catalysts Review α-Glucan Phosphorylase-Catalyzed Enzymatic Reactions Using Analog Substrates to Synthesize Non-Natural Oligo- and Polysaccharides Jun-ichi Kadokawa Department of Chemistry, Biotechnology, and Chemical Engineering, Graduate School of Science and Engineering, Kagoshima University, 1-21-40 Korimoto, Kagoshima 860-0065, Japan; [email protected]; Tel.: +81-99-285-7743 Received: 9 October 2018; Accepted: 16 October 2018; Published: 19 October 2018 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. Keywords: analog substrate; α-glucan phosphorylase; non-natural oligo- and polysaccharides 1. Introduction Oligo- and polysaccharides are widely distributed in nature and enact specific important biological functions in accordance with their chemical structures [1]. Owing to a variety of sugar unit structures and various types of linkages among them in accordance with regio- and stereoarrangements, so-called glycosidic linkages, oligo- and polysaccharides typically have very complicated structures [2]. Accordingly, it has been well accepted that a profound effect on their properties and functions is often produced by a subtle change in the type of glycosidic linkage and the unit structure. The precise synthesis of structurally well-defined oligo- and polysaccharides through stereo- and regio-controlled glycosidic linkages among desired sugar units has therefore attracted much attention in the development of new functional carbohydrate-related materials. However, common organic reactions do not easily provide well-defined oligo- and polysaccharides under perfect control conditions in regio- and stereoarrangements [3–5]. Compared to such common organic reactions, enzymatic reactions exhibit significant advantages in terms of stereo- and regioselectivities. Enzymatic reaction, furthermore, can be operated under mild conditions, eliminating undesired side reactions [6,7]. Of the six main classes of enzymes, transferase (glycosyl transferase, GT) and hydrolase (glycosyl hydrolase, GH) have been used successfully as catalysts in practical synthesis of well-defined saccharide chains via the formation of glycosidic linkages [8,9]. In the enzymatically formation of glycosidic linkages, so-called ‘enzymatic glycosylation’, two substrates, that is, a glycosyl donor and Catalysts 2018, 8, 473; doi:10.3390/catal8100473 www.mdpi.com/journal/catalysts Catalysts 2018, 8, x FOR PEER REVIEW 2 of 11 Catalysts 2018, 8, 473 2 of 11 substrates, that is, a glycosyl donor and a glycosyl acceptor are used, generally, in their unprotected forms in aqueous media. A glycosidic linkage is enzymatically formed by the reaction of a glycosyl adonor glycosyl at the acceptor anomeric are used, position generally, with inthe their hydrox unprotectedy group formsof a glycosyl in aqueous acceptor media. in A regio- glycosidic and linkagestereocontrolled is enzymatically fashion. formedFrom the by viewpoint the reaction of of polymerization a glycosyl donor chemistry, at the anomeric moreover, position oligo- withand thepolysaccharides hydroxy group are of theoretically a glycosyl acceptor produced in regio-by repeated and stereocontrolled enzymatic formation fashion. of Fromglycosidic the viewpoint linkages of(enzymatic polymerization polymerization). chemistry, moreover, oligo- and polysaccharides are theoretically produced by repeatedPhosphorylases enzymatic formation are one of of GTs, glycosidic which linkages have been (enzymatic used as polymerization). catalysts for practical synthesis of oligo-Phosphorylases and polysaccharides are one of GTs,[10–12]. which Phosphoryl have beenases used ascatalyze catalysts the for practicalin vivo synthesis phosphorolysis of oligo- and(phosphorolytic polysaccharides cleavage [10– 12in]. the Phosphorylases presence of inorga catalyzenic phosphate the in vivo (Pi))phosphorolysis of glycosidic (phosphorolytic linkages at the cleavagenon-reducing in the end presence of each of specific inorganic saccharide phosphate chai (Pi))n, to ofproduce glycosidic 1-phosphat linkagese of at a the monosaccharide non-reducing endresidue of eachand specificto simultaneously saccharide chain, generate to produce the saccharide 1-phosphate chain of a monosaccharidewith one lower residuedegree and of topolymerization simultaneously (DP). generate Phosphorylases the saccharide exert chainstrict regio- with oneand lowerstereospecificities, degree of polymerization which catalyze (DP). the Phosphorylasesphosphorolysis exertof a specific strict regio- glycosidic and stereospecificities, linkage under whichether retention catalyze the or phosphorolysisinversion manner of a to specific form glycosidicmonosaccharide linkage 1-phosphates under ether retentionwith the or same, inversion or opposite, manner to anomeric form monosaccharide stereoarrangement 1-phosphates as the withglycosidic the same, linkage or opposite, (Figure 1). anomeric stereoarrangement as the glycosidic linkage (Figure1). OH OH O O HO + Inorganic phosphate HO HO OH HO O α OH O -Glucan phosphorylase (GP) O P O α-Glycosidic linkage O α-Glucose 1-phosphate (α-Glc-1-P (Glc-1-P)) OH O OH O O HO + Inorganic phosphate HO HO OH HO O P O O OH O α -Glycosidic linkage β-Glucose 1-phosphate (β-Glc-1-P) OH O OH O HO + Inorganic phosphate O HO OH HO HO O β-Glycosidic linkage OH O P O O α-Glucose 1-phosphate α ( -Glc-1-P) Figure 1. Typical reversible reactions catalyzedcatalyzed byby phosphorylases.phosphorylases. Because bondbond energyenergy of of a a glycosidic glycosidic linkage linkage in in the the starting starting saccharide saccharide chain chain is comparable is comparable to that to ofthat a phosphateof a phosphate ester at the anomericester at positionthe anomeric of the phosphorolysis position of product,the phosphorolysis phosphorylase-catalyzed product, reactionsphosphorylase-catalyzed show a reversible reactions nature. Withshow respect a revers to reversibility,ible nature. phosphorylases With respect catalyzeto reversibility, reactions forphosphorylases the manner ofcatalyze glycosidic reactions bond formationfor the manne underr adaptedof glycosidic conditions bond [formation13–17]. In theseunder reactions, adapted aconditions monosaccharide [13–17]. residue In these is transferred reactions, from a monosaccharidemonosaccharide 1-phosphate residue is to transferred the non-reducing from endmonosaccharide of a specific 1-phosphate saccharide chain to the to formnon-reducing a glycosidic end linkageof a specific with strictlysaccharide controlled chain regio-to form and a stereoarrangements,glycosidic linkage with accompanied strictly bycontrolled liberating regio- Pi. Amongand stereoarrangements, a variety of phosphorylases accompanied found by in nature,liberatingα-glucan Pi. Among phosphorylase a variety of (GP, phosphorylases also called glycogen found phosphorylase,in nature, α-glucan or starch phosphorylase phosphorylase) (GP, belongingalso called toglycogen the GT35 phosphorylase, family (EC 2.4.1.1) or starch has beenphosphorylase) widely investigated belonging and to practicallythe GT35 family employed (EC for2.4.1.1) the synthesishas been ofwidely oligo- andinvestigated polysaccharides and practically under the employed retention for manner the synthesis (Figure1 )[of13 oligo-,14,16 ,and18]. Furthermore,polysaccharides as aunder result the of the retention weak specificity manner (Figure for the recognition1) [13,14,16,18]. of non-native Furthermore, (analog) as a substratesresult of the by GP,weak non-natural specificity oligo-for the and recognition polysaccharides of non-native have been (analog) synthesized substrates by catalysis by GP, non-natural of this enzyme oligo- [19 –and21]. Onpolysaccharides the basis of the have above been background, synthesized this by review catalysis article of presentsthis enzyme GP-catalyzed [19–21]. On enzymatic the basis reactions of the usingabove analogbackground, substrates this to review precisely article synthesize presents well-defined GP-catalyzed non-natural enzymatic oligo- reactions and polysaccharides. using analog Assubstrates specific to applications precisely synthesize of amylose-based well-defined materials non-natural have been oligo- investigated and polysaccharides. to enable the As addition specific ofapplications further functionalities of amylose-based and precise materials tuning have of thebeen materials investigated in biotechnology to enable the and addition therapeutics of further [22], thefunctionalities