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

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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 non-natural and materials precise described tuning hereinof the can materi be expectedals in biotechnology to act as new glycomaterialsand therapeutics for cosmetics,[22], the

Catalysts 2018, 8, x FOR PEER REVIEW 3 of 11 Catalysts 2018, 8, 473 3 of 11 non-natural materials described herein can be expected to act as new glycomaterials for cosmetics, biomedical,biomedical, andand pharmaceuticalpharmaceutical applications,applications, and for supplements used used in in animal animal farming farming and and in in foodfood[18]. [18].

2.2. Characteristics Characteristics ofof GP-CatalyzedGP-Catalyzed EnzymaticEnzymatic Reactions GPGP isis thethe enzymeenzyme thatthat catalyzescatalyzes thethe reversiblereversible phosphorolysisphosphorolysis of α(1(1→→4)-glucans4)-glucans at at the the non-reducingnon-reducing end,end, suchsuch asas starchstarch andand glycogen,glycogen, in the presence of Pi, to produce αα--DD-glucose-glucose 1-phosphate1-phosphate (α(α-Glc-1-P,-Glc-1-P, hereafterhereafter simplysimply Glc-1-P)Glc-1-P) (Figure(Figure2 a)2a) [ [10,11].10,11]. Depending Depending on on reaction reaction conditions,conditions, GPGP catalyzescatalyzes thethe reversereverse chain-elongationchain-elongation reaction, that is, is, glycosylation glycosylation of of Glc-1-P Glc-1-P and and → αα(1(1→4)-glucan4)-glucan as as a glycosyla glycosyl donor donor and a glycosyland a acceptor,glycosyl respectively, acceptor, torespectively, form an α(1 →to4)-glycosidic form an → linkage,α(1 4)-glycosidic in which a glucoselinkage, residuein which enzymatically a glucose residue transfers enzymatically from Glc-1-P transfers to the non-reducingfrom Glc-1-P to end the of non-reducing end of α(1→4)-glucans, such as maltooligosaccharides in regio- and stereocontrolled α(1→4)-glucans, such as maltooligosaccharides in regio- and stereocontrolled fashions (Figure2b). fashions (Figure 2b). The catalytic mechanism depends on close contact of the phosphate group in The catalytic mechanism depends on close contact of the phosphate group in the covalently bound the covalently bound cofactor pyridoxal 5′-phosphate to either inorganic phosphate (in the direction cofactor pyridoxal 50-phosphate to either inorganic phosphate (in the direction of phosphrolysis) or of phosphrolysis) or the phosphate group of Glc-1-P (in the direction of glycosylation) [23–26]. the phosphate group of Glc-1-P (in the direction of glycosylation) [23–26]. Speciﬁcally, GP has the Specifically, GP has the smallest DP value in α(1→4)-glucan chains to recognize, and accordingly, smallest DP value in α(1→4)-glucan chains to recognize, and accordingly, maltooligosaccharides maltooligosaccharides with DPs higher than the smallest one should be used as the glycosyl with DPs higher than the smallest one should be used as the glycosyl acceptor. The smallest acceptor. The smallest substrates for the phosphorolysis and glycosylation recognized by the most substrates for the phosphorolysis and glycosylation recognized by the most widely studied GP widely studied GP isolated from potato are maltopentaose (Glc5) and maltotetraose (Glc4), isolated from potato are maltopentaose (Glc ) and maltotetraose (Glc ), respectively. On the other respectively. On the other hand, it has been found5 that the smallest DPs4 of substrates accepted by GP hand, it has been found that the smallest DPs of substrates accepted by GP isolated from thermophilic isolated from thermophilic bacteria sources (thermostable GP) for the former and latter reactions are bacteria sources (thermostable GP) for the former and latter reactions are one smaller than those by one smaller than those by potato GP, i.e., Glc4 and maltotriose (Glc3), respectively [18,27–30]. As potato GP, i.e., Glc and maltotriose (Glc ), respectively [18,27–30]. As enzymes often show weak enzymes often show4 weak specificity for 3the recognition of the substrate structure, GPs have been speciﬁcity for the recognition of the substrate structure, GPs have been found to recognize some found to recognize some analog substrates of Glc-1-P (1-phosphates of different monosaccharide analogresidues) substrates depending of Glc-1-P on their (1-phosphates sources [19–21]. of different Therefore, monosaccharide the extension residues) of dependingthe GP-catalyzed on their sourcesenzymatic [19– 21reactions]. Therefore, has been the extension investigated of the using GP-catalyzed several monosaccharide enzymatic reactions 1-phosphates has been investigated to obtain usingnon-natural several monosaccharideoligosaccharides 1-phosphateshaving the different to obtain monosaccharide non-natural oligosaccharides residues at the havingnon-reducing the different end monosaccharide(Figure 3, vide infra). residues at the non-reducing end (Figure3, vide infra).

HO O HO HO O O HO HO O + HO OH HO HO OH HO OH O O P O O OH OHO OH O n Glc-1-P (glycosyl donor) Maltooligosaccharide (glycosyl acceptor) (a)

GP (c) (b) HO HO O HO O Glc-1-P (monomer) / GP O HO HO O O HO HO OH HO HO OH O HO OH O O O + Pi HO OH HO HO OH O Enzymatic polymerization O OHO OH OH α(14)-Glucan (amylose) α OHO OH (14)-Glycosidic linkage n + Inorganic phosphate (Pi) FigureFigure 2.2. α-Glucanα-Glucan phosphorylase phosphorylase (GP)-catalyzed (GP)-catalyzed (a) phosphorolysis, (a) phosphorolysis, (b) glycosylation, (b) glycosylation, and (c) andpolymerization. (c) polymerization.

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HO O HO O O HO O + HO OH HO HO HO OH O O P O O OH OHO OH O n Analog substrates Glycosyl acceptor (glycosyl donors) O HO O HO HO O GP O HO OH HO + Pi HO OH O O OH OHO OH n

OH O OH OH OH O O O O O O O HO HO HO HO HO = HO HO HO HO OH NHR HO OH HO O GlcA Man dGlc Xyl R = H , H Recognized by potato GP GlcN (GlcNF) Recognized by thermostable GP (from Aquifex aeolicus VF5)

Figure 3.3. GP-catalyzedGP-catalyzed enzymatic enzymatic glycosylations glycosylations using using analog substratessubstrates (monosaccharide(monosaccharide 1-phosphates) as glycosyl donorsdonors toto produceproduce non-naturalnon-natural oligosaccharides.oligosaccharides.

When an excessexcess donor/acceptordonor/acceptor (Glc-1-P/maltooligosaccharide) molar molar ratio ratio is is employed in the reaction system,system, GP GP catalyzes catalyzes consecutive consecutive glycosylations glycosylations as the as manner the manner of polymerization of polymerization to produce to theproduceα(1→ the4)-glucan α(1→4)-glucan polymer, polymer, i.e., amylose i.e., amylose (Figure2 (Figurec) [ 30– 332c)], [30–33], which spontaneously which spontaneously forms a forms double a helicaldouble assemblyhelical assembly as the precipitateas the precipitate in the reaction in the re mediaaction [34media,35]. [34,35]. It has beenIt has reported been reported that within that thewithin double the double helix, interstrandhelix, interstrand stabilization stabilization is achieved is achieved without without any stericany steric conﬂict conflict and and through through the occurrencethe occurrence of O(2) of··· O(2)O(6)⋯ typeO(6) of type hydrogen of hydrogen bonds [35 ].bonds The GP-catalyzed [35]. The enzymaticGP-catalyzed polymerization enzymatic ofpolymerization Glc-1-P as a monomer of Glc-1-P belongs as a to monomer chain-growth belong polymerizations to chain-growth because polymerization the reaction is initiated because at the non-reducingreaction is initiated end of theat the acceptor non-reducing and the propagation end of the consecutivelyacceptor and progresses the propagation from the consecutively non-reducing endprogresses of the producingfrom the non-reducingα(1→4)-glucan end chain. of the Accordingly, producing theα(1 glycosyl→4)-glucan acceptor chain. is Accordingly, often referred the to theglycosyl “primer” acceptor of the is polymerization. often referred to As the the “prime GP-catalyzedr” of the polymerization polymerization. is conceived As the GP-catalyzed analogously topolymerization a living polymerization, is conceived theanalogously molecular to weight a living of polymerization, the produced amylose the molecular can be weight controlled of the by theproduced monomer/primer amylose can feedbe controlled ratio, and by the the distribution monomer/ ofprimer the polymeric feed ratio, product and the isdistribution typically narrow of the (polymericMw/Mn < product 1.2) [36]. is typically narrow (Mw/Mn < 1.2) [36]. Polymers having pluralplural non-reducing α(1→4)-glucan chain ends have been used as polymeric primers for GP-catalyzed enzymatic polymerization of Glc-1-P to obtain amylose-grafted and amylose-branched polymeric materials [22,37–43]. [22,37–43]. For example, glycogen, which is a water-soluble highly branched branched natural natural polysaccharide polysaccharide composed composed of of α(1α→(1→4)-glucan4)-glucan chains chains further further interlinked interlinked by byα(1α→(16)-branching→6)-branching points, points, has has been been used used as asthe the polymeric polymeric primer primer for for GP-catalyzed enzymatic polymerization owing to the presence of a numbernumber ofof non-reducingnon-reducing αα(1(1→4)-glucan chain ends (Figure4 4a)a) [[44].44]. When the GP-catalyzed enzymatic polymerization of Glc-1-P from non-reducingnon-reducing ends of glycogenglycogen waswas conductedconducted inin acetateacetate buffer, buffer, and and the the reaction reaction mixture mixture was was then then left left standing standing at roomat room temperature temperature for 24for h, 24 it turnedh, it turned completely completely into a hydrogel into a form.hydrogel Hydrogelation form. Hydrogelation is reasonably is explainedreasonably by explained the double-helix by the formationdouble-helix by theformation elongated by amylose the elongated chains amongamylose glycogens chains among [34,35], whichglycogens act as[34,35], cross-linking which act points as cross-linking for construction points of for network construction structure. of network structure.

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FigureFigure 4. 4.GP-catalyzed GP-catalyzed( a(a)) polymerization polymerization toto produceproduce glycogenglycogen hydrogel,hydrogel, (b(b)) glucuronylation glucuronylation and and subsequentsubsequent glucosaminylation glucosaminylation to to produce produce amphoteric amphoteric glycogen, glycogen, and and ( c()c) following following polymerization polymerization to to produceproduce amphoteric amphoteric glycogen glycogen hydrogel. hydrogel.

3.3. SynthesisSynthesis of of Non-Natural Non-Natural Oligosaccharides Oligosaccharides by by Phosphorylase-Catalyzed Phosphorylase-Catalyzed Enzymatic Enzymatic GlycosylationsGlycosylations Using Using Analog Analog Substrates Substrates PotatoPotato GPGP hashas been found to to recognize recognize several several 1-phosphates 1-phosphates of ofmonosaccharide monosaccharide residues residues as the as the analog substrates of Glc-1-P, such as α-D-mannose (Man), α-D-xylose (Xyl), α-D-glucosamine analog substrates of Glc-1-P, such as α-D-mannose (Man), α-D-xylose (Xyl), α-D-glucosamine (GlcN), (GlcN), and N-formyl-α-D-glucosamine (GlcNF) 1-phosphates (Figure3)[ 45–49]. Potato GP-catalyzed and N-formyl-α-D-glucosamine (GlcNF) 1-phosphates (Figure 3) [45–49]. Potato GP-catalyzed glycosylations using such glycosyl donors have been reported to take place with Glc4 to produce glycosylations using such glycosyl donors have been reported to take place with Glc4 to produce non-naturalnon-natural pentasaccharides pentasaccharides having having the the respective respective monosaccharide monosaccharide residues residues at the atnon-reducing the non-reducing end. α end.-Glucosaminylated α-Glucosaminylated pentasaccharide pentasaccharide obtained from obtained glycosylation from using glycosylation GlcN-1-P (glucosaminylation) using GlcN-1-P is(glucosaminylation) a basic oligosaccharide is a basic having oligosaccharide an amino having group atan theamino C-2 group position at the of C-2 a GlcNposition unit of ata GlcN the non-reducingunit at the non-reducing end. end. 2-Deoxy-α-D-glucose 1-phosphate (dGlc-1-P) was also found to be recognized by potato GP 2-Deoxy-α-D-glucose 1-phosphate (dGlc-1-P) was also found to be recognized by potato GP (Figure(Figure3 )[3) 50[50].]. Interestingly,Interestingly, this this substrate substrate could could be be generated generated byby two-steptwo-step processesprocesses inin situsitu using using D-glucal as a substrate. In a ﬁrst step, D-glucal is transferred as a 2-deoxy-D-glucose unit to the D-glucal as a substrate. In a first step, D-glucal is transferred as a 2-deoxy-D-glucose unit to the non-reducing end of α(1→4)-glucan in the presence of Pi. In a second step, a 2-deoxy-D-glucose residue non-reducing end of α(1→4)-glucan in the presence of Pi. In a second step, a 2-deoxy-D-glucose isresidue released is from released the non-reducing from the non-reducing end of the product end of by the potatoproduct GP-catalyzed by the potato phosphorolysis GP-catalyzed to α → producephosphorolysis dGlc-1-P, andto produce consequently, dGlc-1-P,(1 4)-glucanand consequently, remained intact. α(1 Accordingly,→4)-glucan Dremained-glucal has intact. been employed as a glycosyl donor in the GP-catalyzed enzymatic glycosylation. In the enzymatic reaction Accordingly, D-glucal has been employed as a glycosyl donor in the GP-catalyzed enzymatic glycosylation. In the enzymatic reaction in the presence of D-glucal, Glc4, and a small amount of Pi,

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in the presence of D-glucal, Glc4, and a small amount of Pi, consecutive glycosylations progressed to produce 2-deoxy-α-D-glucosylated penta-heptasaccharides and higher molecular weight products with an average DP of 12. Thermostable GP has been found to show more tolerance in the recognition speciﬁcities for monosaccharide 1-phosphates than those of potato GP. For example, potato GP does not recognize α-D-glucuronic acid 1-phosphate (GlcA-1-P), while thermostable GP isolated from Aquifex aeolicus VF5 recognizes GlcA-1-P in addition to the abovementioned analog glycosyl donors (Figure3)[ 51]. Accordingly, the thermostable GP catalyzes glycosylation using GlcA-1-P with Glc3 (glucuronylation) to produce a tetrasaccharide having a GlcA residue at the non-reducing end, which is an acidic oligosaccharide owing to the presence of a carboxylate group at the C-6 position of a GlcA unit. By means of the above-mentioned thermostable GP-catalyzed glucosaminylation and glucuronylation, amphoteric polysaccharides with highly branched structure, such as an amphoteric glycogen, having both the basic GlcN and acidic GlcA residues at the non-reducing ends, have been synthesized (Figure4b) [ 52,53]. The thermostable GP-catalyzed glucuronylation using GlcA-1-P with highly branched polysaccharides has been conducted to produce acidic materials [54]. Then, the thermostable GP-catalyzed glucosaminylation using GlcN-1-P with the acidic products was performed to obtain amphoteric highly branched polysaccharides. The produced amphoteric glycogen was converted into its hydrogel form by the thermostable GP-catalyzed enzymatic polymerization of Glc-1-P from the pure non-reducing α(1→4)-glucan chain ends without the functionalization by GlcA or GlcN units (Figure4c) [ 53]. The elongated amylose chains formed double helical assemblies among the amphoteric glycogen molecules, which acted as cross-linking points for hydrogelation. The resulting amphoteric glycogen and its hydrogel showed pH-responsive properties.

4. Thermostable GP-Catalyzed Enzymatic Oligomerization and Polymerization of Analog Monomers As mentioned above, the potato GP-catalyzed enzymatic polymerization produces the α(1→4)-glucan chain when the native glycosyl donor, Glc-1-P, is employed. A series of studies on the potato GP-catalyzed glycosylations using analog substrates, on the other hand, suggested that after a monosaccharide residue is transferred from the analog substrates to the non-reducing end of the maltooligosaccharide acceptor, further glycosylations do not take place owing to the loss of recognition of the produced non-reducing end structure being different from the Glc residue by the enzyme [19–21]. Taking the different recognition behavior of thermostable GP (from Aquifex aeolicus VF5) from potato into account, it has been found that the former enzyme catalyzed a different reaction manner from the latter enzyme when Man-1-P or GlcN-1-P was used as a glycosyl donor. When the thermostable GP-catalyzed glycosylation of Glc3 acceptor with Man-1-P or GlcN-1-P was carried out in acetate buffer, consecutive glycosylations took place to obtain non-natural heterooligosaccharides composed of α(1→4)-linked Man/GlcN chains [55]. The MALDI-TOF mass spectra of the products with 10:1 donor/acceptor feed ratio showed several peaks corresponding to the molecular masses of tetra~hepta-octa-saccharides having one–four or ﬁve Man or GlcN residues with Glc3 (Figure5). The production of longer chains in higher donor/acceptor feed ratios, however, has been inhibited by Pi produced from the glycosyl donors, due to the fact that it is a native substrate for phosphorolysis by the GP catalysis. Catalysts 2018, 8, x FOR PEER REVIEW 7 of 11 Catalysts 2018, 8, 473 7 of 11 Catalysts 2018, 8, x FOR PEER REVIEW 7 of 11

Figure 5. Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrum of crudeFigure product 5. Matrix from assisted thermostable laser desorption/ionization-time GP-catalyzed glucosaminylations of ﬂight (MALDI-TOF)using GlcN-1-P mass in acetate spectrum buffer of Figure 5. Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrum of (10:1crude donor/acceptor product from thermostablefeed ratio). GP-catalyzed glucosaminylations using GlcN-1-P in acetate buffer crude product from thermostable GP-catalyzed glucosaminylations using GlcN-1-P in acetate buffer (10:1 donor/acceptor feed ratio). (10:1An donor/acceptorattempt has been feed ratio).made to remove Pi as a precipitate in the thermostable GP-catalyzed consecutiveAn attempt glucosaminylations has been made using to remove ammonium Pi as abuffer precipitate (0.5 M, in pH the 8.6) thermostable containing GP-catalyzed MgCl2 as a An attempt has been made to remove Pi as a precipitate in the thermostable GP-catalyzed reactionconsecutive solvent glucosaminylations system, as the fact using that Pi ammonium is reported buffer to form (0.5 an M, insoluble pH 8.6) salt containing with ammonium MgCl2 asand a consecutive glucosaminylations using ammonium buffer (0.5 M, pH 8.6) containing MgCl2 as a magnesiumreaction solvent ions system,[56]. As as a theresult, fact thatthe thermostable Pi is reported GP-catalyzed to form an insoluble enzymatic salt polymerization with ammonium of reaction solvent system, as the fact that Pi is reported to form an insoluble salt with ammonium and GlcN-1-Pand magnesium with the ions Glc [563 ].primer As a result,(30:1) in the such thermostable buffer sy GP-catalyzedstem successfully enzymatic occurred polymerization to produce magnesium ions [56]. As a result, the thermostable GP-catalyzed enzymatic polymerization of αof(1 GlcN-1-P→4)-linked with non-natural the Glc3 (amyloseprimer (30:1) analog) in suchaminopolysaccharide buffer system successfully with a DP occurredof the GlcN to produceunits of GlcN-1-P with the Glc3 primer (30:1) in such buffer system successfully occurred to produce ~20,α(1→ which4)-linked was non-natural named ‘amylosamine’ (amylose analog) (Figure aminopolysaccharide 6) [57]. The withproduced a DP of amylosamine the GlcN units shows of ~20, α(1→4)-linked non-natural (amylose analog) aminopolysaccharide with a DP of the GlcN units of water-solubilitywhich was named because ‘amylosamine’ it has not (Figure formed6)[ a regula57]. Ther higher-order produced amylosamine assembly in shows water water-solubilityas observed for ~20, which was named ‘amylosamine’ (Figure 6) [57]. The produced amylosamine shows thebecause amylose it has double not formed helix. a However, regular higher-order the cationic assembly amylosamine in water formed as observed a double for thehelix amylose with anionic double water-solubility because it has not formed a regular higher-order assembly in water as observed for amylouronichelix. However, acid the (α cationic(1→4)-linked amylosamine GlcA polymer), formed aanother double helixamylose with analog anionic polysaccharide, amylouronic acid by the amylose double helix. However, the cationic amylosamine formed a double helix with anionic electrostatic(α(1→4)-linked interaction GlcA polymer), between another amino amylose and carb analogoxylate polysaccharide, groups [58]. by Furthermore, electrostatic interactionreductive amylouronic acid (α(1→4)-linked GlcA polymer), another amylose analog polysaccharide, by aminationbetween amino of amylosamine and carboxylate formed groups hierarchically [58]. Furthermore, controlled reductive assemblies amination to of obtain amylosamine nanoparticles, formed electrostatic interaction between amino and carboxylate groups [58]. Furthermore, reductive microaggregates,hierarchically controlled and macrohydrogels assemblies to obtain depending nanoparticles, on reaction microaggregates, conditions and[59]. macrohydrogelsThe reductive amination of amylosamine formed hierarchically controlled assemblies to obtain nanoparticles, aminationdepending of on amylosamine reaction conditions was carried [59]. The out reductive in the presence amination of NaBH of amylosamine3CN as a reductant was carried in 0.1 out mol/L in the microaggregates, and macrohydrogels depending on reaction conditions◦ [59]. The reductive aceticpresence acid of aq. NaBH at 603CN °C asfor a 1 reductant h. When the in 0.1 lower mol/L feed acetic ratios acid of aq.reductant/redu at 60 C forcing 1 h. Whenend of the the lower substrate feed amination of amylosamine was carried out in the presence of NaBH3CN as a reductant in 0.1 mol/L wereratios ofemployed reductant/reducing for the reaction, end of the nanoparticles substrate were were employed formed. for theAs reaction, the feed nanoparticles ratios of werethe acetic acid aq. at 60 °C for 1 h. When the lower feed ratios of reductant/reducing end of the substrate reductant/reducingformed. As the feed ratiosend increased, of the reductant/reducing a larger number end of increased,α(1→4)-linked a larger GlcN number chains of αassembled(1→4)-linked to were employed for the reaction, nanoparticles were formed. As the feed ratios of the furtherGlcN chains form assembledthe water-insoluble to further microaggregates form the water-insoluble and macrohydrogels. microaggregates and macrohydrogels. reductant/reducing end increased, a larger number of α(1→4)-linked GlcN chains assembled to OH OH further form the water-insoluble microaggregatesO and macrohydrogels.O OH O OH GlcN-1-P (monomer) + HO OH OH OH OH O O OH O O OH O HO HO HO OH GlcN-1-P (monomer) + OH OH OH HO GlcO 3 (primer) O HO HO HO Glc (primer) OH O OH O OH O Thermostable GP OH 3 OH OH O O (from Aquifex aeolicus VF5) OH OH OH OH ONH2 O HO O O OH O Thermostable GP OH ONH2 O HO HO OH Ammonia buffer / MgCl HO OH O NH2 O HO 2 (from Aquifex aeolicus VF5)2 HO m-2 OH OH NH2 O HO O NH2 O HO HO Ammonia buffer / MgCl HO NH2 O HOAmylosamine + Ammonium2 magnesium Pi (precipitate) 2 HO m-2 Figure 6. Thermostable GP-catalyzed enzymatic enzymaticAmylosamine polymerization polymerization of of GlcN-1-P GlcN-1-P+ Ammonium with with magnesium removal removal Pi (precipitate)of of Pi to produce amylosamine. Figure 6. Thermostable GP-catalyzed enzymatic polymerization of GlcN-1-P with removal of Pi to produce amylosamine.

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The thermostablethermostable GP-catalyzedGP-catalyzed enzymatic enzymatic copolymerizations copolymerizations of Glc-1-Pof Glc-1-P with with GlcN-1-P, GlcN-1-P, and withand withMan-1-P, Man-1-P, have successfully have successfully progressed progressed under the conditions under ofthe removal conditions of Pi inof ammonia/MgCl removal of 2 Pibuffer in ammonia/MgClto obtain non-natural2 buffer glucosaminoglucan to obtain non-natural comprising glucosaminoglucan Glc/GlcN units comprising and mannoglucan Glc/GlcN composed units and of mannoglucanGlc/Man units composed [60,61]. The of thermostable Glc/Man units GP-catalyzed [60,61]. The enzymatic thermostable polymerization GP-catalyzed of GlcN-1-P enzymatic using polymerizationmaltooligosaccharide-functionalized of GlcN-1-P using amylouronic maltooligosaccharide-functionalized acid (amylouronic acid with a shortamylouronicα(1→4)-linked acid (amylouronicGlc chain at the acid non-reducing with a short end) α(1 as→ the4)-linked primer underGlc chain the sameat the operation non-reducing yielded end) an amyloseas the primer analog underamphoteric the same block operation polysaccharide yielded an composed amylose ofanalog GlcN amphoteric and GlcA chains block polysaccharide (Figure7)[ 62]. composed The product of GlcNshowed and pH-responsive GlcA chains (Figure properties. 7) [62]. The product showed pH-responsive properties.

FigureFigure 7. ThermostableThermostable GP-catalyzed GP-catalyzed synthesis synthesis of amphoteric bloc blockk polysaccharide. 5. Conclusions 5. Conclusions This review article demonstrated that the GP-catalyzed enzymatic glycosylations and This review article demonstrated that the GP-catalyzed enzymatic glycosylations and polymerizations using analog substrates are powerful tools to precisely synthesize α(1→4)-linked polymerizations using analog substrates are powerful tools to precisely synthesize α(1→4)-linked non-natural oligo- and polysaccharides. As GP shows weak specificity for the recognition of non-natural oligo- and polysaccharides. As GP shows weak specificity for the recognition of substrates, several monosaccharide 1-phosphates could be employed to enzymatically introduce substrates, several monosaccharide 1-phosphates could be employed to enzymatically introduce the the corresponding monosaccharide units into the saccharide chains. Such non-natural oligo- and corresponding monosaccharide units into the saccharide chains. Such non-natural oligo- and polysaccharides have a high potential to be employed as practical functional materials in the biological, polysaccharides have a high potential to be employed as practical functional materials in the medicinal, and pharmaceutical research fields. Because the analog substrates have been provided biological, medicinal, and pharmaceutical research fields. Because the analog substrates have been efficiently by organic synthetic techniques, the approach from the viewpoint of the synthetic organic provided efficiently by organic synthetic techniques, the approach from the viewpoint of the synthetic chemistry, is in demand for conducting the studies presented in this review. The author, therefore, organic chemistry, is in demand for conducting the studies presented in this review. The author, claims the efficient collaboration between the research fields of synthetic organic chemistry and therefore, claims the efficient collaboration between the research fields of synthetic organic chemistry enzymatic technology will exhibit synergistic effects to contribute to further development in the and enzymatic technology will exhibit synergistic effects to contribute to further development in the enzymatic synthesis of functional non-natural oligo- and polysaccharides in the future. enzymatic synthesis of functional non-natural oligo- and polysaccharides in the future. Funding: This research was partly funded by a Grant-in-Aid for Scientific Research from Ministry of Education, Funding:Culture, Sports, This research and Technology, was partly Japan funded (Nos. by 25,620,177a Grant-in-Aid and 17K06001).for Scientific Research from Ministry of Education, Culture,Acknowledgments: Sports, and Technology,The author isJapan indebted (Nos. to 25,620,177 the co-workers, and 17K06001). whose names are found in references from his Acknowledgments:papers, for their enthusiastic The author collaborations. is indebted to the co-workers, whose names are found in references from his papers,Conflicts for of their Interest: enthusiasticThe author collaborations. declares no conflict of interest.

Conflicts of Interest: The author declares no conflict of interest. References

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