Enzymatic Building‐Block Synthesis for Solid‐Phase Automated Glycan Assembly

Abstract

Automated chemical oligosaccharide synthesis is an attractive concept that has been successfully applied to a large number of target structures, but requires excess quantities of suitably protected and activated building blocks. Herein we demonstrate the use of biocatalysis to supply such reagents for automated synthesis. By using the promiscuous NmLgtB‐B β1‐4 galactosyltransferase from Neisseria meningitidis we demonstrate fast and robust access to the LacNAc motif, common to many cell‐surface glycans, starting from either lactose or sucrose as glycosyl donors. The enzymatic product was shown to be successfully incorporated as a complete unit into a tetrasaccharide target by automated assembly.

Immobilization Screening and Characterization of an Alcohol Dehydrogenase and its Application to the Multi-Enzymatic Selective Oxidation of 1,-Omega-Diols

“…Immobilization of His-tagged enzymes is one of the most preferred strategies to control the enzyme orientation due to its versatility to purify and immobilize recombinantly expressed enzymes on a great variety of carries and in one-pot (Mateo et al, 2006;Patel et al, 2017). Paradisi's and Flitch's groups (Contente et al, 2019;Marchesi et al, 2020) are exploiting His-tagged enzymes to control their orientation upon immobilization on porous carriers functionalized with metal-complexes. These immobilized systems enable to carry out telescoped synthetic schemes in flow.…”

Biocatalytic Approaches to Building Blocks for Enzymatic and Chemical Glycan Synthesis

“…Scheme highlights recent methods utilized to prepare NDP-sugars in situ for subsequent use with glycosyltransferases. This includes sucrose synthase (SuSy), which provides UDP- d -Glc to then undergo C4-epimerization and provide cheap access to UDP- d -Gal (Scheme A) . The use of galactokinase (GalK) or N -acetylhexosamine 1-kinase (NahK) has provided the corresponding anomeric phosphates, followed by pyrophosphorylative coupling using UDP-pyrophosphorylase (USP) or GlcNAc-1P uridylyltransferase (GlmU/AGX1) to grant UDP-hexose/hexosamine or uronate sugars (Scheme B–E). − Due to the reversible nature of this pyrophosphate formation, the inclusion of inorganic pyrophosphatase (iPPase) hydrolyzes the inorganic pyrophosphate produced (to inorganic phosphate), thus rendering the coupling reaction irreversible.…”

Recent Developments in Solid-Phase Glycan Synthesis

“…Recently, a biocatalytic approach assisted the preparation of a selectively protected N -acetyllactosamine (LacNAc) motif, which, after a few chemical manipulations, was employed in the solid-phase synthesis of a tetrasaccharide. 59 This enzymatic strategy combined the efficiency and scalability of biocatalysis with the flexibility of chemical synthesis; if implemented to a wider range of BBs, this approach could speed up SPGS significantly. For many years, BB preparation has been the rate-determining step of SPGS; rapid access to properly protected BBs will fuel the synthesis of new structures and the implementation of new solid-phase reactions.…”