Strategies for chemoenzymatic synthesis of carbohydrates

Abstract

Carbohydrates are structurally complex but functionally important biomolecules. Therefore, they have been challenging but attractive synthetic targets. While substantial progress has been made on advancing chemical glycosylation methods, incorporating enzymes into carbohydrate synthetic schemes has become increasingly practical as more carbohydrate biosynthetic and metabolic enzymes as well as their mutants with synthetic application are identified and expressed for preparative and large-scale synthesis. Chemoenzymatic strategies that integrate the flexibility of chemical derivatization with enzyme-catalyzed reactions have been extremely powerful. Briefly summarized here are our experiences on developing one-pot multienzyme (OPME) systems and representative chemoenzymatic strategies from others using glycosyltransferase-catalyzed reactions for synthesizing diverse structures of oligosaccharides, polysaccharides, and glycoconjugates. These strategies allow the synthesis of complex carbohydrates including those containing naturally occurring carbohydrate postglycosylational modifications (PGMs) and nonnatural functional groups. By combining these strategies with facile purification schemes, synthetic access to the diverse space of carbohydrate structures can be automated and will not be limited to specialists.

The role of 9-O-acetylated glycan receptor moieties in the typhoid toxin binding and intoxication

“…9-O-Acetylated derivatives of those glycans were synthesized using one-pot three-enzyme sialylation systems in the Xi Chen laboratory [22]. In brief, sialosides containing 9-O-acetylated Neu5Ac (Neu5,9Ac 2 α2-3Galβ1-4GlcNAcβProN 3 and Neu5,9Ac 2 α2-6Galβ1-4GlcNAcβProN 3 ), and 4-O-acetylated Neu5Ac (Neu4,5Ac 2 α2-3Galβ1-4GlcNAcβProN 3 ) were synthesized from Galβ1-4GlcNAcβProN 3 (LacNAcβProN 3 ) [33] as described previously using Neu5,9Ac 2 [34] and Neu4,5Ac 2 [35], respectively, as the donor precursors in an one-pot two-enzyme sialylation system [36] containing Neisseria meningitidis CMP-sialic acid synthetase (NmCSS) [37] and a sialyltransferase. Pasteurella multocida α2-3 sialyltransferase 1 M144D (PmST1 M144D) mutant [38] was used for the synthesis of Neu5,9Ac 2 α2-3Galβ1-4GlcNAcβProN 3 and Photobacterium sp.…”

Glycosyltransferase Co‐Immobilization for Natural Product Glycosylation: Cascade Biosynthesis of the C‐Glucoside Nothofagin with Efficient Reuse of Enzymes

“…Like other enzymatic reactions that require (co)‐substrates (e. g., nicotinamide coenzymes for oxidoreductions; [5] nucleoside triphosphates for phosphorylation [6] or nucleotide transfer; [7] S ‐ adenosylmethionine for methylation [8] ) which for reasons of atom economy and costs cannot be used stoichiometrically, the overall glycosyltransferase reaction typically consists of a multistep cascade transformation in which the immediate enzymatic glycosylation is supplied in situ with the corresponding sugar nucleotide donor substrate [1,9] . Besides synthesis from free monosaccharide via phosphorylation and nucleotide transfer cascades, [9a] sugar nucleotides are often prepared through a second glycosyltransferase reaction, run in reverse direction and operated in parallel to the synthetic reaction [1a,3,9b] . A suitably activated glycoside serves as the donor for in situ sugar nucleotide synthesis.…”

Cell-Free Synthetic Glycobiology: Designing and Engineering Glycomolecules Outside of Living Cells

“…The utilization of completely enzyme-catalyzed synthesis of nucleotide sugars from simple monosaccharides in a single-pot reaction has become an attractive platform due to its simplicity, versatility, and ability to be coupled with GT-catalyzed reactions. One-pot multienzyme (OPME) synthesis technology was developed to realize this goal and has now become a widely used system (Li W. et al, 2019 ). In its simplest system, OPME involves modification of the monosaccharide with a nucleotide pytophosphate group using multiple enzymes in a single-pot reaction.…”