Highly efficient one-pot multienzyme (OPME) synthesis of glycans with fluorous-tag assisted purification

Abstract

Oligo(ethylene glycol)-linked light fluorous tags have been found to be optimal for conjugating to glycans for both high-yield enzymatic glycosylation reactions using one-pot multienzyme (OPME) systems and quick product purification using fluorous solid-phase extraction (FSPE) cartridges. The combination of OPME glycosylation systems and the FSPE cartridge purification scheme provides a highly effective strategy for facile synthesis and purification of glycans.

Highly efficient chemoenzymatic synthesis and facile purification of α-Gal pentasaccharyl ceramide Galα3nLc4βCer

“…The method allows the enzyme-catalyzed glycosylation reactions to occur in aqueous solutions and facile purifications by solid-phase extraction. 23 An additional advantage is that intermediates for long chain complex glycosphingosines can be acylated to form other naturally occurring glycosphingolipids.…”

“…Lactosyl sphingosine (LacβSph, 5 ) was readily soluble in aqueous solution for up to 30 mM, allowing it be used efficiently as a starting glycosyltransferase acceptor for enzymatic extension using one-pot multienzyme (OPME) reactions 23, 40-45 for the synthesis of more complex structurally diverse glycosphingosines. The sphingosine (lipid) component of the acceptor and the product can be used as an anchor to allow facile purification of the glycosphingosines by reverse phase column chromatography such as simple C18 cartridge-based purification.…”

“…The last OPME reaction was carried out to convert the obtained nLc 4 βSph ( 3 ) to Galα3nLc 4 βSph ( 2 ) using a galactose activation and transfer system containing SpGalK, BLUSP, PmPpA, and a recombinant bovine α1–3-galactosyltransferase (Bα1–3GalT) 23, 52 in Tris-HCl buffer (100 mM, pH 7.5) at 37 °C for 48 hours. The donor substrate UDP-Gal was generated from galactose in situ as described in the previous step for stereo-selective production of the desired Galα3nLc 4 βSph ( 2 ).…”

Assembly of a Complex Branched Oligosaccharide by Combining Fluorous‐Supported Synthesis and Stereoselective Glycosylations using Anomeric Sulfonium Ions

“…[9] Several fluorous versions of protecting groups have been developed for a variety of functional groups, and thus tags can easily be installed. [10] Additionally, it is possible to array fluorous-tagged glycans, thereby eliminating the necessity to install reactive functional groups for glycan immobilization. [11] …”

“…[10] This limited application is most likely due to the difficulties of controlling anomeric selectivities in glycosylations and challenges to install branching points in high yield. [5a, 6a, 11] In this respect, many complex oligosaccharides are branched and due to steric crowding, the corresponding glycosylations are often low yielding.…”