of a diverse array of complex carbohydrates, glycoproteins, antibiotics, and herbal extracts with medicinal value [20][21][22][23][24][25][26]. The potential implications of these bioengineered molecules are profound, particularly in the pharmaceutical industry, where they play pivotal roles in drug discovery and therapeutic developments [27][28][29].…”