α-Glucan Phosphorylase-Catalyzed Enzymatic Reactions Using Analog Substrates to Synthesize Non-Natural Oligo- and Polysaccharides

Abstract

As natural oligo- and polysaccharides are important biomass resources and exhibit vital biological functions, non-natural oligo- and polysaccharides with a well-defined structure can be expected to act as new functional materials with specific natures and properties. α-Glucan phosphorylase (GP) is one of the enzymes that have been used as catalysts for practical synthesis of oligo- and polysaccharides. By means of weak specificity for the recognition of substrates by GP, non-natural oligo- and polysaccharides has precisely been synthesized. GP-catalyzed enzymatic glycosylations using several analog substrates as glycosyl donors have been carried out to produce oligosaccharides having different monosaccharide residues at the non-reducing end. Glycogen, a highly branched natural polysaccharide, has been used as the polymeric glycosyl acceptor and primer for the GP-catalyzed glycosylation and polymerization to obtain glycogen-based non-natural polysaccharide materials. Under the conditions of removal of inorganic phosphate, thermostable GP-catalyzed enzymatic polymerization of analog monomers occurred to give amylose analog polysaccharides.

Chemoenzymatic Synthesis of Fluorinated Cellodextrins Identifies a New Allomorph for Cellulose‐Like Materials**

“…As an alternative, enzymatic synthesis presentsa na t-tractive approach, [11,12] enabling the bottom-up preparation of site-specifically modified oligo-and polysaccharides in ar egioand stereocontrolled manner. [13][14][15][16] Specifically in relationt o glucose-based materials, glycoside phosphorylases( GPs) [17][18][19][20][21] have shown substantial potentialf or the synthesis of amyloseand cellulose-like materials. In particular,c ellodextrin phosphorylase (CDP,E C2 .4.1.49) [22][23][24] has emerged as ap owerful tool for the synthesiso fd ifferently functionalised cellulose oligomers, giving rise to av ariety of nanostructures (sheets, [25,26] rods, [27] or ribbons [28] )d epending on the nature of the substrate.…”

“…[29] In connection with this study,w eh ad an eed for the site-specific introduction of probesi nto cellulose to report on local structure and solvation, and potentially to modulate materialp roperties.F luorine is well known for its unique physicochemical properties, such as small size, high electronegativity,g reat polarity and stability of the CÀFb ond. [30] In addition, the absence of fluorine in biological systems and int he majority of materials makes the introduction of 19 Fn uclei ap owerful reportero fl ocal structure and environment. For instance, 19 FNMR spectroscopy has been used to monitorc rystallisation in nanoporous materials [31] and fibril formation of intrinsically disordered proteins, [32] to characterise polymeric biomaterials, [33] and to map the interactions of fluorinated oligosaccharides with protein targets.…”

“…[30] In addition, the absence of fluorine in biological systems and int he majority of materials makes the introduction of 19 Fn uclei ap owerful reportero fl ocal structure and environment. For instance, 19 FNMR spectroscopy has been used to monitorc rystallisation in nanoporous materials [31] and fibril formation of intrinsically disordered proteins, [32] to characterise polymeric biomaterials, [33] and to map the interactions of fluorinated oligosaccharides with protein targets. [34] Nonetheless,t he use of fluoriner emains under-explored with respectt oc arbohydrate-basedm aterials.…”

Thermostable α-Glucan Phosphorylase-Catalyzed Enzymatic Copolymerization to Produce Partially 2-Deoxygenated Amyloses

“…Since it is well recognized that the enzymatic method is a useful approach to generate well-defined oligo-and polysaccharides [6][7][8][9][10], we investigated α-glucan phosphorylase-catalyzed enzymatic reactions using several monosaccharide 1-phosphates as substrates to obtain non-natural oligoand polysaccharides [11][12][13][14][15]. α-Glucan phosphorylase catalyzes the enzymatic polymerization of α-d-glucose 1-phosphate (Glc-1-P) monomers from a maltooligosaccharide primer to produce α(1→4)-glucan-i.e., amylose-upon the liberation of inorganic phosphate (Pi) [16][17][18][19][20][21][22][23].…”

“…This enzymatic 2 of 8 polymerization corresponds to the following reversible chain-elongation (glycosylation) and phosphorolysis: [α(1→4)-Glc] n + Glc-1-P [α(1→4)-Glc] n+1 + Pi. Owing to the weak specificity for the substrate recognition of this enzyme, some substrate analogs of Glc-1-P (1-phosphates of different monosaccharides) can be used in enzymatic reactions to yield amylose analogs [11][12][13][14][15]. For example, we previously reported the synthesis of an aminopolysaccharide amylose analog composed of α(1→4)-linked glucosamine (GlcN) (named "amylosamine") as a repeating unit upon the enzymatic polymerization of α-d-glucosamine 1-phosphate (GlcN-1-P), as catalyzed by a thermostable α-glucan phosphorylase (isolated from Aquifex aeolicus VF5 thermophilic bacteria) [24].…”