Precision Synthesis of Functional Polysaccharide Materials by Phosphorylase-Catalyzed Enzymatic Reactions

polymers

Review Precision Synthesis of Functional Polysaccharide Materials by Phosphorylase-Catalyzed Enzymatic Reactions

Jun-ichi Kadokawa

Graduate School of Science and Engineering, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065, Japan; [email protected]; Tel.: +81-99-285-7743; Fax: +81-99-285-3253

Academic Editors: Katja Loos and Alexander Böker Received: 22 December 2015; Accepted: 13 January 2016; Published: 11 April 2016

Abstract: In this review article, the precise synthesis of functional polysaccharide materials using phosphorylase-catalyzed enzymatic reactions is presented. This particular enzymatic approach has been identified as a powerful tool in preparing well-defined polysaccharide materials. Phosphorylase is an enzyme that has been employed in the synthesis of pure amylose with a precisely controlled structure. Similarly, using a phosphorylase-catalyzed enzymatic polymerization, the chemoenzymatic synthesis of amylose-grafted heteropolysaccharides containing different main-chain polysaccharide structures (e.g., chitin/chitosan, cellulose, alginate, xanthan gum, and carboxymethyl cellulose) was achieved. Amylose-based block, star, and branched polymeric materials have also been prepared using this enzymatic polymerization. Since phosphorylase shows a loose specificity for the recognition of substrates, different sugar residues have been introduced to the non-reducing ends of maltooligosaccharides by phosphorylase-catalyzed glycosylations using analog substrates such as α-D-glucuronic acid and α-D-glucosamine 1-phosphates. By means of such reactions, an amphoteric glycogen and its corresponding hydrogel were successfully prepared. Thermostable phosphorylase was able to tolerate a greater variance in the substrate structures with respect to recognition than potato phosphorylase, and as a result, the enzymatic polymerization of α-D-glucosamine 1-phosphate to produce a chitosan stereoisomer was carried out using this enzyme catalyst, which was then subsequently converted to the chitin stereoisomer by N-acetylation. Amylose supramolecular inclusion complexes with polymeric guests were obtained when the phosphorylase-catalyzed enzymatic polymerization was conducted in the presence of the guest polymers. Since the structure of this polymeric system is similar to the way that a plant vine twines around a rod, this polymerization system has been named “vine-twining polymerization”. Through this approach, amylose supramolecular network materials were fabricated using designed graft copolymers. Furthermore, supramolecular inclusion polymers were formed by vine-twining polymerization using primer–guest conjugates.

Keywords: amylose; enzymatic reaction; phosphorylase; polysaccharide; polymerization; supramolecule

1. Introduction Polysaccharides are commonly present in nature and have been identiﬁed as vital materials for important in vivo functions [1]. These molecules are composed of monosaccharide residues linked together through glycosidic bonds, a type of covalent linkage that joins a monosaccharide residue at its anomeric position to another group, typically another saccharide moiety, such that it is structurally a result of dehydrative condensation between the components’ two hydroxy groups (Figure1)[ 2–4]. Natural polysaccharides are constructed by a wide variety of monosaccharide repeating units linked through the different stereo- and region-types of glycosidic bonds, giving rise

Polymers 2016, 8, 138; doi:10.3390/polym8040138 www.mdpi.com/journal/polymers Polymers 2016,, 8,, 138 138 2 of 19 20 through the different stereo- and region-types of glycosidic bonds, giving rise to quite complicated tochemical quite complicated structures. The chemical structural structures. diversity The of structuralpolysaccharides diversity leads of polysaccharidesto the provision leadsof a whole to the provisionrange of biological of a whole functions, range of biological and a subtle functions, change and in a subtletheir structure change in has their a structureprofound has effect a profound on the effectproperties on the and properties functions and functionsof these ofmolecules these molecules [5]. For [5]. Forexample, example, two two representative representative natural polysaccharides, cellulose and star starch,ch, are both composed of glucose repeating units, but are linked through stereo-oppositelystereo-oppositely β(1Ñ4)-4)- and α(1Ñ4)-glucosidic4)-glucosidic bonds, bonds, respecti respectivelyvely [6,7]. [6,7]. Owing Owing to the differences inin thethe stereochemistrystereochemistry of of the the linkages linkages between between the the same same glucose glucose repeating repeating units, units, the the roles roles of celluloseof cellulose and and starch starch in naturein nature are are completely completely different, different, such such that, that, the formerthe former is a structuralis a structural material material and theand latterthe latter acts acts as an as energy an energy provider provider as a as component a component of starch,of starch, respectively. respectively. The The precise precise synthesis synthesis of structurallyof structurally well-deﬁned well-defined polysaccharides polysaccharides composed composed of stereo- of stereo- and regio-controlled and regio-controlled glycosidic glycosidic linkages haslinkages therefore has attractedtherefore much attracted attention much in attention carbohydrate in carbohydrate and materials and sciences materials in the developmentsciences in the of newdevelopment functional of bio-related new function materials.al bio-related materials.

O O - H2O + HO O OH

Glycosidic linkage Figure 1. Formation of glycosidic linkage between anomeric hydroxy group of sugar residue and Figure 1. Formation of glycosidic linkage between anomeric hydroxy group of sugar residue and hydroxy group of another sugar residue.

To provide well-defined chain structures from monosaccharide repeating units, the stereo- and regio-selectiveTo provide formation well-defined of glycosidic chain structures linkages from between monosaccharide two glycosyl repeating donor and units, acceptor the stereo-substrates, and regio-selectivea so-called ”glycosylation”, formation of glycosidicis in demand linkages [2–4]. between In a typical two glycosyl glycosylation donor and reaction, acceptor an substrates,anomeric acarbon so-called of the ”glycosylation”, donor substrate is inis activated demand [by2–4 the]. In in atroduction typical glycosylation of a leaving reaction, group to an the anomeric molecule, carbon such ofthat the it donorcan react substrate with the is activatedfree hydroxy by the group introduction of the acceptor of a leaving substrate. group Itto is theimportant molecule, that such the thatother it canhydroxy react groups with the of free the hydroxyglycosyl groupdonor ofand the acceptor acceptor are substrate. protected It to is importantprevent unwanted that the otherside reactions hydroxy groupsfrom occurring. of the glycosyl For the donor stereo- and and acceptor regio-selective are protected formation to prevent of the glycosidic unwanted linkage, side reactions the leaving from occurring.group, protecting For the stereo-groups and, catalyst, regio-selective and solvent formation are therefore of the glycosidic important linkage, and themust leaving be selected group, protectingappropriately. groups, The catalyst,control of and the solventstereo- areand therefore regio-selectivities important of and the mustresulting be selected glycosidic appropriately. linkages in Thegeneral control chemical of the glycosylations stereo- and regio-selectivities is still a challenging of the research resulting topic glycosidic in glycoscience. linkages in general chemical glycosylationsAn alternative is still to a challengingchemical glycosylations research topic that in glycoscience. has also been developed involves an in vitro approach,An alternative the so-called to chemical “enzymatic glycosylations glycosylation”, that haswhich also offers been advantages developed involvesbecause enzymatic an in vitro approach,reactions are the superior so-called to chemical “enzymatic catalysis glycosylation”, in terms of which stereo- offers and regio-select advantagesivities because [8,9].enzymatic Of the six reactionsmain classes are of superior enzymes, to transferase chemical catalysis (glycosyl in transferase) terms of stereo- and hydrolase and regio-selectivities (glycosyl hydrolase) [8,9]. Of have the sixbeen main used classes successfully of enzymes, as catalysts transferase in enzymatic (glycosyl glycosylations transferase) [10,11]. and hydrolase In these (glycosylreactions, hydrolase)a glycosyl havedonor been and useda glycosyl successfully acceptor as are catalysts employed in enzymatic in their glycosylationsunprotected forms, [10,11 whereby]. In these the reactions, reaction aprogresses glycosyl donor in a stereo- and a glycosyl and regio-controlled acceptor are employed fashion by in enzymatic their unprotected catalysis forms, in aqueous whereby media the reaction [9]. In progressesthe same fashion in a stereo- as andthe regio-controlledaforementioned fashionchemical by enzymaticglycosylation, catalysis the inenzymatic aqueous mediaformation [9]. In of the a sameglycosidic fashion linkage as the occurs aforementioned between chemicalthe anomeric glycosylation, position theof enzymaticone monosaccharide formation of residue, a glycosidic the linkageactivated occurs glycosyl between donor, the and anomeric the hydroxy position group of oneof the monosaccharide second monosaccharide residue, the residue, activated the glycosyl donor,acceptor and (Figure the hydroxy 2). In groupthe course of the of second the reaction, monosaccharide the anomeric residue, position the glycosyl of the acceptorglycosyl(Figure donor2 is). Ininitially the course recognized of the reaction,by and interacts the anomeric with positionthe catalytic of the center glycosyl of the donor enzyme is initially to form recognized a glycosyl– by andenzyme interacts complex. with Next, the catalyticthe hydroxy center group of the of enzymethe glycosyl to form acceptor a glycosyl–enzyme attacks the anomeric complex. carbon Next, of the complex hydroxy in group a stereo- of the and glycosyl regio-selective acceptor manner, attacks according the anomeric to the carbon specificity of the of complex the enzyme, in a stereo-which andgives regio-selective rise to the direct manner, formation according of the unprotec to the specificityted glycoside. of the As enzyme, mentioned which previously, gives rise the to the enzymes direct formationthat have been of the employed unprotected to catalyze glycoside. these As glyc mentionedosylations previously, are primarily the enzymesglycosyl transferase that have beenand employedglycosyl hydrolase. to catalyze Additionally, these glycosylations the former are can primarily be sub-classified glycosyl transferase into the synthetic and glycosyl enzymes hydrolase. (Leloir Additionally,glycosyl transferase) the former phosphorolytic can be sub-classified enzyme (phosphorylase), into the synthetic and enzymes sucrase (Leloir [12,13]. glycosyl transferase) phosphorolytic enzyme (phosphorylase), and sucrase [12,13]. Polymers 2016, 8, 138 3 of 20 Polymers 2016, 8, 138 3 of 19

O HO X + OH O + HX HO Glycosyl donor Active center Active center of enzyme of enzyme

Glycosyl-enzyme complex

HO O O Glycosyl acceptor + OH HO O Active center Glycoside of enzyme

Figure 2. Reaction scheme for enzymatic glycosylation. Figure 2. Reaction scheme for enzymatic glycosylation.

Due to the fact that polysaccharides are theoretically produced by repeated glycosylations, severalDue enzymatic to the fact approaches that polysaccharides have been are employed theoretically inproduced the efficient by repeated syntheses glycosylations, of polysaccharides several enzymatic(enzymaticapproaches polymerizations) have been [14,15]. employed Phosphorylas in the efficiente is an syntheses enzyme ofthat polysaccharides has been employed (enzymatic to polymerizations)synthesize amylose [14 ,15with]. Phosphorylase a precise stereo- is an enzymeand regio-controlled that has been employedstructure to[16]. synthesize This enzymatic amylose withapproach a precise provides stereo- a andpure regio-controlled amylose sample, structure which [16is ].useful This enzymaticas the separation approach of provides amylose a from pure amyloseamylopectin sample, in natural which starch is useful is quite as the difficult. separation Furthermore, of amylose as froma result amylopectin of the loose in specificity natural starch for the is quiterecognition difficult. of Furthermore,substrates by asph aosphorylase, result of the loosefunctional specificity non-natu for theral recognitionpolysaccharides of substrates other than by phosphorylase,amylose have also functional been synthesized non-natural by polysaccharides phosphorylase othercatalysis. than In amylose this review have article, also been on synthesizedthe basis of bythe phosphorylase above viewpoints catalysis. and background, In this review the article, precise on thesyntheses basis of of the functional above viewpoints polysaccharide and background, materials theby phosphorylase-catalyzed precise syntheses of functional enzymatic polysaccharide reactions are materials reviewed. by phosphorylase-catalyzed enzymatic reactions are reviewed. 2. Characteristic Features of Phosphorylase Catalysis 2. Characteristic Features of Phosphorylase Catalysis Phosphorylase, belonging to the GT35 family (EC 2.4.1.1), is often called starch phosphorylase, Phosphorylase, belonging to the GT35 family (EC 2.4.1.1), is often called starch phosphorylase, glycogen phosphorylase, or α-glucan phosphorylase. It is well accepted, however, that this enzyme glycogen phosphorylase, or α-glucan phosphorylase. It is well accepted, however, that this enzyme is simply referred to as “phosphorylase”, because it is the most extensively studied and widely used is simply referred to as “phosphorylase”, because it is the most extensively studied and widely enzyme as the catalyst for reactions in many of the phosphorylases found in nature. In nature, used enzyme as the catalyst for reactions in many of the phosphorylases found in nature. In nature, phosphorylase catalyzes the phosphorolytic cleavage of α(14)-glucans, such as amylose and phosphorylase catalyzes the phosphorolytic cleavage of α(1Ñ4)-glucans, such as amylose and glycogen, glycogen, at their non-reducing ends in the presence of inorganic phosphate (Pi) to produce α-D- at their non-reducing ends in the presence of inorganic phosphate (Pi) to produce α-D-glucose glucose 1-phoshate (Glc-1-P) (Figure 3a) [17–19]. Because of the reversibility of this reaction, 1-phoshate (Glc-1-P) (Figure3a) [ 17–19]. Because of the reversibility of this reaction, phosphorylase phosphorylase also catalyzes enzymatic glucosylation reaction using Glc-1-P as the glycosyl donor also catalyzes enzymatic glucosylation reaction using Glc-1-P as the glycosyl donor (Figure3a) [ 13], (Figure 3a) [13], whereby a glucose residue is transferred from Glc-1-P to the non-reducing end of the whereby a glucose residue is transferred from Glc-1-P to the non-reducing end of the glycosyl acceptor glycosyl acceptor to form an α-(14)-glucosidic linkage. Maltooligosaccharides with degrees of to form an α-(1Ñ4)-glucosidic linkage. Maltooligosaccharides with degrees of polymerization (DPs) polymerization (DPs) higher than the smallest one, which are recognized by phosphorylase, are higher than the smallest one, which are recognized by phosphorylase, are typically used as glycosyl typically used as glycosyl acceptors. The smallest substrates used in phosphorolysis and acceptors. The smallest substrates used in phosphorolysis and glucosylation reactions catalyzed by the glucosylation reactions catalyzed by the most common phosphorylase isolated from potato (potato most common phosphorylase isolated from potato (potato phosphorylase) are typically maltopentaose phosphorylase) are typically maltopentaose (Glc5) and maltotetraose (Glc4). On the other hand, Glc4 (Glc5) and maltotetraose (Glc4). On the other hand, Glc4 and maltotriose (Glc3) are the smallest and maltotriose (Glc3) are the smallest substrates recognized in phosphorolysis and glucosylation substrates recognized in phosphorolysis and glucosylation reactions by the catalysis of phosphorylase reactions by the catalysis of phosphorylase isolated from thermophilic bacterial sources isolated from thermophilic bacterial sources (thermostable phosphorylase) [20–23]. These observations (thermostable phosphorylase) [20–23]. These observations suggest that the sources of phosphorylases suggest that the sources of phosphorylases strongly affect recognition behavior of the substrates. strongly affect recognition behavior of the substrates. Polymers 2016, 8, 138 4 of 20 Polymers 2016, 8, 138 4 of 19

(a) HO Reducing end O HO HO O Phosphorylase O HO HO + HO OH HO O HO OH O (Glusocylation) HO OH O O P O OH Non-reducing end OHO OH O n (Phosphorolysis) α -D-Glucose 1-phosphate (Glc-1-P) Maltooligosaccharide (glycosyl donor) (glycosyl acceptor)

HO O HO O HO + Inorganic phosphate (Pi) HO OH HO HO OH O O OH OHO OH n+1 α(14)-Glucosidic linkage

(b) HO O HO HO O O HO m HO O + HO OH HO HO OH HO OH O O P O O OH OHO OH O n

Glc-1-P (monomer)) Maltooligosaccharide (primer)

HO Phosphorylase O HO O HO (Enzymatic polymerization) HO OH HO HO OH O + m Pi O OH OHO OH m+n α(14)-Glucan (amylose) Figure 3. Phosphorylase-catalyzed (a) phosphorolysis and glucosylation and (b) enzymatic polymerization. Figure 3. Phosphorylase-catalyzed (a) phosphorolysis and glucosylation and (b) enzymatic polymerization.

When an excess molar ratio of Glc-1-P with respect to the maltooligosaccharide acceptor is presentWhen in the an reaction excess molarsystem, ratio the ofsuccessive Glc-1-P glucos with respectylations to in the the maltooligosaccharidepresence of phosphorylase acceptor occur is presentas a propagation in the reaction of polymerization system, the to successive yield the glucosylationsα(14)-glucan inpolymer, the presence i.e., amylose of phosphorylase (Figure 3b) α occur[20,24–26]. as a The propagation reaction of ofsuch polymerization phosphorylase-catalyzed to yield the successive(1Ñ4)-glucan glucosylations polymer, is a chain-growthi.e., amylose (Figurepolymerization,3b) [ 20,24 and–26 the]. The glycosyl reaction acceptor of such is often phosphorylase-catalyzed referred to as the “primer” successive of the glucosylations polymerization is abecause chain-growth the reaction polymerization, is initiated andat the the non-redu glycosylcing acceptor end of is the often acceptor. referred Since to as the “primer”phosphorylase- of the polymerizationcatalyzed polymerization because the is conceived reaction is analogously initiated at theto a non-reducingliving polymerization, end of the the acceptor. molecular Since weight the phosphorylase-catalyzedof the generated amylose polymerizationcan be controlled is conceivedby the monomer analogously (Glc-1-P)/primer to a living polymerization,feed ratios, and the molecular weight of the generated amylose can be controlled by the monomer (Glc-1-P)/primer feed distribution of polymeric products is typically narrow (Mw/Mn < 1.2) [27]. ratios,The and phosphorylase-catalyzed the distribution of polymeric enzymatic products polyme is typicallyrization narrow can be (M wconducted/Mn < 1.2) using [27]. modified maltooligosaccharideThe phosphorylase-catalyzed primers, which enzymatic are covalently polymerization immobilized can on other be conducted substances using at its modiﬁedreducing maltooligosaccharideends, such as a polymeric primers, chain, which which are does covalently not take immobilized part in the onpolymerization other substances (Figure at its 4) reducing [28–34]. ends,Such a such modified as a polymeric primer is typically chain, which multifunctional does not take because part inof the presence polymerization of multiple (Figure non-reducing4)[ 28–34]. Suchmaltooligosaccharide a modiﬁed primer chain is typically ends on multifunctional the substanc becausee, and has of the produced presence various of multiple amylose-grafted non-reducing maltooligosaccharidepolymeric materials, such chain as endsamylose-grafted on the substance, polystyrene, and has polyacetylene, produced various poly(dimethylsiloxane), amylose-grafted polymeric materials, such as amylose-grafted polystyrene, polyacetylene, poly(dimethylsiloxane), poly(vinyl alcohol), and poly(L-glutamic acid) [35–41]. Amylose-grafted brushes were also obtained poly(vinylby the phosphorylase-catalyzed alcohol), and poly(L-glutamic enzymatic acid) polyme [35–41rization]. Amylose-grafted of Glc-1-P from brushes maltoheptaose were also obtained primers bycovalently the phosphorylase-catalyzed attached to Au and Si enzymaticsurfaces [42]. polymerization of Glc-1-P from maltoheptaose primers covalently attached to Au and Si surfaces [42]. Polymers 2016, 8, 138 5 of 20 Polymers 2016, 8, 138 5 of 19 Polymers 2016, 8, 138 5 of 19 HO O HO HO O HO O HO HO OH O HO HO HO OH O HO OH O HO Non-reducing end HO OH O O OHO OH Non-reducing end n HO OHO OH O Modified primer n Reducing end HO m HO O O HO OH Modified primer Reducing end m HO O OP O HO OH O PO O Glc-1-P O Phosphorylase Glc-1-P Phosphorylase

m Pi HO m Pi O HO HO O HO HO OH O HO HO HO HO OH O O O HO OH HO O O HO HO OH HO OH O O HO m HO OH O O OHO OH m n OHO OH n

Figure 4. Phosphorylase-catalyzed enzymatic polymerization using modiﬁedmodified primer. Figure 4. Phosphorylase-catalyzed enzymatic polymerization using modified primer. Phosphorylase has demonstrated loose specificity for the recognition of the substrate structures of interest,Phosphorylase and therefore, has demonstrated the extension loose of phos specificity speciﬁcityphorylase-catalyzed for the recognition enzymatic of the glucosylations substrate structures using ofseveral interest, interest, monosaccharide and and therefore, therefore, 1-phosphates, the the extension extension i.e. of, phosanalog of phosphorylase-catalyzedphorylase-catalyzed substrates of Glc-1-P, enzymatic as enzymatic glycosyl glucosylations donors glucosylations to obtain using i.e. usingseveralnon-natural several monosaccharide oligosaccharides monosaccharide 1-phosphates, comprising 1-phosphates, i.e. , theanalog corresponding, substrates analog substrates of monosaccharide Glc-1-P, of as Glc-1-P, glycosyl residues as donors glycosyl at tothe donors obtain non- tonon-naturalreducing obtain ends non-natural oligosaccharides has been oligosaccharides investigated comprising [43,44]. comprisingthe co rrespondingFor theexample, corresponding monosaccharide potato phosphorylase monosaccharide residues athas the residues shown non- at the non-reducing ends has been investigated [43,44]. For example, potato phosphorylase reducingrecognition ends for αhas-D-mannose, been investigated 2-deoxy-α -D[43,44].-glucose, For α -Dexample,-xylose, αpotato-D-glucosamine, phosphorylase and N -formyl-has shownα-D- has shown recognition for α-D-mannose, 2-deoxy-α-D-glucose, α-D-xylose, α-D-glucosamine, and recognitionglucosamine for 1-phosphates α-D-mannose, (Man-1-P, 2-deoxy- dGlc-1-P,α-D-glucose, Xyl-1-P, α-D-xylose, GlcN-1-P, α-D-glucosamine, and GlcNF-1-P, and respectively) N-formyl-α- Das- N-formyl-α-D-glucosamine 1-phosphates (Man-1-P, dGlc-1-P, Xyl-1-P, GlcN-1-P, and GlcNF-1-P, glucosamineglycosyl donors 1-phosphates in glycosylations (Man-1-P, using dGlc-1-P, Glc4 asXyl-1-P, a glycosyl GlcN-1-P, acceptor, and GlcNF-1-P,to produce respectively)non-natural αas- respectively) as glycosyl donors in glycosylations using Glc as a glycosyl acceptor, to produce glycosylmannosylated, donors 2-deoxy-in glycosylationsα-glucosylated, using Glcα-xylosylated,4 as a glycosyl α-glucosaminylated, acceptor,4 to produce and non-natural N-formyl- α- α α α α non-naturalmannosylated,glucosaminylated-mannosylated, 2-deoxy- pentasaccharides,α-glucosylated, 2-deoxy- respectively -glucosylated,α-xylosylated, (Figure 5) α-xylosylated,[45–50].-glucosaminylated, -glucosaminylated, and N-formyl- andα- Nglucosaminylated-formyl-α-glucosaminylated pentasaccharides, pentasaccharides, respectively respectively(Figure 5) [45–50]. (Figure 5)[45–50]. HO O HO O HO O HO O + HO OH HO HO O O HO OOH O HO HO O O OP O + HO OH HO OH HO HO OH OHOO OH O PO O O 2 OH Analogue substrates O Glc (glycosyl acceptor)OHO OH (glycosyl donor) 4 2 Analogue substrates Glc (glycosyl acceptor) (glycosyl donor) 4 O HO Potato phosphorylase O O HO HO HO + Pi Potato phosphorylase HO OOH O O HO HO OH + Pi HO OH O O OHO OH 3 OH OHO OH OH OH OH OH 3 OH O OH O OH O O O OH O HO HO HO HO OH HO = OH HO O HO O HO O O HO O HO O HO OH HO NH HO NH HO HO HO 2 = HO HO HO H HO HO OH NH NH HO Man 2 O dGlc Xyl GlcN H GlcNF Man dGlc Xyl GlcN O GlcNF Figure 5. Phosphorylase-catalyzed enzymatic glycosylations using analogue substrates as glycosyl Figuredonors 5.to Phosphorylase-catalyzedproduce non-natural pentasaccharides enzymatic glycosylations using analogue substrates as glycosyl donors to produce non-naturalnon-natural pentasaccharides.pentasaccharides

Polymers 2016, 8, 138 6 of 20

Polymers 2016, 8, 138 6 of 19 3. Synthesis of Amylose-Containing Functional Polysaccharide Materials by Phosphorylase-Catalyzed3. Synthesis of Amylose-Containing Enzymatic PolymerizationFunctional Polysaccharide Materials by Phosphorylase- Catalyzed Enzymatic Polymerization Besides linear natural polysaccharides such as cellulose, chitin/chitosan, and amylose, branched Besides linear natural polysaccharides such as cellulose, chitin/chitosan, and amylose, branched or grafted structures, where a main-chain polysaccharide is functionalized with other polysaccharide or grafted structures, where a main-chain polysaccharide is functionalized with other polysaccharide side chains by covalent linkages [51], have also often been observed in naturally occurring side chains by covalent linkages [51], have also often been observed in naturally occurring polysaccharides. Such chemical structures likely contribute to promising and specific functions polysaccharides. Such chemical structures likely contribute to promising and specific functions and and demonstrate their important roles in natural processes. Efficient methods for the preparation of demonstrate their important roles in natural processes. Efficient methods for the preparation of well- well-defined branched or grafted artificial polysaccharides have therefore attracted increasing attention defined branched or grafted artificial polysaccharides have therefore attracted increasing attention in in the glycomaterial research fields. the glycomaterial research fields. On the basis of the above viewpoint, the phosphorylase-catalyzed enzymatic polymerization of On the basis of the above viewpoint, the phosphorylase-catalyzed enzymatic polymerization of Glc-1-P has been employed for the synthesis of amylose-grafted functional heteropolysaccharide Glc-1-P has been employed for the synthesis of amylose-grafted functional heteropolysaccharide materials by chemoenzymatic approaches (Figure6)[ 29–34]. One such synthesis was achieved materials by chemoenzymatic approaches (Figure 6) [29–34]. One such synthesis was achieved using using the aforementioned modified primer, where the reducing ends of maltooligosaccharides were the aforementioned modified primer, where the reducing ends of maltooligosaccharides were covalently connected to polysaccharide chains, such that, in this case, appropriate chemical reactions covalently connected to polysaccharide chains, such that, in this case, appropriate chemical reactions werewere carried carried out out to to immobilize immobilize maltooligosaccharides maltooligosaccharides ontoonto thethe main-chainmain-chain polysaccharides, and and the the resultingresulting modified modified primers primers were thenwere used then for theused phosphorylase-catalyzed for the phosphorylase-catalyzed enzymatic polymerization. enzymatic Twopolymerization. types of chemical Two reactionstypes of havechemical been reactions employed have to prepare been employed the modified to prepare primers: the the modified reductive aminationprimers: usingthe reductive reductants amination of maltooligosaccharides using reductants with basicof maltooligosaccharides polysaccharides containing with aminobasic groups,polysaccharides and the condensation containing amino using condensinggroups, and agentsthe condensation of amine-functionalized using condensing maltooligosaccharides agents of amine- withfunctionalized acidic polysaccharides maltooligosaccharides containing with carboxylate acidic polysaccharides groups. containing carboxylate groups.

FigureFigure 6. Chemoenzymatic6. Chemoenzymatic synthesis synthesis of amylose-grafted of amylose-grafted heteropolysaccharides heteropolysaccharides via reductive via aminationreductive andamination condensation, and condensation, followed by followed phosphorylase-catalyzed by phosphorylase-catalyzed enzymatic polymerization.enzymatic polymerization.

Using the former approach, chitosan, a basic polysaccharide with amino groups at the C-2 Using the former approach, chitosan, a basic polysaccharide with amino groups at the C-2 position in its glucosamine repeating units, was functionalized to form a maltooligosaccharide- position in its glucosamine repeating units, was functionalized to form a maltooligosaccharide-grafted grafted chitosan. This material was then converted into a maltooligosaccharide-grafted chitin by N- N chitosan.acetylation, This using material acetic was anhydride, then converted of the intoamino a maltooligosaccharide-grafted groups in the chitosan main-chain. chitin Finally, by -acetylation, from the usingmaltooligosaccharide acetic anhydride, primer of theends amino of the groupschitin/chi intosan the derivatives, chitosan main-chain. amylose chains Finally, were elongated from the maltooligosaccharideby the phosphorylase-catalyzed primer ends ofenzymatic the chitin/chitosan polymerization derivatives, of Glc-1-P amylose to yield chains amylose-grafted were elongated bychitin/chitosan the phosphorylase-catalyzed [52,53]. When the enzymaticpolymerization polymerization mixture was ofslowly Glc-1-P dried to at yield 40–50 amylose-grafted °C, a hydrogel ˝ chitin/chitosanof the amylose-grafted [52,53]. Whenchitosan the was polymerization produced. mixture was slowly dried at 40–50 C, a hydrogel of the amylose-graftedAn amylose-grafted chitosan cellulose was produced. was also synthesized using a similar synthetic approach [54]. A celluloseAn amylose-grafted derivative with cellulose partialwas conversion also synthesized of its C-6 using hydroxy a similar groups synthetic to amine approach groups [54 was]. A celluloseinitially derivativeprepared with by the partial successive conversion partial of itstosylation C-6 hydroxy of its groupsC-6 hydroxy to amine groups, groups displacement was initially of prepared the tosylates by the successiveby azido partial groups, tosylation and their of its subsequent C-6 hydroxy reduction groups, displacement to amine groups. of the tosylates This amine-functionalized by azido groups, and theircellulose subsequent was then reduction reacted to aminewith a groups.maltooligosaccharide This amine-functionalized by reductive cellulose amination was to then give reacted a primer with amaltooligosaccharide-grafted maltooligosaccharide by reductive cellulose, amination which to was give further a primer reacted maltooligosaccharide-grafted with Glc-1-P in a phosphorylase- cellulose, whichcatalyzed was further enzymatic reacted polymerization with Glc-1-P in ato phosphorylase-catalyzed produce the amylose-grafted enzymatic cellulose polymerization of interest. to produce The product formed has a very unique structure because it is made up of two representative glucose Polymers 2016, 8, 138 7 of 20 the amylose-grafted cellulose of interest. The product formed has a very unique structure because it is made up of two representative glucose polymers, cellulose and amylose, which are composed of the same repeating units but linked through stereoregular and opposite glucosidic bonds, β(1Ñ4)– and α(1Ñ4)–, respectively. When the polymerization solution was kept on a Petri dish in an ambient atmosphere for several days, it was converted entirely to a hydrogel, which was stronger than the aforementioned amylose-grafted chitosan hydrogel. This drying technique produceda solid (or film) material. Moisturizing the film returned the molecule to its hydrogel form again, a process that could be reversibly switched by the subsequent drying and wetting of the system. Alternatively, the latter condensation reaction was also used to prepare modified primers from acidic polysaccharides, such as alginate, xanthan gum, and carboxymethyl cellulose (CMC) [55–58]. The reaction of a maltooligosaccharide lactone with 2-azidoethylamine and the subsequent reduction of the azido group using NaBH4 gave an amine-functionalized maltooligosaccharide at the reducing end. Next, maltooligosaccharide-grafted alginate, xanthan gum, and CMC were obtained by condensation with the carboxylates of the corresponding polysaccharides using the condensing agents of water-soluble carbodiimide (WSC) and N-hydroxysuccinimide (NHS). Finally, the phosphorylase-catalyzed enzymatic polymerization of Glc-1-P with these maltooligosaccharide primer chains was carried out to obtain amylose-grafted alginate, xanthan gum, and CMC. The resulting amylose-grafted CMC formed a film upon the drying of a thinly spread alkaline solution (0.040 g in 0.50 mol/L aqueous NaOH (1.50 mL)). Scanning electron microscopy (SEM) imaging of the film showed a surface morphology comprised of highly entangled nanofibers. After removal of the residual NaOH from the film by immersion in water, further SEM imaging showed that the nanofibers had merged at the interface, while the fiber arrangement was retained in the bulk of the material. This method offers an efficient self-assembling generative (bottom-up) route that achieves control over the morphology and self-assembly of heteropolysaccharides to fabricate new nanofibrillated materials. Glycogen is known to be a water soluble and high molecular-weight natural polysaccharide, composed of linear α(1Ñ4)-glucan chains containing an average of 10 to 14 glucose residues, which are further interlinked by α(1Ñ6)-glucosidic linkages that lead to a highly branched structure [59,60]. Aside from glycogen’s role in in vivo phosphorolysis with glycogen phosphorylase, it is also used as a multifunctional primer for phosphorylase-catalyzed enzymatic polymerizations because of the presence of a number of non-reducing α(1Ñ4)-glucan chain ends [61]. When the phosphorylase-catalyzed enzymatic polymerization of Glc-1-P with glycogen was carried out in aqueous acetate buffer solution, followed by the standing of the reaction mixture under ambient atmosphere for 24 h, the solution was fully converted into a hydrogel (Figure7). This hydrogelation was induced by the formation of cross-links as a result of the double helix conformation of the elongated amylose chains among the glycogen molecules [62,63]. The stress-strain curves under compressive mode of the hydrogels obtained by employing different Glc-1-P/glycogen ratios showed that the gels were strengthened and then became brittle with increasing amounts of glycogen. The formation of more double helix cross-linking points from the larger number of elongated amylose chains induced the increase of the gel strength when the amounts of glycogen increased. However, the further larger number of cross-linking points most likely led to the brittle nature of the gels. The hydrogels were readily converted to cryogels by lyophilization. The stress-strain curves under compressive mode of these cryogels showed that harder cryogels were obtained with increases in the amount of glycogen used for the initial hydrogelation. This result is probably due to the formation of smaller networks when larger numbers of cross-linking points were present with larger amounts of glycogen in the system. Furthermore, a transparent ﬁlm could be obtained by casting the alkaline solution of the cryogel on a glass plate, followed by drying. Polymers 2016, 8, 138 8 of 20 Polymers 2016, 8, 138 8 of 19

Figure 7.7. Phosphorylase-catalyzedPhosphorylase-catalyzed enzymatic enzymatic polymerization polymerization using using glycogen glycogen as multifunctional as multifunctional primer primerto produce to produce hydrogel. hydrogel.

Glc5-block-alkyl chain surfactants (octyl, C8Glc5; dodecyl, C12Glc5; hexadecyl, C16Glc5) formed Glc5-block-alkyl chain surfactants (octyl, C8Glc5; dodecyl, C12Glc5; hexadecyl, C16Glc5) formed micelles in water, which dissociated upon phosphorylase-catalyzed enzymatic polymerization. micelles in water, which dissociated upon phosphorylase-catalyzed enzymatic polymerization. Moreover, the micelle-to-vesicle transition of the mixed lipid/primer systems was controlled by the Moreover, the micelle-to-vesicle transition of the mixed lipid/primer systems was controlled by enzymatic polymerization. An enzyme-responsive artificial chaperone system using C12Glc5 and the enzymatic polymerization. An enzyme-responsive artiﬁcial chaperone system using C12Glc5 phosphorylase b was also designed to facilitate protein refolding. After both guanidine and phosphorylase b was also designed to facilitate protein refolding. After both guanidine hydrochloride and heat denaturation of carbonic anhydrase B, the efficient refolding of the protein hydrochloride and heat denaturation of carbonic anhydrase B, the efﬁcient refolding of the protein was observed upon the controlled association of the protein molecules and the C12Glc5 micelle after was observed upon the controlled association of the protein molecules and the C12Glc5 micelle after enzymatic polymerization [64,65]. The poly(L-lysine) moiety with pendant maltooligosaccharide enzymatic polymerization [64,65]. The poly(L-lysine) moiety with pendant maltooligosaccharide primers and cholesterols formed positively-charged nanogels via self-assembly in water, which after primers and cholesterols formed positively-charged nanogels via self-assembly in water, which phosphorylase-catalyzed enzymatic polymerization with Glc-1-P gave amylose-conjugated nanogels after phosphorylase-catalyzed enzymatic polymerization with Glc-1-P gave amylose-conjugated [66]. A series of amylose-based star polymers (1, 2, 4, and 8 arms) was also synthesized by nanogels [66]. A series of amylose-based star polymers (1, 2, 4, and 8 arms) was also synthesized by phosphorylase-catalyzed enzymatic polymerization using Glc5-functionalized poly(ethylene glycol) phosphorylase-catalyzed enzymatic polymerization using Glc5-functionalized poly(ethylene glycol) (PEG) [67]. The eight-arm primer formed was able to act as a gelator when triggered enzymatically. (PEG) [67]. The eight-arm primer formed was able to act as a gelator when triggered enzymatically.

4. Synthesis Synthesis of of Non-Natural Non-Natural Functional Functional Polysa Polysaccharideccharide Materials Materials by by Phosphorylase-Catalyzed Phosphorylase-Catalyzed Enzymatic Reactions Using Analog Substrates In addition to the introduction of GlcN unitsunits containing an amine functional group to maltooligosaccharides byby phosphorylase-catalyzedphosphorylase-catalyzed glycosylationglycosylation (glucosaminylation) (glucosaminylation) using using GlcN-1-P GlcN-1- Pas as a a glycosyl glycosyl donor donor [ 49[49],], an an attempt attempt has has alsoalso beenbeen mademade toto enzymaticallyenzymatically produce a different functional oligosaccharideoligosaccharide by by phosphorylase phosphorylase catalysis catalysis using using the the analog analog substrate, substrate,α-D -glucuronicα-D-glucuronic acid 1-phosphateacid 1-phosphate (GlcA-1-P), (GlcA-1-P), in which in a carboxylatewhich a groupcarboxylate can be introducedgroup can to thebe maltooligosaccharide.introduced to the maltooligosaccharide.Potato phosphorylase Potato did not phosphorylase recognize GlcA-1-P, did not recognize however, GlcA-1-P, and thus however, the desired and functional thus the desiredoligosaccharide functional was oligosaccharide not produced by was catalysis not produc with thised particular by catalysis enzyme. with Interestingly, this particular it was enzyme. found Interestingly,that thermostable it was phosphorylase found that thermostable isolated from phosphorylaseAquifex aeolicus isolatedVF5 [68 from] did Aquifex recognize aeolicus GlcA-1-P VF5 [68] and didcould recognize catalyze GlcA-1-P the glycosylation and could (glucuronylation) catalyze the glycosylation of Glc3, the (glucuronylation) smallest glycosyl of acceptor Glc3, the recognized smallest glycosylby this enzyme, acceptor to recognized produce an acidicby this tetrasaccharide enzyme, to produce containing an aacidic carboxylate tetrasaccharide group (Figure containing8)[69]. a carboxylate group (Figure 8) [69]. Polymers 2016, 8, 138 9 of 20 PolymersPolymers 2016 2016, ,8 8, ,138 138 99 of of 19 19

OO OO OO HOHO OO HOHO OO HOHO OO HOHO OO ++ OHOH HOHO OHOH OHOH OHOH OHOH OO PP OO HOHO OO O HOHO HOHO OHOHO O O Glc (glycosyl acceptor) GlcA-1-PGlcA-1-P Glc33 (glycosyl acceptor) (glycosyl(glycosyl donor) donor)

Thermostable phosphorylase OO Thermostable phosphorylase OO O O (Aquifex aeolicus VF5) OO HOHO O HOHO O (Aquifex aeolicus VF5) OHOH ++ Pi Pi OHOH OHOH OHOH HOHO OO OO HOHO HOHO HOHO 22 Figure 8. Thermostable phosphorylase-catalyzed enzymatic glucuronylation using GlcA-1-P as FigureFigure 8.8. Thermostable phosphorylase-catalyzed enzymatic glucuronylation using GlcA-1-P asas glycosyl donor to produce acidic tetrasaccharide. glycosylglycosyl donordonor toto produceproduce acidicacidic tetrasaccharide.tetrasaccharide.

ThroughThrough the the use use of of the the above above phosphorylase-ca phosphorylase-catalyzedtalyzed glucosaminylation glucosaminylation and and glucuronylation glucuronylation Through the use of the above phosphorylase-catalyzed glucosaminylation and glucuronylation reactionsreactions usingusing analoganalog substratessubstrates (GlcN-1-P(GlcN-1-P andand GlcA-1-P),GlcA-1-P), highlyhighly branchedbranched amphotericamphoteric reactions using analog substrates (GlcN-1-P and GlcA-1-P), highly branched amphoteric polysaccharides, polysaccharides,polysaccharides, suchsuch asas amphotericamphoteric glycogensglycogens havihavingng bothboth basicbasic GlcNGlcN andand acidicacidic GlcAGlcA residues,residues, such as amphoteric glycogens having both basic GlcN and acidic GlcA residues, could also be couldcould alsoalso bebe synthesizedsynthesized (Figure(Figure 9)9) [70,71].[70,71]. TheThe thermostablethermostable phosphorylase-catalyzedphosphorylase-catalyzed synthesized (Figure9) [ 70,71]. The thermostable phosphorylase-catalyzed glucuronylation of highly glucuronylationglucuronylation of of highly highly branched branched polysaccharides polysaccharides using using GlcA-1-P GlcA-1-P was was first first conducted conducted to to give give rise rise branched polysaccharides using GlcA-1-P was first conducted to give rise to acidic materials [72], toto acidic acidic materials materials [72], [72], followed followed by by thermostable thermostable phosphorylase-catalyzed phosphorylase-catalyzed glucosaminylation glucosaminylation of of the the followed by thermostable phosphorylase-catalyzed glucosaminylation of the products using GlcN-1-P productsproducts using using GlcN-1-P GlcN-1-P to to obtain obtain amphoteric amphoteric polysaccharides. polysaccharides. The The functionalities functionalities of of the the GlcA/GlcN GlcA/GlcN to obtain amphoteric polysaccharides. The functionalities of the GlcA/GlcN residues depended on residuesresidues depended depended on on the the feed feed ratios ratios employed employed in in the the reaction. reaction. The The electrokinetic electrokinetic ζ ζ-potential-potential values values the feed ratios employed in the reaction. The electrokinetic ζ-potential values of the amphoteric ofof thethe amphotericamphoteric productsproducts changedchanged fromfrom positivepositive toto negativenegative asas thethe pHpH ofof thethe solutionsolution increased.increased. products changed from positive to negative as the pH of the solution increased. The values of the TheThe values values of of the the isoelectric isoelectric points points of of the the amphoter amphotericic products products could could be be calc calculatedulated by by the the pH pH values values isoelectric points of the amphoteric products could be calculated by the pH values observed when the observedobserved whenwhen thethe ζζ-potential-potential becamebecame zero,zero, andand notnot surprisingly,surprisingly, theythey werewere dependentdependent onon thethe ζ-potential became zero, and not surprisingly, they were dependent on the GlcA/GlcN ratios present GlcA/GlcNGlcA/GlcN ratios ratios present present in in the the products. products. Amphoter Amphotericic glycogen glycogen hydrogels hydrogels could could be be prepared prepared by by the the in the products. Amphoteric glycogen hydrogels could be prepared by the formation of cross-links formationformation of of cross-links cross-links between between the the double-helical double-helical conformations conformations of of the the el elongatedongated amylose amylose chains chains between the double-helical conformations of the elongated amylose chains upon the thermostable uponupon the the thermostable thermostable phosphorylase-catalyzed phosphorylase-catalyzed en enzymaticzymatic polymerization polymerization of of Glc-1-P Glc-1-P with with the the non- non- phosphorylase-catalyzed enzymatic polymerization of Glc-1-P with the non-functionalized, non-reducing functionalized,functionalized, non-reducingnon-reducing endsends ofof thethe amphotericamphoteric glycogensglycogens (Figure(Figure 9)9) [71].[71]. TheThe producedproduced ends of the amphoteric glycogens (Figure9) [ 71]. The produced hydrogels showed pH-responsive hydrogelshydrogels showedshowed pH-responsivepH-responsive behavior,behavior, wherewhere theythey werewere solublesoluble inin alkalinealkaline conditionsconditions andand behavior, where they were soluble in alkaline conditions and returned to hydrogels upon acidification of returnedreturned toto hydrogelshydrogels uponupon acidificationacidification ofof thethe system.system. Furthermore,Furthermore, thethe hydrogelshydrogels exhibitedexhibited pH-pH- the system. Furthermore, the hydrogels exhibited pH-dependent shrinking/swelling behavior. dependentdependent shrinking/swelling shrinking/swelling behavior. behavior.

FigureFigureFigure 9. 9. 9. Phosphorylase-catalyzed Phosphorylase-catalyzedPhosphorylase-catalyzed successive successive successive enzymati enzymati enzymaticcc reactions reactions reactions to to produce produce to amphoteric produceamphoteric amphoteric glycogen glycogen hydrogel.glycogenhydrogel. hydrogel.

AA series series of of studies studies on on the the potato potato phosphorylase- phosphorylase-catalyzedcatalyzed glycosylations glycosylations using using analog analog substrates substrates havehave suggested suggested that that if if a a single single mo monosaccharidenosaccharide residue residue is is transferred transferred from from the the analog analog substrates substrates to to Polymers 2016, 8, 138 10 of 20

Polymers 2016, 8, 138 10 of 19 A series of studies on the potato phosphorylase-catalyzed glycosylations using analog substrates thehave non-reducing suggested that end if of a singlethe maltooligosaccharide monosaccharide residue acceptor, is transferred further glycosylations from the analog will substratesnot occur becauseto the non-reducing the new structure end ofdifferent the maltooligosaccharide from the Glc residue acceptor, at the furthernon-reducing glycosylations end is no will longer not recognizedoccur because by the the enzyme new structure (Figure 10) different [43,44] from. When the the Glc thermostable residue at thephosphorylase non-reducing (from end Aquifex is no longer recognized by the enzyme (Figure 10)[43,44]. When the thermostable phosphorylase (from aeolicus VF5) was instead employed, reactions of Glc3 using excess molar ratios of Man-1-P and GlcN- Aquifex1-P, successive aeolicus VF5) mannosylations/glucosaminylat was instead employed, reactionsions of Glctook3 using place excess to molar obtain ratios non-natural of Man-1-P heterooligosaccharidesand GlcN-1-P, successive composed mannosylations/glucosaminylations of α(14)-linked mannose/glucosamine took place chains to obtain (Figure non-natural 10) [73]. α Theheterooligosaccharides MALDI-TOF mass composed spectra of of the(1 Ñproducts4)-linked of mannose/glucosamine this thermostable ph chainsosphorylase-catalyzed (Figure 10)[73]. Theenzymatic MALDI-TOF glycosylation mass spectra with of a the donor products to ofacceptor this thermostable feed ratio phosphorylase-catalyzed of 10:1 showed several enzymatic peaks correspondingglycosylation with to the a donor molecular to acceptor masses feed of ratiotetra-octa-saccharides of 10:1 showed several having peaks one–five corresponding Man or toGlcN the molecular masses of tetra-octa-saccharides having one–ﬁve Man or GlcN residues with Glc . Further residues with Glc3. Further chain-elongation for higher DPs, however, was inhibited by Pi produced3 fromchain-elongation the glycosyl for donors, higher DPs,which however, is a naturall was inhibitedy occurring by Pi producedsubstrate from for thephosphorolysis glycosyl donors, by whichphosphorylase is a naturally catalysis. occurring substrate for phosphorolysis by phosphorylase catalysis.

HO R2 O HO O HO O HO O 10 HO OH HO O + OH OH OH R1 HO O O P O HO HO OHO n O Man-1-P; R1 = H, R2 = OH Glcn (Glycosyl acceptor) GlcN-1-P; R1 = NH2, R2 = H (Glycosyl donor)

R2 O O O HO HO HO OH + Pi R1 OH OH lase HO O O hory HO HO HO hosp to p n+1 Pota uffer ate b n = 2 Acet The rmos ( table Aqui pho fex a spho eolic ryla us se R2 VF5) O HO O HO O Ac HO + m Pi etate OH buf fer R1 OH OH H O O O HO HO HO m n+1 n = 1, m = 1~5

Man; R1 = H, R2 = OH GlcN; R1 = NH2, R2 = H

Figure 10. Differences in glycosylationglycosylation using Man-1-P/GlcN-1-PMan-1-P/GlcN-1-P as as glycosyl glycosyl donors donors by potato and thermostable phosphorylase catalyses.

An attempt was made to remove this Pi as a precipitate in the thermostable phosphorylase- An attempt was made to remove this Pi as a precipitate in the thermostable catalyzed glucosaminylation by conducting the reaction in an ammonium buffer (0.5 M, pH 8.6) phosphorylase-catalyzed glucosaminylation by conducting the reaction in an ammonium buffer containing MgCl2, as it has been reported that Pi forms an insoluble salt with ammonium and (0.5 M, pH 8.6) containing MgCl , as it has been reported that Pi forms an insoluble salt with magnesium ions [74]. Consequently,2 the thermostable phosphorylase-catalyzed polymerization in ammonium and magnesium ions [74]. Consequently, the thermostable phosphorylase-catalyzed the buffer system at 40 °C for 7 days of GlcN-1-P˝ with the Glc3 primer (30:1) successfully occurred to polymerization in the buffer system at 40 C for 7 days of GlcN-1-P with the Glc3 primer (30:1) produce the α(14)-linked glucosamine polymer with a DP of the GlcN units of ~20 (Mn = 3760 ), successfully occurred to produce the α(1Ñ4)-linked glucosamine polymer with a DP of the GlcN units which corresponded to the structure of a chitosan stereoisomer, a non-natural aminopolysaccharide of ~20 (M = 3760 ), which corresponded to the structure of a chitosan stereoisomer, a non-natural (Figure 11)n [75]. This product was then converted into a chitin stereoisomer, also a non-natural aminopolysaccharide (Figure 11)[75]. This product was then converted into a chitin stereoisomer, aminopolysaccharide, by N-acetylation using acetic anhydride (Figure 11). The enzymatic also a non-natural aminopolysaccharide, by N-acetylation using acetic anhydride (Figure 11). The copolymerization of Glc-1-P with GlcN-1-P using thermostable phosphorylase catalysis was also enzymatic copolymerization of Glc-1-P with GlcN-1-P using thermostable phosphorylase catalysis performed under the same conditions to obtain a non-natural heteroaminopolysaccharide composed was also performed under the same conditions to obtain a non-natural heteroaminopolysaccharide of Glc/GlcN units [76]. composed of Glc/GlcN units [76]. Polymers 2016, 8, 138 11 of 20 Polymers 2016, 8, 138 11 of 19

OH OH OH O O O OH O OH 30 HO HO + OH OH OH O HO O O NH3 HO HO HO O P OH O- Glc3 (primer) GlcN-1-P (monomer)

OH O OH O OH O Thermostable α-glucan phosphorylase OH OH OH O O (Aquifex aeolicus VF5) OH OH NH2 O HO O NH2 O HO HO Ammonia buffer HO NH2 O HO 2 HO m-2 (0.5 M, pH 8.6) / MgCl2 o 40 C, 7 days Chitosan stereoisomer; m = ~18 + ammonium magnesium Pi (precipitate)

OH O OH O OH O OH O OH Acetic anhydride OH O OH OH NHAc O O NHAc O HO HO HO Aqueous Na2CO3 HO NHAc O HO 2 HO m-2

Chitin stereoisomer Ac = C CH3 O

Figure 11. ThermostableThermostable phosphorylase-cata phosphorylase-catalyzedlyzed enzymatic enzymatic polymerization polymerization of ofα-αD-glucosamine-D-glucosamine 1- 1-phosphatephosphate to toproduce produce chitosan chitosan stereoisomer stereoisomer and andsubsequent subsequent N-acetylationN-acetylation to produce to produce chitin chitinstereoisomer. stereoisomer.

5. Preparation Preparation of of Amylose Amylose Supramolecular Supramolecular Mate Materialsrials by by Phosphorylase-Catalyzed Phosphorylase-Catalyzed Enzymatic EnzymaticPolymerization Polymerization Amylose constructs left-handed helical conformation and forms inclusion complexes with various guest molecules, typically monomeric and oligomeric low low molecular molecular weight weight compounds compounds [77]. [77]. Accordingly it is suitablesuitable for incorporationincorporation intointo high-performancehigh-performance polymeric materials. The driving force forfor thisthis binding binding of of guest guest molecules molecules is hydrophobicis hydrophobic interactions, interactions, as the as insidethe inside of the of amylose the amylose helix ishelix hydrophobic, is hydrophobic, and therefore, and therefore, in aqueous in solvents,aqueous hydrophobic solvents, hydrophobic guest molecules guest will molecules be spontaneously will be includedspontaneously in the included amylose cavity.in the amylose On the other cavity. hand, On littlethe other has been hand, reported little has regarding been reported the formation regarding of inclusionthe formation complexes of inclusion between complexes amylose between and higher amylose molecular and weighthigher molecular polymeric weight guest molecules polymeric [78 guest–87]. Themolecules difﬁculty [78–87]. in the The inclusion difficulty of thesein the polymericinclusion of guest these molecules polymeric into guest the molecules amylose cavity into the arises amylose from thecavity necessity arises forfrom a weak the necessity hydrophobic for a interaction weak hydr toophobic drive the interaction complexation, to drive and the amylose complexation, does therefore and notamylose have does sufﬁcient therefore power not to have direct sufficient the inclusion power of to long direct polymeric the inclusion chains of intolong its polymeric cavity. chains into its cavity.It has been reported that phosphorylase-catalyzed enzymatic polymerization to form the shorter α(1ÑIt4)-glucan has been reported (maltooligosaccharide) that phosphorylase-catalyzed to the longer enzymaticα(1Ñ4)-glucan polymerization (amylose) to induces form the inclusion shorter complexationα(14)-glucan with (maltooligosaccharide) hydrophobic guest polymersto the longer [15,16 α,34(1,884)-glucan–93]. Such (amylose) an efficient induces method inclusion for the formationcomplexation of amylose-polymerwith hydrophobic inclusion guest polymers complexes [15,16,34,88–93]. was achieved Such by the an phosphorylase-catalyzedefficient method for the enzymaticformation of polymerization amylose-polymer of amylose inclusion in complexe a dispersions was of achieved hydrophobic by the polymers phosphorylase-catalyzed in aqueous buffer solvents.enzymatic The polymerization process of this syntheticof amylose method in a disper for thesion generation of hydrophobic of such inclusion polymers complexes in aqueous is similar buffer to thesolvents. way that The vines process of plants of this grow synthetic and twine method around for a rodthe (Figuregeneration 12), whichof such is whyinclusion this polymerization complexes is approachsimilar to the has way been that named vines “vine-twiningof plants grow polymerization”.and twine around a Hydrophobic rod (Figure 12), polyethers, which is why such this as poly(tetrahydrofuran)polymerization approach (PTHF), has been poly(oxetane) named “vine-twining (POXT)) [94,95 polymerization”.]; polyesters, such Hydrophobic as, poly(δ-valerolactone) polyethers, (PVL),such as poly( poly(tetrahydrofuran)ε-caprolactone) (PCL), (PTHF), poly(glycolic poly(oxetane) acid-co-ε -caprolactone))(POXT)) [94,95]; [96 polyesters,–98]; polycarbonates, such as, suchpoly( as,δ- poly(tetramethylenevalerolactone) (PVL), carbonate) poly(ε-caprolactone) [99]; and poly(ester-ether)s (PCL), poly(glycolic (–CH2CH 2acid-C(C=O)OCHco-ε-caprolactone))2CH2CH2CH [96–98];2–) [97] havepolycarbonates, been observed such to actas, aspoly(tetramethylene guest polymers and carbonate) have been included[99]; and into poly(ester-ether)s amylose using this(– polymerizationCH2CH2C(C=O)OCH technique2CH2CH (Figure2CH2 –)12 [97]). have Vine-twining been observed polymerization to act as guest does polymers not produce and have inclusion been complexesincluded into with amylose hydrophilic using this polymers, polymerization such as technique PEG and (Figure the other 12). Vine-twining poly(ester–ether)s, polymerization owing to insufficientdoes not produce interactions inclusion between complexes the polymer with and hydrophilic the cavity ofpolymers, the amylose. such In as addition, PEG and the the inclusion other viapoly(ester–ether)s, vine-twining polymerization owing to insufficient has not interact been achievedions between with stronglythe polymer hydrophobic and the polymerscavity of likethe poly(oxepane),amylose. In addition, due to their the aggregationinclusion via in aqueousvine-twi bufferning polymerization solvents as a result has of not their been longer achieved alkyl chains with whenstrongly compared hydrophobic to PTHF polymers and POXT. like It poly(oxepane), can therefore be due concluded to their that aggregation the moderate in aqueous hydrophobicity buffer solvents as a result of their longer alkyl chains when compared to PTHF and POXT. It can therefore be concluded that the moderate hydrophobicity of guest polymers is the important factor in Polymers 2016, 8, 138 12 of 20 Polymers 2016, 8, 138 12 of 19 Polymers 2016, 8, 138 12 of 19 determiningof guest polymers whether is the or important not amylose factor inwill determining form inclusion whether complexes or not amylose during will a formvine-twining inclusion determiningpolymerization.complexes duringwhether a vine-twiningor not amylose polymerization. will form inclusion complexes during a vine-twining polymerization.

FigureFigure 12. 12. ImageImage of of vine-twining vine-twining polymerization polymerization to to produce produce amylose–polymer amylose–polymer inclusion inclusion complexes complexes Figure 12. Image of vine-twining polymerization to produce amylose–polymer inclusion complexes andand typical typical guest guest polymers polymers that that have have been been employed employed in in this system. and typical guest polymers that have been employed in this system. An attempt at a parallel enzymatic polymerization system was made to achieve the formation An attempt at a parallel enzymatic polymerization system was made to achieve the formation of anAn inclusion attempt atcomplex a parallel with enzymatic a strongly polymerization hydrophobic polyestersystem was [100]. made In thisto achieve system, the two formation enzymatic of ofan an inclusion inclusion complex complex with with a strongly a strongly hydrophobic hydrophobic polyester polyester [100]. [100 ].In In this this system, system, two two enzymatic enzymatic polymerizations, the phosphorylase-catalyzed enzymatic polymerization of Glc-1-P from the Glc7 polymerizations, the phosphorylase-catalyzed enzymatic polymerization of Glc-1-P from the Glc polymerizations,primer to produce the amylose,phosphorylase-catalyzed and the lipase-catalyzed enzymatic polycondensation polymerization of of a diolGlc-1-P (1,8-octanediol) from the Glc and7 7 primer to produce amylose, and the lipase-catalyzed polycondensation of a diol (1,8-octanediol) primera dicarboxylic to produce acid amylose, (sebabic and acid) the to lipase-catalyzed produce a strongly polycondensation hydrophobic aliphaticof a diol (1,8-octanediol)polyester as the andguest and a dicarboxylic acid (sebabic acid) to produce a strongly hydrophobic aliphatic polyester as the a dicarboxylicpolymer [101,102], acid (sebabic were simultaneously acid) to produce conducted a strongly in hydrophobican aqueous buffer aliphatic solvent polyester (Figure as 13). the Successguest guest polymer [101,102], were simultaneously conducted in an aqueous buffer solvent (Figure 13). polymerwas achieved [101,102], using were this simultaneously approach, and conducted consequently, in an aqueous the analytical buffer solventresults (Figureof the reaction13). Success fully Success was achieved using this approach, and consequently, the analytical results of the reaction fully wassupported achieved the using formation this approach, of an inclusion and consequently,complex between the amyloseanalytical and results the polyester. of the reaction fully supportedsupported the the formation formation of ofan an inclusion inclusion complex complex between between amylose amylose and and the the polyester. polyester.

OH OH O HO HO O O O HO O Glc7 HO OH HOO C (CH ) O HO (CH ) OH OO P O Glc7 2 8 C OH 2 8 OH HO C (CH ) C OH HO (CH2)8 OH O P OO 2 8 Glc-1-PO Glc-1-P

Phosphorylase Lipase Phosphorylase Lipase

O O O C(CH2)8O CO (CH ) O 2 8 m C(CH2)8 CO (CH ) O 2 8 m amylose-polyester inclusion complexes amylose-polyester inclusion complexes Figure 13. Parallel enzymatic polymerization system to produce inclusion complex from amylose and FigurestronglyFigure 13. 13. ParallelhydrophobicParallel enzymatic enzymatic polyester. polymerization polymerization system system to produce to produce inclusion inclusion complex complex from from amylose amylose and and stronglystrongly hydrophobic hydrophobic polyester. polyester. Amylose showed the selective inclusion behavior in certain structures, molecular weight distributions,Amylose showed and chiralities the selective of the guesinclusiont polymers. behavi [103–105].or in certain For example,structures, poly( molecularL-lactide) weight (PLLA) distributions, and chiralities of the guest polymers. [103–105]. For example, poly(L-lactide) (PLLA) with lower Mn, ca. 1000–3000 was included in the cavity of amylose in the vine-twining with lower Mn, ca. 1000–3000 was included in the cavity of amylose in the vine-twining Polymers 2016, 8, 138 13 of 20

Amylose showed the selective inclusion behavior in certain structures, molecular weight Polymersdistributions, 2016, 8, 138 and chiralities of the guest polymers. [103–105]. For example, poly(L-lactide) (PLLA)13 of 19 with lower Mn, ca. 1000–3000 was included in the cavity of amylose in the vine-twining polymerization polymerizationprocess, to form process, the amylose-PLLA to form the inclusion amylose-PL complex,LA inclusion while PLLA complex, with while higher PLLAMn, ca. with>6000, higher and M itsn, ca.stereoisomers, >6000, and its poly( stereoisomers,D-lactide) (PDLA) poly(D-lactide) and poly( (PDLA)DL-lactide), and poly( wereDL not-lactide), recognized were asnot guest recognized polymers as guestand were polymers therefore and not were taken therefore up by no thet taken amylose up [by106 the]. amylose [106]. Amylose supramolecular supramolecular network network hydrogels hydrogels were were fabricated fabricated through through the formation the formation of vine- of twinedvine-twined inclusion inclusion complexes complexes of amylose of amylose during duringthe phosphorylase-catalyzed the phosphorylase-catalyzed polymerization. polymerization. In these studies,In these the studies, designed the graft designed copolymers, graft copolymers, such as poly(acrylic such as acid poly(acrylic sodium salt- acidgraft sodium-δ-valerolactone) salt-graft-δ- (PAA-Na-valerolactone)g-PVL), (PAA-Na- CMC-graftg-PVL),-PCL CMC- (CMC-graftg-PCL),-PCL (CMC-and poly(g-PCL),γ-glutamic and poly( acid-γ-glutamicgraft-ε-caprolactone) acid-graft-ε- (PGA-caprolactone)g-PCL) were (PGA- employedg-PCL) were as guest employed polymers as (Figur gueste polymers 14a) [107–109], (Figure while 14a) the [107 hydrophilic–109], while main- the chains,hydrophilic PAA, main-chains, CMC, and PGA, PAA, acted CMC, as and the PGA,main actedcomponents as the main of the components hydrogels. When of the hydrogels.the vine-twining When polymerizationthe vine-twining was polymerization carried out in was the carriedpresence out of in these the presence guest graft of thesecopolymers guest graft in aqueous copolymers acetate in bufferaqueous solution, acetate bufferthe reaction solution, mixtures the reaction yielded mixtures the yieldedsupramolecular the supramolecular network hydrogels network hydrogels through inclusionthrough inclusion of the graft of the chains graft with chains amylose with amylose to form to supramolecular form supramolecular copolymers copolymers (Figure (Figure 14b). 14Theb). intermolecularThe intermolecular inclusion inclusion complexes complexes could could act actas ascross-linking cross-linking points points to to construct construct higher-order supramolecular network structures in the hydrogels.

Figure 14.14.( a)(a Preparation) Preparation of amylose of amylose supramolecular supramolecular network materialsnetwork by materials vine-twining by polymerizationvine-twining polymerizationusing graft copolymers using graft having copolymers hydrophilic having main-chains hydrophilic andmain-chains hydrophobic and hydrophobic guest graft chainsguest graft and chains(b) photographs and (b) photographs before and afterbefore vine-twining and after vine-twining polymerization. polymerization.

The ability of the hydrogel of PAA-Na-g-PVL to reversibly facilitate enzymatic disruption and The ability of the hydrogel of PAA-Na-g-PVL to reversibly facilitate enzymatic disruption reproduction was successfully demonstrated by the combined use of β-amylase-catalyzed hydrolysis and reproduction was successfully demonstrated by the combined use of β-amylase-catalyzed of the amylose component in the hydrogel, followed by its reformation by subsequent hydrolysis of the amylose component in the hydrogel, followed by its reformation by subsequent phosphorylase-catalyzed enzymatic polymerization [107]. By the two successive enzymatic reactions, phosphorylase-catalyzed enzymatic polymerization [107]. By the two successive enzymatic reactions, therefore, enzymatically recyclable behavior of the hydrogel at the molecular level was observed. A therefore, enzymatically recyclable behavior of the hydrogel at the molecular level was observed. film was further formed by adding water followed by drying to a powdered sample prepared by A ﬁlm was further formed by adding water followed by drying to a powdered sample prepared by lyophilization of the hydrogel from CMC-g-PCL [108]. lyophilization of the hydrogel from CMC-g-PCL [108]. The macroscopic interfacial healing of a supramolecular hydrogel consisting of PGA-g-PCL was The macroscopic interfacial healing of a supramolecular hydrogel consisting of PGA-g-PCL was observed through phosphorylase-catalyzed enzymatic polymerization [109]. The hydrogel formed observed through phosphorylase-catalyzed enzymatic polymerization [109]. The hydrogel formed initially from vine-twining polymerization was cut into two pieces, and a sodium acetate buffer initially from vine-twining polymerization was cut into two pieces, and a sodium acetate buffer solution containing Glc-1-P and phosphorylase was dropped on the surfaces of the hydrogel pieces. solution containing Glc-1-P and phosphorylase was dropped on the surfaces of the hydrogel pieces. After the surfaces were placed in contact with one another, the materials were left standing at 40 °C After the surfaces were placed in contact with one another, the materials were left standing at 40 ˝C for for 6 h, over which time the enzymatic polymerization proceeded, such that the two hydrogel pieces 6 h, over which time the enzymatic polymerization proceeded, such that the two hydrogel pieces fused fused at the point of contact. The healing of the gels on a macroscopic level was induced by the at the point of contact. The healing of the gels on a macroscopic level was induced by the complexation complexation of the enzymatically-produced amyloses with the PCL graft chains at the interface. of the enzymatically-produced amyloses with the PCL graft chains at the interface. Porous cryogels Porous cryogels were fabricated by the lyophilization of the hydrogel, while ion gels could be prepared by soaking the hydrogels in an ionic liquid of 1-butyl-3-methylimidazolium chloride. Supramolecular polymers comprised of amylose-PTHF and amylose-PLLA inclusion complexes were successfully fabricated by vine-twining polymerizations using Glc7-block-PTHF and Glc7-block- PLLA, respectively, as primer–guest conjugates (Figure 15a) [110,111]. In these systems, a propagating amylose chain initiated by phosphorylase catalysis from a Glc7 conjugate could Polymers 2016, 8, 138 14 of 20 were fabricated by the lyophilization of the hydrogel, while ion gels could be prepared by soaking the hydrogels in an ionic liquid of 1-butyl-3-methylimidazolium chloride. Supramolecular polymers comprised of amylose-PTHF and amylose-PLLA inclusion complexes were successfully fabricated by vine-twining polymerizations using Glc7-block-PTHF and GlcPolymers7-block 2016-PLLA,, 8, 138 respectively, as primer–guest conjugates (Figure 15a) [110,111]. In these systems,14 of 19 a propagating amylose chain initiated by phosphorylase catalysis from a Glc7 conjugate could potentiallypotentially includeinclude aa guestguest polymerpolymer segmentsegment fromfrom another conjugate, whereby whereby the the successive successive occurrenceoccurrence of of such such an an eventevent wouldwould givegive riserise toto linearlinear inclusion supramolecular polymers.

FigureFigure 15. 15.Preparation Preparation ofof ((aa)) linearlinear andand (b) hyperbranched supramolecular supramolecular polymers polymers by by vine-twining vine-twining polymerizationpolymerization using using primer–guestprimer–guest conjugates.conjugates.

Similarly, vine-twining polymerization using a branched Glc7-PLLA2 conjugate would produce Similarly, vine-twining polymerization using a branched Glc -PLLA conjugate would a hyperbranched inclusion supramolecular polymer composed of7 amylose-PLLA2 inclusion producecomplexes a hyperbranched (Figure 15b) [112]. inclusion This hyperbranched supramolecular pr polymeroduct could composed then form of amylose-PLLAan ion gel with inclusion1-butyl- complexes3-methylimidazolium (Figure 15 b)chloride, [112]. which This could hyperbranched be further converted product could into a thenhydrogel form upon an ionexchange gel with of 1-butyl-3-methylimidazoliumthe dispersion media. The lyophilization chloride, whichof the hydrogel could be resulted further in converted the fabrication into aof hydrogela cryogel with upon exchangea porous ofmorphology. the dispersion The media.gelation The behavior lyophilization of the hyperbranched of the hydrogel supramolecular resulted in the polymer fabrication was of aa cryogelresult of with its highly a porous extended morphology. structure, The wherea gelations the behavior aforementioned of the hyperbranchedlinear supramolecular supramolecular polymer polymerdid not exhibit was asuch result gelation of its behavior. highly extended structure, whereas the aforementioned linear supramolecularThe relative polymer chain orientations did not exhibit of amylose such gelation and the behavior. two stereoisomers of PLA in the inclusion complexesThe relative formed chain in phosphorylase-catalyzed orientations of amylose andenzymatic the two polymerizations stereoisomers of were PLA investigated in the inclusion by complexesusing four formeddifferent in primer–guest phosphorylase-catalyzed conjugates, which enzymatic consisted polymerizations of a Glc7 moiety werefunctionalized investigated at the by usingcarboxylate four different or hydroxy primer–guest termini of both conjugates, PLLA an whichd PDLA consisted [113]. The of aamylose-PLLA Glc7 moiety functionalized supramolecular at thepolymers carboxylate formed or hydroxy in the terminienzymatic of both polymerization PLLA and PDLA in the [113 presence]. The amylose-PLLA of both of the supramolecular two PLLA polymersconjugates, formed suggesting in the enzymaticthat, regardless polymerization of the chain in orientation the presence of ofPLLA, both the of the amylose two PLLA cavity conjugates, took up suggestingthe guest polymer. that, regardless Interestingly, of the chainthe amylose–PD orientationLA of PLLA,diblock the copolymers amylose cavitydid not took form up inclusion the guest polymer.complexes, Interestingly, because of the amylose–PDLAeffects of chirality diblock on the copolymers recognition didbehavior not form of amylose, inclusion which complexes, were becauseirrespective of the of effects the PDLA of chirality chain orientation. on the recognition The similar behavior left-handed of amylose, helical which direction were of irrespective amylose and of thePLLA PDLA imply chain that orientation. an inclusion The complex similar left-handedwill form, whereas helical direction the directions of amylose of methyl and PLLA substituents imply thatin anPLA inclusion toward complex amylose, will which form, are whereas oppositely the changed directions depending of methyl on substituents the relative in chain PLA towardorientation, amylose, are whichnot the are important oppositely factor changed for the depending complexation. on the relative chain orientation, are not the important factor for the complexation. 6. Conclusions 6. Conclusions This review presents evidence that phosphorylase-catalyzed enzymatic reactions efficiently provideThis reviewvarious presentspolysaccharide evidence materials that phosphorylase-catalyzed with precisely controlled enzymatic structures. reactions Such efficientlyartificial providepolysaccharides various exhibit polysaccharide specific and materials unique withfunctions, precisely which controlled are very different structures. to those Such of artificialtypical polysaccharidessynthetic polymers. exhibit Furthermore, specific and this unique enzymatic functions, approach which is commonly are very different identified to thoseas a green of typical and sustainable process because it is generally conducted in aqueous media under mild conditions and proceeds without the production of harmful by-products. Accordingly, the phosphorylase-catalyzed methods described herein have the potential to be applied in the environmentally benign production of useful and functional polysaccharide materials in various fields, such as biomedicine and tissue engineering. The author strongly believes that the further development of these enzymatic methods, including phosphorylase catalysis, will provide unique functional materials in the future. Polymers 2016, 8, 138 15 of 20 synthetic polymers. Furthermore, this enzymatic approach is commonly identified as a green and sustainable process because it is generally conducted in aqueous media under mild conditions and proceeds without the production of harmful by-products. Accordingly, the phosphorylase-catalyzed methods described herein have the potential to be applied in the environmentally benign production of useful and functional polysaccharide materials in various fields, such as biomedicine and tissue engineering. The author strongly believes that the further development of these enzymatic methods, including phosphorylase catalysis, will provide unique functional materials in the future.

Acknowledgments: The author is indebted to the co-workers, whose names are found in references from his papers, for their enthusiastic collaborations. The authors also gratefully thank for financial supports from a Grant-in-Aid for Scientific Research from Ministry of Education, Culture, Sports, and Technology, Japan (Nos. 14550830, 17550118, 19550126, 24350062, and 25620177). Conflicts of Interest: The author declares no conflict of interest.

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