molecules

Review Synthesis of Glycosides by Glycosynthases

Marc R. Hayes 1 and Jörg Pietruszka 1,2,* ID

1 Institut für Bioorganische Chemie, Heinrich-Heine-Universität Düsseldorf im Forschungszentrum Jülich, 52426 Jülich, Germany; [email protected] 2 Forschungszentrum Jülich, IBG-1: Biotechnology, 52426 Jülich, Germany * Correspondence: [email protected]; Tel.: +49-2461-61-4158

Received: 26 July 2017; Accepted: 28 August 2017; Published: 30 August 2017

Abstract: The many advances in glycoscience have more and more brought to light the crucial role of glycosides and glycoconjugates in biological processes. Their major influence on the functionality and stability of peptides, cell recognition, health and immunity and many other processes throughout biology has increased the demand for simple synthetic methods allowing the defined syntheses of target glycosides. Additional interest in glycoside synthesis has arisen with the prospect of producing sustainable materials from these abundant polymers. Enzymatic synthesis has proven itself to be a promising alternative to the laborious chemical synthesis of glycosides by avoiding the necessity of numerous protecting group strategies. Among the biocatalytic strategies, glycosynthases, genetically engineered glycosidases void of hydrolytic activity, have gained much interest in recent years, enabling not only the selective synthesis of small glycosides and glycoconjugates, but also the production of highly functionalized polysaccharides. This review provides a detailed overview over the glycosylation possibilities of the variety of glycosynthases produced until now, focusing on the transfer of the most common glucosyl-, galactosyl-, xylosyl-, mannosyl-, fucosyl-residues and of whole glycan blocks by the different glycosynthase enzyme variants.

Keywords: biocatalysis; glycoside; polysaccharide; glycosidase; glycosynthase; synthesis; glycosylation

1. Introduction With the increasingly growing knowledge of the major role glycosides and glycoconjugates play in biological processes, the demand for simple methods for the synthesis of defined glycosides is constantly rising. Their high biological functionality and complex structure is a result of their numerous functional moieties, diverse stereochemistry, and numerous linkage possibilities. Glycosidic structures are ubiquitous throughout Nature, covering the surface of all cellular organisms and even the macromolecules inside these. Therefore, glycosides fulfill many different functions in biological systems providing on the one hand, function, structure and stability for cells and enzymes, but also acting as recognition motifs, as for example, in blood group antigens. Many bacterial and viral infections are mediated by glycoside recognition, clearly demonstrating the importance of glycosides for the pharmaceutical industry [1]. Additionally, specific oligosaccharides, such as human milk oligosaccharides have been described to have probiotic and antimicrobial effects making them highly desired targets for the food and nutrition industry [2]. Polysaccharides also play an important role in discovering new sustainable materials as they originate from renewable resources. Synthesis of glycoside structures would therefore greatly improve the possibility in producing new pharmaceuticals and materials, and allow targeted research on functional and structural properties of these compounds. The chemical synthesis of glycosides is well developed, but remains a laborious task, as many protection and selective deprotection steps are required to solely address single functional groups in order to avoid side product formation [3–6]. Complete anomeric control of the newly formed bond is also a prerequisite to avoid lengthy separation methods. Throughout nature the synthesis of

Molecules 2017, 22, 1434; doi:10.3390/molecules22091434 www.mdpi.com/journal/molecules Molecules 2017, 22, 1434 2 of 20

Molecules 2017, 22, 1434 2 of 20 glycosidic structures is accomplished by the enzyme group of glycosyltransferases. These enzymes transferalso activateda prerequisite sugar to nucleotide avoid lengthy donor separation molecules methods. selectively Throughout onto an acceptor nature molecule.the synthesis Their of use in biocatalysisglycosidic structures has been extensivelyis accomplished researched, by the enzy butme is stillgroup limited of glycosyltransferases. for a large scale byThese the enzymes high cost of transfer activated sugar nucleotide donor molecules selectively onto an acceptor molecule. Their use the nucleotide-phosphate donors and difficulty in expression and handling of these mostly membrane in biocatalysis has been extensively researched, but is still limited for a large scale by the high cost of bound enzymes [7]. Alternative carbohydrate active enzymes are glycohydrolases or glycosidases, the nucleotide-phosphate donors and difficulty in expression and handling of these mostly of whichmembrane a high bound variety enzymes are readily [7]. Alternative available due carboh to theirydrate occurrence active enzymes in the metabolicare glycohydrolases pathways or of all organismsglycosidases, [8]. This of which group a ofhigh enzymes variety naturallyare readily degrade available glycosidic due to their structures occurrence and in the are metabolic defined into twopathways groups depending of all organisms on their [8]. catalytic This group mechanism of enzymes (Scheme naturally1A,B) degrade [ 9]. Retaining glycosidic glycosidases structures and follow a doubleare defined displacement into two catalyzedgroups depending by a nucleophilic on their catalytic and acid/base mechanism residue (Scheme in the 1A,B) enzymes [9]. Retaining active site resultingglycosidases in a retention follow ofa double the anomeric displacement configuration catalyzed of by the a nucleophilic substrate in and the yieldedacid/base product. residue Invertingin the glycosidasesenzymes active in comparison site resulting follow in a retention a single of displacementthe anomeric configuration mechanism of with the substrate two catalytic in the yielded acid/base residuesproduct. supporting Inverting the glycosidases nucleophilic in substitutioncomparison follow at the anomerica single displacement center of the mechanism substrate. Glycosidaseswith two cancatalytic be utilized acid/base in synthetic residues reactions supporting by the reverse nucleoph hydrolysisilic substitution or transglycosylation at the anomeric methodscenter of [the10, 11]. Nevertheless,substrate. Glycosidases the ability ofcan the be utilized glycosidase in synthetic in hydrolyzing reactions by thereverse produced hydrolysis product or transglycosylation leads to strongly methods [10,11]. Nevertheless, the ability of the glycosidase in hydrolyzing the produced product leads diminished yields. To overcome the disadvantage of product hydrolysis Mackenzie et al. and also Malet to strongly diminished yields. To overcome the disadvantage of product hydrolysis Mackenzie et al. et al. (working on exo- and endo-glycosidases, respectively) reported the production of genetically and also Malet et al. (working on exo- and endo-glycosidases, respectively) reported the production engineeredof genetically glycosidases, engineered namely glycosidases, glycosynthases, namely glycosynthases, which were which lacking were a nucleophilic lacking a nucleophilic residue and thereforeresidue void and oftherefore hydrolytic void of activity hydrolytic [12, 13activity]. However, [12,13]. However, the intact the structure intact structure of the enzymeof the enzyme allowed the formationallowed the of formation glycosidic of glycosidic bonds in highbonds yields in high in yields the presence in the presence of activated of activated glycosyl glycosyl donors donors such as glycosylsuch fluoridesas glycosyl (Scheme fluorides1C). (Scheme 1C).

SchemeScheme 1. Mechanisms 1. Mechanisms of glycosyl of glycosyl hydrolases hydrolases and and synthases. synthases. (A) Retaining(A) Retaining hydrolases hydrolases follow follow a double a displacementdouble displacement mechanism mechanism in which the in enzymewhich the forms enzyme a glycosyl-enzyme-intermediate forms a glycosyl-enzyme-intermediate by attacking by the attacking the anomeric centre with a nucleophilic amino acid residue. The anomeric configuration of anomeric centre with a nucleophilic amino acid residue. The anomeric configuration of the substrate is the substrate is retained in the product; (B) Inverting hydrolases follow a single displacement retained in the product; (B) Inverting hydrolases follow a single displacement mechanism, which is mechanism, which is supported by acid/base residues. The anomeric configuration of the product is supported by acid/base residues. The anomeric configuration of the product is inverted compared inverted compared to the substrate; (C) ‘Classic’ glycosynthase mechanism involving an activated to the substrate; (C) ‘Classic’ glycosynthase mechanism involving an activated glycosyl donor in glycosyl donor in opposite configuration to the natural substrate. The product cannot be hydrolyzed due oppositeto the configurationlack of a nucleophilic to the residue; natural (D substrate.) Alternative The glycosynthase product cannot mechanism be hydrolyzed producing the due activated to the lack of adonor nucleophilic in situ with residue; an external (D) Alternativenucleophile (azide, glycosynthase formate or acetate mechanism ion); (E producing) Glycosynthases the activated derived from donor in situendo with-β-N-acetylglucosaminidases an external nucleophile transfer (azide, oxazoline formate glycoside or acetate donors ion); to (variableE) Glycosynthases acceptors. derived from endo-β-N-acetylglucosaminidases transfer oxazoline glycoside donors to variable acceptors.

Since the introduction of this ‘classical’ glycosynthase method mostly restricted to retaining glycosidases, alternative methods such as in situ generation of donor molecules have been reported, Molecules 2017, 22, 1434 3 of 20 expanding the repertoire of glycosynthases greatly [14]. Glycosidases of various origins have been transformed into glycosynthases enabling the production of α- and β-glycosyl linkages with glucosyl, galactosyl-, fucosyl-, arabinosyl-, mannosyl- or even lactosyl-residues [15]. Most recently, the method has been transferred to endo-β-N-acetylglucosaminidases, such as Endo-M of M. hiemalis, which play important roles in protein post-translational glycosylation. Elucidation of the mechanistic properties of these enzymes led to the development of synthase-like mutants, which transfer oxazoline glycoside structures onto acceptors enabling the en bloc transfer of large glycans [16]. This has resulted in a powerful tool for modifying peptides with high pharmaceutical interest with defined glycan structures. There are many excellent reviews on the topic of enzymatic glycoside synthesis, which give a great overview of the biocatalytic methods but manly focus on the production and mechanistic studies of glycosynthases [10,17–19]. This review will focus on the synthesis of glycosides employing glycosynthase methods, only, summarizing in each section the findings of a specific glycosyl residue also giving an overview of described products, which have been synthesized, rather than focusing on the production of the glycosynthase itself.

2. Glycoside Syntheses Using Glycosynthase Methods

2.1. Glucosynthases Since the introduction of the genetically engineered glycosynthases by Mackenzie et al. in 1998, glucosynthases derived from glucosidases (exo or endo) originating from a variety of organisms have been the most commonly produced type of these enzymes [12]. This type of variant is also often named generally ‘glycosynthase’ throughout literature, due to the often-observed promiscuity in regard to the donor glycoside. The Abg E358A glycosynthase, for example, catalyzed not only the transfer of glucosyl but also galactosyl residues to para-nitrophenyl (pNP) glycosides. Further modulation of this enzyme broadened the scope of donor substrates as reported by Shim et al. and Kim et al. [20,21]. The use of the glycosynthase variant Abg NNT was thereby extended to C3-modified (methylated) gluco- and galactosyl donors (producing β-1,4 linkages) and Abg 2F6 to a xylosyl donor (Section 2.3), giving a much broader range of synthesizable glycosides. The promiscuity of glycosynthases to accept different glycosyl fluoride donors was also impressively demonstrated by Wei et al. with the β-glycosidase mutant TnG E338A of T. nonproteolyticus [22]. The glycosynthase showed high variability towards the donor molecule, transferring α-D-glucosyl, α-D-galactosyl, and α-D-fucosyl fluoride donors (1, 2 and 3) to different acceptors 4–8 in yields for 9–13 varying between 15–100% depending on the donor and acceptor combination (Table1). Interestingly the enzyme also exhibited activity towards dihydroisoandrosterone (5), an unusually large, lipophilic acceptor, with α-D-glucosyl fluoride (αGlcF, 1) as the donor glycoside (49% yield). An even broader donor scope was exhibited by the glycosynthase BGlu1 E386G originating from the β-glucosidase of rice examined by Pengthaisong et al. [23]. The glycosynthase was shown to transfer glycoside residues of α-D-glucosyl (1), α-D-galactosyl (2), α-D-fucosyl (3), α-D-arabinosyl, α-D-xylosyl (14), and α-D-mannosyl (15) fluoride donor to pNP-cellobioside 8 producing yields of 57%, 59%, 42%, 99%, 3% and 79%, respectively. The use of the Abg E358S glycosynthase in the synthesis of natural or unnatural glycosides was also demonstrated efficiently by Fairwether et al. in the synthesis of glycosylated versions of methyl β-acarviosin (16), an inhibitor of cellulases (Scheme2a) [ 24]. The inhibitor was glucosylated (β-1,4) to the tri- 17 and tetrasaccharide 18 in yields of 42% and 6%, respectively. Further putative cellulase inhibitors, variants of isofagomine and tetrahydrooxazine were produced by MacDonald et al. producing a mixture of cello-oligosaccharide inhibitor variants with different lengths [25]. The reported glucosylation of erythromycin A (19) by Jakeman et al. showed the possibility of transferal of a glucosyl residue (β-1,2) to a more complex type of acceptor in a yield of 14% (20, Scheme2b) [26]. Molecules 2017, 22, 1434 4 of 20

MoleculesMolecules 2017 2017, 22, ,22 1434, 1434 4 of4 of20 20 MoleculesMolecules 2017 2017, 22, ,22 1434, 1434 4 of4 of20 20 MoleculesMolecules 20172017,, 2222,, 14341434 44 ofof 2020 MoleculesTableMolecules 1. 2017Examples 20172017, 22,, ,2222 1434,, 14341434 of synthetic glycosylation products catalyzed by the TnG E338A [22]. Reactions4 of44 of20of 2020 MoleculesMoleculesTableTable 2017 2017 1.,, 221.Examples, ,22, Examples14341434, 1434 αof ofsynthetic synthetic glycosylationα glycosylation products productsα catalyzed catalyzed by by the the TnG TnG E338A E338A [22]. [22]. Reactions Reactions4 of4 of20 20 wereTable carriedTable 1. 1. outExamples Examples with of- D ofsynthetic- GlcFsynthetic (1), glycosylation glycosylation-D-GalF (2 ),products orproducts-D- FucFcatalyzed catalyzed (3) as by theby the the donor. TnG TnG E338A DonorE338A [22]. and[22]. Reactions acceptor Reactions TableTable 1.1. ExamplesExamples ofof syntheticsynthetic glycosylationglycosylation productsproducts catalyzedcatalyzed byby thethe TnGTnG E338AE338A [22].[22]. ReactionsReactions moleculeswereTablewereTable carried were1. carried 1.Examples ExamplesExamples employedout out with withof of ofsyntheticα in -syntheticsyntheticDα-GlcF- equalD-GlcF glycosylation(1amounts ),glycosylationglycosylation(1 α),- Dα-GalF-D-GalF unless (products2 ),(productsproducts2 or), otherwise orα- Dα catalyzed-FucF-D catalyzedcatalyzed-FucF noted.(3 )( 3asby) asbyby thethe Yields thethethe donor.TnG donor.TnGTnG were E338A Donor E338AE338A Donor determined [22]. and [22].[22]. and Reactions acceptor ReactionsReactions acceptor after TablewereTablewere carried 1. carried 1.ExamplesExamples Examples out out with ofofwith of synthetic syntheticα synthetic-αD--GlcFD-GlcF glycosylationglycosylation( 1 glycosylation),(1 ),α -αD--GalFD-GalF productsproducts(2 products),(2 ),or or α - α Dcatalyzedcatalyzed--FucF Dcatalyzed-FucF (3 )( 3bybyas) byas thethe thethe donor.TnGTnG donor.TnG E338AE338A DonorE338A Donor [22].[22]. and[22]. and ReactionsReactions acceptorReactions acceptor Moleculesmoleculeswereweremolecules 2017 carriedcarried, 22, 1434were wereoutout employed withwith employed αα--DD-GlcF -GlcFin inequal equal((11),), amountsαα --amountsDD-GalF-GalF unless((22 ),unless), oror otherwiseαα --DotherwiseD-FucF-FucF ((noted.33) ) noted. asas thethe Yields donor.Yieldsdonor. were wereDonorDonor determined determined andand acceptoracceptor after4 ofafter 20 purificationmoleculeswerewere carried carried or were isolation. out outemployed with with α -αD --GlcF-Din-GlcF-GlcF equal (1 ),((1 amountsα),), - αD--GalF-D-GalF-GalF unless(2 ),((2 or),), or orotherwiseα - αD--FucF-D-FucF-FucF (noted.3 )(( 3as)) as asthe Yields thethe donor. donor.donor. were Donor Donor Donordetermined and andand acceptor acceptoracceptor after weremoleculesweremolecules carried carried were wereout out employed with employed with α -αD--GlcF Din-GlcF inequal equal(1 ),(1 α),amounts -αDamounts--GalFD-GalF unless(2 ),unless(2 or), or αotherwise -αDotherwise--FucFD-FucF ( 3noted. )( 3noted.as) as the Yieldsthe donor.Yields donor. were wereDonor Donor determined determined and and acceptor acceptor after after purificationmoleculesmoleculespurification werewere or orisolation. employed employedisolation. inin equalequal amountsamounts unlessunless otherwiseotherwise noted.noted. YieldsYields werewere determineddetermined afterafter purificationmoleculespurificationmolecules were orwere or isolation. employedisolation. employed in inequal equal amounts amounts unless unless otherwise otherwise noted. noted. Yields Yields were were determined determined after after Tablemoleculespurificationpurificationmolecules 1. Examples were ororwere isolation.isolation. employed ofemployed synthetic in in equalglycosylation equal amounts amounts products unless unless otherwise catalyzed otherwise noted.by noted. the TnGYields Yields E338A were were [22]. determined determined Reactions after after purificationpurificationDonor or orisolation. isolation. Acceptor Product Yield (%) purificationpurificationDonorDonor oror isolation.isolation. AcceptorAcceptor Product Product Yield Yield (%) (%) werepurification carriedDonorDonor orout isolation. with α-D -GlcF (1),Acceptor αAcceptor-D-GalF (2), or α-D-FucF (3) as Productthe Product donor. Yield Donor Yield (%) and(%) acceptor Donor Acceptor Product Yield (%) moleculesDonor wereDonor employed in equalAcceptor amountsAcceptor unless otherwise noted. Product Yields Product were Yield Yield determined (%) (%) after DonorDonorDonor AcceptorAcceptorAcceptor Product Product Product Yield Yield Yield (%) (%)(%) purification or isolation.

Donor1 1 Acceptor4 4 Product Yield9 80%9 80% (%) 1 1 4 4 9 9 80%9 80% 11 4 9 80% 1 1 4 4 9 80%9 80% 1 1 4 44 9 80%80%99 80%80%

1 4 9 80%

5 5 1010 49% 49% 5 5 1010 49% 49% 5 5 10 10 49% 5 5 1010 49% 49% 5 55 101010 49%49% 49%49%

5 10 49% 2 2 6 6 1111 a 14% a 14% 2 2 6 6 1111 a 14% a 14% 22 66 1111 aa 14%14% 2 2 6 66 11 11 a 14% aa 14%14% 2 2 6 66 1111 a 14% 14%a 14%

2 11 a 14% 6 7 7 1212 32% 32% 7 7 1212 32% 32% 77 1212 32%32% 7 77 12 1212 32% 32%32% 7 7 1212 32%32% 32%

7 12 32%

3 3 1313 91% 91% 3 3 8 8 1313 91% 91% 33 8 8 1313 91%91% 3 3 a Donora Donor88 was was used used in in 2 equivalents.2 equivalents. 1313 91% 91% 3 a a Donor8 8 was used in 2 equivalents. 13 91% 3 3 aa Donor8 8 was used in 2 equivalents. 13 1313 91%91% 91% a DonorDonoraa waswas usedused inin 22 equivalents.equivalents. a Donora DonorDonor was waswas used usedused in inin2 equivalents. 22 equivalents.equivalents. 3 a Donor DonorDonor8 Donor was waswas was used usedused used in in 2in equivalents. in 22 equivalents.equivalents.2 equivalents. 13 91%

a Donor was used in 2 equivalents.

(b)( b) (a)( a) (b()b ) (a)( a) ((bb)) ((aa)) (b()(b )) (a)(( a)) (b()b ) SchemeScheme 2. 2.Glucosylation Glucosylation(a)( a) of ofnatural natural and and unnatural unnatural glycosides. glycosides. (a )( aSynthesis) Synthesis of ofglucosylated glucosylated methyl methyl β- β- SchemeScheme 2. 2.Glucosylation Glucosylation of ofnatural natural and and unnatural unnatural glycosides. glycosides. (a )( aSynthesis)( bSynthesis) of ofglucosylated glucosylated methyl methyl β- β- acarviosinSchemeSchemeacarviosin 2.2. ( Glucosylation16Glucosylation ()a16 )by ) by Fairweather Fairweather ofof naturalnatural et etal. al. [24]. andand [24]. The unnaturalunnatural The mono- mono- glycosides.glycosides.and and diglycosylated diglycosylated ((aa)) SynthesisSynthesis products products ofof glucosylatedglucosylated 17 ,17 18, 18were were isolated methylmethyl isolated βinβ-- in acarviosinSchemeacarviosinScheme 2. (2.Glucosylation16 ( Glucosylation16Glucosylation) by) by Fairweather Fairweather of ofofnatural etnaturalnatural etal. al. [24].and [24]. andand Theunnatural The unnaturalunnatural mono- mono- glycosides.and glycosides.glycosides.and diglycosylated diglycosylated (a )(( aSynthesis)) SynthesisSynthesis products products of ofofglucosylated 17 glucosylatedglucosylated 17, 18, 18 were were isolatedmethyl isolated methylmethyl βin- βin-- SchemeacarviosinacarviosinScheme 2. (2.(GlucosylationGlucosylation1616 Glucosylation)) byby FairweatherFairweather ofof ofnaturalnatural naturaletet al.al. [24]. [24].andand and TheTheunnaturalunnatural unnatural mono-mono- glycosides.glycosides. andand glycosides. diglycosylateddiglycosylated ((a )( aSynthesis) Synthesis productsproducts of ofglucosylated 17 17glucosylated,, 1818 werewere isolatedisolatedmethyl methyl βinin- β - yieldsacarviosinyieldsacarviosin 42% 42% (and16 (and16) by 6%;)) byby Fairweather6%; (FairweatherFairweatherb )( bGlucosylation) Glucosylation et etal.et al. al.[24]. [24].[24].of ofTheerythromycin TheTheerythromycin mono- mono-mono- and andand A diglycosylated A( diglycosylated19diglycosylated ()19 by) by the the glucosynthase productsglucosynthase productsproducts 17 17, EryBI18,, 18 EryBIwere werewere D257G isolatedD257G isolatedisolated [26]. [26].in inin Schemeacarviosinyieldsacarviosinyields 42% 2. 42% Glucosylation( 16and ( 16)and by )6%; by Fairweather6%; Fairweather(b ()b Glucosylationof) Glucosylation natural et etal. andal. [24]. [24]. ofunnatural The oferythromycin The erythromycin mono- mono- glycosides. and and A diglycosylated A (diglycosylated19 ((19)a )by) Synthesisby the the glucosynthase productsglucosynthase productsof glucosylated 17 17,, 18EryBI, 18 EryBIwerewere weremethyl D257G isolatedisolatedD257G isolated β -[26]. [26].inin in SchemeTheyieldsyieldsThe enzyme 2. enzyme42%42%Glucosylation andand showed showed 6%;6%; ( (bhighb)) of high GlucosylationGlucosylation naturaltolerance tolerance and towards towardsofof unnatural erythromycinerythromycin the the bulk bulk glycosides.y dimethylated yAA dimethylated ((1919)) byby (a the)the Synthesis amino glucosynthaseglucosynthase amino group group of glucosylated in EryBIinEryBIthe the C3 D257GC3D257G position. position. methyl [26].[26]. acarviosinTheyieldsTheyields enzyme enzyme42% 42% (16 and )showed andby showed 6%; Fairweather 6%; ( bhigh )( b highGlucosylation)) GlucosylationGlucosylationtolerance tolerance et al. [24]. towards towards ofThe ofoferythromycin mono- erythromycinerythromycin the the bulk and bulky diglycosylated dimethylated yA dimethylated AA(19 ((19) by)) byby the the theproductsamino glucosynthase amino glucosynthaseglucosynthase group 17group, 18 in were inEryBIthe EryBIEryBIthe C3isolated C3D257G position. D257GD257G position. in [26]. [26].[26]. β-acarviosinyieldsTheTheyields enzymeenzyme 42% 42% (16 and) showed showed byand 6%; Fairweather 6%; ( bhighhigh )(b Glucosylation) Glucosylation tolerancetolerance et al. [ towards24towards of]. Theoferythromycin erythromycin thethe mono- bulkbulk andyy dimethylated dimethylatedA diglycosylated A(19 (19) by) by the the amino aminoglucosynthase glucosynthase products groupgroup inin17 EryBI thethe, 18EryBI C3C3were D257G position.position. D257G isolated [26]. [26]. yieldsTheThe enzyme 42% enzyme and showed 6%;showed (b )high Glucosylation high tolerance tolerance towardsof towards erythromycin the the bulk bulk yA dimethylated y(19 dimethylated) by the glucosynthase amino amino group group EryBI in inthe theD257G C3 C3 position. position.[26]. in yieldsTheInTheIn addition enzyme 42%addition enzyme and showedto 6%;showed tothe the (b ‘classical’ )high Glucosylation‘classical’ high tolerance tolerance glycosynthase glycosynthase towards oftowards erythromycin the the bulkapproach bulkapproachy dimethylatedy A dimethylated (19 catalyzing) bycatalyzing the amino glucosynthase amino the the group transfer group transfer in in EryBIthe of the ofC3glycosyl C3 glycosylD257Gposition. position. fluoride [26 fluoride]. TheInIn addition enzyme addition showed to to the the high‘classical’ ‘classical’ tolerance glycosynthase glycosynthasetowards the bulk approach yapproach dimethylated catalyzing catalyzing amino thegroup the transfer transferin the C3 of ofposition. glycosyl glycosyl fluoride fluoride donors,Thedonors, enzymeInIn addition additionPozzo Pozzo showed et toettoal. al.the theimproved high improved ‘classical’‘classical’ tolerance glycosylation glycosylation glycosynthaseglycosynthase towards the yields yields bulky approachapproach by dimethylatedby utilizing utilizing catalyzingcatalyzing the the aminochemical chemical thethe grouptransfertransfer rescue rescue in of theof method glycosylglycosyl C3method position. for fluoridefluoridefor in insitu situ donors,donors,InInIn addition Pozzo addition additionPozzo et ettoal. to toal.the improved the theimproved ‘classical’ ‘classical’‘classical’ glycosylation glycosylation glycosynthase glycosynthaseglycosynthase yields yields approach approachapproach by by utilizing utilizing catalyzing catalyzingcatalyzing the the chemical chemical the thethe transfer transfertransfer rescue rescue of methodof ofglycosyl method glycosylglycosyl for fluoridefor influoridefluoride in situ situ donors,InIn addition Pozzo addition et toal. to the improved the ‘classical’ ‘classical’ glycosylation glycosynthase glycosynthase yields approach approach by utilizing catalyzing catalyzing the chemical the the transfer transfer rescue of methodof glycosyl glycosyl for fluoride influoride situ donordonors,donordonors,In productionaddition Pozzoproduction Pozzo et to etal. thewith al. improvedwith ‘classical’improved the the β-glucosynthase β glycosylation-glucosynthase glycosynthase glycosylation yields(TnBgl1A approach(TnBgl1Ayields by by utilizing E349G; catalyzingutilizing E349G; GH1)the GH1)the thechemical ofchemical transfer ofT. T.neapolitana neapolitanarescue rescueof glycosyl method method[27]. [27]. fluoride The for The for groupin group insitu situ donors,donordonors,donordonors, production Pozzo production PozzoPozzo et et etal. withal.al. improved with improvedimproved the the β -glucosynthaseβ glycosylation-glucosynthase glycosylationglycosylation yields(TnBgl1A yieldsyields(TnBgl1A by byby utilizing E349G; utilizingutilizing E349G; theGH1) GH1)thethe chemical chemical chemicalof of T. T. neapolitana rescueneapolitana rescuerescue method method method[27]. [27]. The for The forfor ingroup in groupinsitu situsitu achieveddonordonorachieved productionproduction a 3.7×a 3.7× higher higherwithwith theyieldthe yield ββ-glucosynthase -glucosynthaseduring during the the synthesis synthesis (TnBgl1A(TnBgl1A of of quercetin-3,4 E349G;E349G; quercetin-3,4 GH1)GH1)′-di- ofof′-di- T.OT.- O neapolitanaβneapolitana-βD--glucopyranosideD-glucopyranoside [27].[27]. TheThe groupgroup with with donors,achieveddonorInachieveddonor addition production Pozzo production a a3.7× to 3.7×et theal.higher higher withimproved ‘classical’ with theyield theyield β -glucosynthaseglycosylationβduring glycosynthase-glucosynthase -glucosynthaseduring the the synthesis yields synthesis (TnBgl1A approach (TnBgl1A(TnBgl1A by ofutilizing of quercetin-3,4E349G; quercetin-3,4catalyzing E349G;E349G; the GH1) chemical GH1)GH1)′ the-di- of′-di- of ofT.O transfer rescueT.- Oneapolitanaβ- -neapolitanaβD--glucopyranosideD -glucopyranosidemethod of glycosyl [27]. [27].[27]. for The in TheThe fluoride situ group with groupgroup with donorachievedachieveddonor production production aa 3.7×3.7× higherhigher with with theyieldyield the β -glucosynthase βduringduring-glucosynthase thethe synthesissynthesis (TnBgl1A (TnBgl1A ofof quercetin-3,4quercetin-3,4E349G; E349G; GH1) GH1)′′-di- -di-of of T.OO T. --neapolitanaββ --neapolitanaDD-glucopyranoside-glucopyranoside [27]. [27]. The The group withwithgroup donororthoachievedorthoachieved-NPGlc production-NPGlc a a3.7× and 3.7× and higherformate with higherformate the yield for yieldβ for-glucosynthase in during in situduring situ donor donorthe the synthesisproduction, (TnBgl1A synthesisproduction, of E349G;of comparedquercetin-3,4 comparedquercetin-3,4 GH1) to of tothe′-di- T. the′′-di--di- synthesisneapolitanaO synthesis-Oβ--βD---glucopyranosideD-glucopyranoside-glucopyranoside using [27]. using αTheGlcF αGlcF group (1 , ( with110% , withwith10% donors,achievedorthoachievedortho Pozzo-NPGlc-NPGlc a eta3.7× and al.3.7× and higher improvedformate higher formate yield foryield for glycosylation in during in situduring situ donor donorthe the synthesis yieldsproduction, synthesis production, by of utilizing of quercetin-3,4compared quercetin-3,4compared the to chemical to ′′the-di- ′the-di- synthesisO synthesis-Oβ-- rescueβD--glucopyranosideD-glucopyranoside using methodusing α GlcFαGlcF for (1 in (,with 110% , situwith 10% yield).orthoorthoyield).-NPGlc-NPGlc This This approach andand approach formateformate was was forfor subseq insubseqin situsituuently donordonoruently transferred production,production, transferred efficiently comparedcomparedefficiently to toto tothe the thethe TnBgl3B synthesissynthesis TnBgl3BD D242A usingusingD242A αW243Fα GlcFGlcFW243F (( 1(GH1;1,, 10%(GH1;10% donorachievedyield).orthoyield).ortho production-NPGlc -NPGlc-NPGlcThis Thisa 3.7× approach and approach and withandhigher formate formateformate the wasyield wasβ for-glucosynthase subseq forfor duringsubseqin in insitu situsituuently donoruentlythe donordonor synthesis transferred production, (TnBgl1Atransferred production,production, of quercetin-3,4efficiently E349G; comparedefficiently comparedcompared GH1) to ′toto-di- the to to the oftheO thetheTnBgl3B-T. synthesis βTnBgl3B - synthesis neapolitanasynthesis-glucopyranoside D242A usingD242A usingusing[27 W243Fα ]. GlcF W243Fα TheGlcF with ( 1(GH1; group (, 1(GH1;10% ,, 10%10% orthoyield).ortho-NPGlc -NPGlcThis approach and and formate formate was for subseqfor in in situ situuently donor donor transferred production, production, efficiently compared compared to to the to the the TnBgl3B synthesis synthesis D242A using using αW243F GlcFαGlcF ( 1(GH1; (,, 110%10%, 10% orthoT.yield). T.yield).neapolitana -NPGlcneapolitana This This andapproach )approach )glycosynthaseformate glycosynthase was forwas subseq in subseq situ which which uentlydonoruently exhibited production, exhibitedtransferred transferred transglu transglu compared efficiently efficientlycosylationcosylation to to the tothe thesynthesis activityTnBgl3B 0activity TnBgl3B usingto D242A to D242Aquercetin-3- quercetin-3-αGlcF W243F W243F (1, 10%O (GH1;-O β(GH1; -βD--D - achievedyield).T.yield).T.yield). neapolitana neapolitana aThis 3.7ThisThis ×approach approach)approach higher)glycosynthase glycosynthase was yield waswas subseq subseqsubseq during which whichuentlyuentlyuently theexhibited transferredexhibited transferred synthesistransferred transglu transglu efficiently of efficientlyefficiently quercetin-3,4cosylationcosylation to to tothe thethe activityTnBgl3B activityTnBgl3B-di-TnBgl3BO -β to D242A- to DD242AD242Aquercetin-3- -glucopyranosidequercetin-3- W243F W243FW243F O(GH1; -O (GH1;β(GH1;--βD--D - yield).glucopyranosideT.T. glucopyranoside neapolitananeapolitana This approach)) glycosynthaseglycosynthase (30%), (30%), was quercetin-3- subseq quercetin-3- whichwhichuentlyO - Oexhibitedexhibitedβtransferred-βD--galactopyranosideD-galactopyranoside transglutransglu efficientlycosylationcosylation to (40%), the (40%), TnBgl3B activityactivityand and quercetin D242Aquercetin toto quercetin-3-quercetin-3- W243F (15%) (15%) (GH1;[28]. [28].OO-- ββThe -- DDThe-- withglucopyranosideT.orthoglucopyranosideT. neapolitana neapolitana-NPGlc) and)glycosynthase) (30%),glycosynthaseglycosynthase (30%), formate quercetin-3- quercetin-3- forwhich whichwhich inO situ-Oexhibitedβ --exhibitedexhibitedβD--galactopyranoside donorD-galactopyranoside transglu production,transglutransglucosylationcosylation (40%), compared(40%), activityand activityand quercetin toquercetin to theto quercetin-3- quercetin-3- synthesis(15%) (15%) [28]. [28].O using-O βThe-- βDThe--D -- T.glucopyranosideglucopyranosideT. neapolitana neapolitana) )glycosynthase (30%),glycosynthase(30%), quercetin-3-quercetin-3- which whichOO --exhibitedβ β-exhibited-DD-galactopyranoside-galactopyranoside transglu transglucosylationcosylation (40%),(40%), activityand andactivity quercetinquercetin to to quercetin-3- quercetin-3- (15%)(15%) [28].[28].O-O βTheThe--βD--D - T.glucopyranoside glucopyranosideneapolitana) glycosynthase (30%), (30%), quercetin-3- quercetin-3- which Oexhibited-Oβ--βD---galactopyranosideD-galactopyranoside-galactopyranoside transglucosylation (40%), (40%),(40%), activity and andand quercetinto quercetinquercetin quercetin-3- (15%) (15%)(15%)O [28].- β [28].[28].-D -The TheThe glucopyranoside glucopyranoside (30%), (30%), quercetin-3- quercetin-3-O-Oβ--βD--galactopyranosideD-galactopyranoside (40%), (40%), and and quercetin quercetin (15%) (15%) [28]. [28]. The The glucopyranoside (30%), quercetin-3-O-β-D-galactopyranoside (40%), and quercetin (15%) [28]. The

Molecules 2017, 22, 1434 5 of 20

αGlcF (1, 10% yield). This approach was subsequently transferred efficiently to the TnBgl3B D242A W243F (GH1; T. neapolitana) glycosynthase which exhibited transglucosylation activity to quercetin-3-O-β-D-glucopyranoside (30%), quercetin-3-O-β-D-galactopyranoside (40%), and quercetin (15%)Molecules [28]. The 2017 chemical, 22, 1434 rescue method was also applied to the α-glucosynthase variant of AglA5 of 20 from T. acidophilum (AglA D408G) enabling the synthesis of the putative tyrosinase inhibitor α-1,4-D-glucosyl arbutinchemical in 38% rescue yield method [29]. Though was also most appliedexo-glycosidase to the α-glucosynthase derived glycosynthases variant of AglA arefrom limited T. acidophilum to glycosyl transfer(AglA to D408G) glycosides, enabling Yang the et synthesis al. utilized of the putative the glycosynthase tyrosinase inhibitor of HiCel7B, α-1,4-D HiCel7B-glucosyl arbutin E197S in in the 38% yield [29]. Though most exo-glycosidase derived glycosynthases are limited to glycosyl transfer glycosylation of non-glycosylated flavonoid structures (e.g., 21 and 22, Scheme3)[ 30]. The former to glycosides, Yang et al. utilized the glycosynthase of HiCel7B, HiCel7B E197S in the glycosylation endo-cellulase catalyzed the transfer of lactosyl fluoride (23, LacF) with high selectivity to the 0 of non-glycosylated flavonoid structures (e.g., 21 and 22, Scheme 3) [30]. The former endo-cellulase 4 -O positioncatalyzed (positionthe transfer Y) of oflactosyl four fluoride different (23 flavonoids, LacF) with high with selectivity yields ranging to the 4′- betweenO position72–95% (position (Y)24 /25, Schemeof 3four). different flavonoids with yields ranging between 72–95% (24/25, Scheme 3).

SchemeScheme 3. Direct 3. Direct glycosylation glycosylation of of flavonoids flavonoids21 21and and 22 utilizing the the glycosynthase glycosynthase HiCel7B HiCel7B E197S E197S [30]. [30]. The enzymeThe enzyme exhibited exhibited high high specificity specificity forfor glycosylation of ofthe the hydroxyl hydroxyl function function in the inY position the Y position (as (as forfor flavonoid flavonoid22 22)) onlyonly deviating in in the the case case of ofabsence absence of this of thisgroup group as observed as observed for thefor flavonoid the flavonoid 21. The yields of the flavonoid glycosylation ranged from 72–95%. 21. The yields of the flavonoid glycosylation ranged from 72–95%. A different variant of this enzyme, HiCel7B E197A was recently employed in the synthesis of Aregularly different substituted, variant offunctionalized this enzyme, celluloses HiCel7B by E197ACodera waset al. recently[31]. Polymerization employed of in 6 the′-azido- synthesisα- of regularlycellobiosyl substituted, fluoride by functionalized HiCel7B E197A celluloses resulted byin Coderaregularly et azido al. [ 31substituted]. Polymerization cellulose of 60-azido-oligosaccharidesα-cellobiosyl with fluoride a degree by HiCel7Bof polymerization E197A resulted(DP) up into 32 regularly in a yield azido of 88%. substituted A subsequent cellulose oligosaccharidescopper catalyzed with azide/alkyne a degree of cycloaddition polymerization (Huisgen (DP) reaction) up to 32 with in aalkyne yield functionalized of 88%. A subsequent Alexa copperFluor catalyzed die led to azide/alkyne fluorescent polymers. cycloaddition The synthesis (Huisgen of a reaction) further polysaccharide with alkyne was functionalized performed by Alexa Aragunde et al. [32]. The glycosynthase produced from the β-1,3-1,4-glucanase of B. licheniformis Fluor die led to fluorescent polymers. The synthesis of a further polysaccharide was performed by catalyzed the oligomerization of the Glc-β1,4-Glc-β1,3-GlcαF donor reaching yields of 90% (weight β Aragundepolymer/weight et al. [32]. initial The donor). glycosynthase produced from the -1,3-1,4-glucanase of B. licheniformis catalyzed the oligomerization of the Glc-β1,4-Glc-β1,3-GlcαF donor reaching yields of 90% (weight polymer/weight2.2. Galactosynthases initial donor). Showing the importance of the glycosynthase method in the production of glycoside structures 2.2. Galactosynthases for biological or pharmaceutical research, Kwan et al. described the synthesis of the type 2 blood Showinggroup A oligosaccharide the importance 26 of by the combining glycosynthase the glycosynthase method in Abg the production2F6 in a reaction of glycoside sequence structures with for biologicalglycosyltranfersases or pharmaceutical WbgL and research, BgtA (Scheme Kwan et4) al.[33]. described The enzyme the synthesisefficiently oftransferred the type α 2-D blood- groupgalactosyl A oligosaccharide fluoride (2, α26GalF)by combiningonto methyl umbelliferone the glycosynthase β-N-acetylglucosamine Abg 2F6 in a reaction (27, UM- sequenceβ-GlcNAc)with in yields of 84% (28). By exploitation of the chemical rescue method (Section 1, Scheme 1D) usually glycosyltranfersases WbgL and BgtA (Scheme4)[ 33]. The enzyme efficiently transferred α-D-galactosyl carried out in the case of α-glycosynthases, Strazulli et al. catalyzed the formation of various β- fluoride (2, αGalF) onto methyl umbelliferone β-N-acetylglucosamine (27, UM-β-GlcNAc) in yields of galactosyl glycosides by the β-galactosynthase AaβGal D361G (A. acidocaldarius) and simple donor 28 84% (pNPGal). By exploitationas starting material of the chemical [34]. Though rescue a methodhigh transglycosylation (Section1, Scheme was1 D)achieved, usually the carried enzyme out in the caseshowed of α-glycosynthases, high promiscuity Strazulliproducing et different al. catalyzed regioisomers the formation and polyglycosylated of various β-galactosyl products. glycosides by the β-galactosynthase AaβGal D361G (A. acidocaldarius) and simple donor pNPGal as starting material [34]. Though a high transglycosylation was achieved, the enzyme showed high promiscuity producing different regioisomers and polyglycosylated products.

Molecules 2017, 22, 1434 6 of 20 Molecules 2017, 22, 1434 6 of 20

Molecules 2017, 22, 1434 6 of 20

SchemeScheme 4. Synthesis 4. Synthesis of the of methyl the methyl umbelliferone umbelliferone derivative derivative of the type of 2the blood type group 2 blood A oligosaccharide group A 26 performedoligosaccharide by Kwan 26 performed et al. [33 ].by The Kwan synthesis et al. combined[33]. The thesynthesis engineered combined glycosynthase the engineered Abg 2F6 glycosynthase Abg 2F6 (A19T, E358G, Q248R, M407V) with two glycosyltransferases WbgL and BgtA. (A19T,Scheme E358G, 4. Q248R, Synthesis M407V) of the with methyl twoglycosyltransferases umbelliferone derivative WbgL of and the BgtA. type The2 blood glycosyltranferases group A The glycosyltranferases could also be employed in a one-pot reaction giving a higher yield of 62% couldoligosaccharide also be employed 26 performed in a one-pot by reaction Kwan et giving al. [33]. a higher The yieldsynthesis of 62% combined compared the toengineered the total yield compared to the total yield of 36% for the sequential reaction. of 36%glycosynthase for the sequential Abg 2F6 reaction. (A19T, E358G, Q248R, M407V) with two glycosyltransferases WbgL and BgtA. The glycosyltranferases could also be employed in a one-pot reaction giving a higher yield of 62% Goddard-Borger et al. demonstrated the use of a glycosynthase in the synthesis of psychosine, a compared to the total yield of 36% for the sequential reaction. βGoddard-Borger-D-galactopyranosyl et sphingosine al. demonstrated found thein the use central of a glycosynthase nervous system in [35]. the synthesisAccumulation of psychosine, of this β a -compoundD-galactopyranosylGoddard-Borger can lead to et sphingosine neuralal. demonstrated signaling found thedysfunction in use the of a central glycosynthase often nervousobserved in the systemin synthesisindividuals [35]. of Accumulationpsychosine, with Krabbe a of disease. The β-glycosynthase EGALC E341S (endo-galactosyl ceramidase of R. equi) was successfully this compoundβ-D-galactopyranosyl can lead tosphingosine neural signaling found in dysfunction the central nervous often observed system [35]. in individualsAccumulation with of this Krabbe disease.employedcompound The β in-glycosynthase canthe onelead step to neural synthesis EGALC signaling of E341S psychosine dysfunction (endo -galactosylfrom often αGalF observed ceramidase(2) and sphingosinein individuals of R. equi (30) waswith) in a successfully Krabbeyield of employed21%,disease. the in yieldThe the β onebeing-glycosynthase step limited synthesis mainly EGALCof by psychosine E341Sproblems (endo involving-galactosyl from αGalF the ceramidase low (2) solubility and sphingosineof R. of equi sphingosine) was (successfully30) in(30 a) yieldand of 21%,precipitationemployed the yield beingin theof onethe limited stepenzyme. mainlysynthesis Further by of problems psychosine glycosphingolipids involving from αGalF the were(2 low) and solubilitysynthesized sphingosine of sphingosine by(30 ) thein a yieldeffective (30 of) and combination of glycosyltransferases and endo-glycoceraminidase (EGCase) derived synthases. Rich precipitation21%, the ofyield the being enzyme. limited Further mainly glycosphingolipids by problems involving were the synthesized low solubility by of the sphingosine effective combination(30) and et al. described the synthesis of the glycolipid lyso-GM3 (31) by combining lactosyl fluoride (23) with of glycosyltransferasesprecipitation of the and enzyme.endo-glycoceraminidase Further glycosphingolipids (EGCase) derivedwere synthesized synthases. by Rich the et al.effective described sphingosinecombination ( 30of) glycosyltransferasescatalyzed by EGCase and II endo D351S-glycoceraminidase and subsequent (EGCase) sialylation derived with synthases.the Cst-I αRich-2,3- the synthesis of the glycolipid lyso-GM3 (31) by combining lactosyl fluoride (23) with sphingosine sialyltransferaseet al. described thereaching synthesis an overall of the glycolipidyield of 51% lyso (31-G,M3 Scheme (31) by 5) combining [36]. lactosyl fluoride (23) with (30) catalyzed by EGCase II D351S and subsequent sialylation with the Cst-I α-2,3-sialyltransferase sphingosine (30) catalyzed by EGCase II D351S and subsequent sialylation with the Cst-I α-2,3- reachingsialyltransferase an overall yieldreaching of 51% an overall (31, Scheme yield of5 51%)[36 (].31, Scheme 5) [36].

Scheme 5. Synthesis of the glycolipid lyso-GM3 (31) by Rich et al. [36]. The production of the lactosyl

sphingosine acceptor 32 for the Cst-I α-2,3-sialyltransferase was catalyzed by the EGCase II glycosynthaseScheme 5. Synthesis in an overall of the glycolipidyield of 61%. lyso -Ga YieldM3 (31 encompassing) by Rich et al. the[36]. chemical The production synthesis of theof LacF lactosyl (23 ) Scheme 5. Synthesis of the glycolipid lyso-GM3 (31) by Rich et al. [36]. The production of the andsphingosine the glycosynthase acceptor reaction.32 for the Cst-I α-2,3-sialyltransferase was catalyzed by the EGCase II lactosyl sphingosine acceptor 32 for the Cst-I α-2,3-sialyltransferase was catalyzed by the EGCase glycosynthase in an overall yield of 61%. a Yield encompassing the chemical synthesis of LacF (23) a II glycosynthaseand the glycosynthase in an overall reaction. yield of 61%. Yield encompassing the chemical synthesis of LacF (23) and the glycosynthase reaction.

Molecules 2017, 22, 1434 7 of 20

Yang et al. broadened the same synthesis to a FRET-probe of lyso-GM3 in order to visualize enzymatic processing of the glycolipid [37]. Another combination of glycosyltransferases and a glycosynthase was performed in sequential and also one-pot reactions by Henze et al. [38]. The report demonstrates the synthesis of N-acetyllactosamine type 1 (-3-Gal-β1,3-GlcNAc-1-) and type 2 (-3-Gal-β1,4-GlcNAc-1-) oligomers by combining the glycosynthase His6BgaC D233G (B. circulans) either in a one-pot or sequentially with the glycosyltransferases β3GlcNAcT (H. pylori) and the βGalT-1 (human origin) [39]. By sequential use of these enzymes, it was even possible to create neo-N-acetyllactosylamine (LacNAc) oligomers with alternating type 1 and type 2 units. Even though much research and many advances have occurred in the synthesis of β-Gal linkages, the synthesis of α-Gal linkages utilizing α-galactosynthases is still limited. Cobucci-Ponzano et al. reported the effective conversion of β-D-galactopyranosyl azide (35, βGalN3) to α-galacto- oligosaccharides by the glycosynthase TmGalA D327G derived from T. maritima [40]. The enzyme produced galacto-oligosaccharides in good yields and the method was recently expanded by Okuyama et al. to the in situ formation of a β-D-galactosyl formate donor (36), which exhibited a higher transglycosylation rate compared to the azide donor (35, Scheme6)[ 41]. The method allowed the galactosylation of carbohydrates such as glucose (α-1,1-β), xylose (α-1,4), maltose (α-1,1-β), cellobiose (α-1,1-β; α-1,6), lactose (α-1,1-β), and pNP derivatives of glucose (pNPGlc, 37), and mannose (pNPMan, 6; exclusively α-1,6) in yields ranging from 75 to 95%. Bayón et al. examined the addition of green co-solvents towards the synthesis of α-galactosyl residues with βGalN3 (35) and reported increased conversion and yield for the glycosynthase TmGalA D327G producing α-1,6 bonds with pNPGlc (37) and pNPMan (6)[42].

Scheme 6. In situ formation of the glycosyl donors βGalN3 (35) or βGal formate (36) by incubation of αGalF (2) with glycosynthase BtGH97b D415G the additional external nucleophiles sodium azide of formate [41]. The in situ produced donor can then be subsequently transferred to a suitable acceptor (R2OH).

2.3. Xylosynthases Xylooligosaccharides have gained much interest in the pharmaceutical industry, due to their anti-freezing activity, non-digestibility, non-cariogenic and beneficial properties for the intestinal flora [43]. One of the first approaches to the synthesis of xylosides was carried out by Kim et al. using the β-glycosynthase Abg 2F6 (A19T, E358G, Q248R, M407V) derived from a glucosidase from A. tumefaciens [21]. The glycosynthase variant exhibited a broad donor substrate range and showed an increased specificity to α-D-xylosyl fluoride (αXylF, 14; 34× higher) compared to the original mutant Abg E358G. This was then utilized in 2005 by the same group in the synthesis of xylosaccharides with pNPGlc (37, β-1,4), pNPXyl (38, β-1,3/1,4) and pNPXylobioside (39, Xyl2pNP; β-1,4) as acceptors in yields of 35–98%. Xylosylated 1-deoxyxylanojirimycin (40), a xylose derived nitrogen-containing inhibitor, was also synthesized by this mutant in a yield of 28% (acetylated product; Scheme7)[44].

1

Molecules 2017, 22, 1434 8 of 20 MoleculesMolecules 20172017,, 2222,, 14341434 88 ofof 2020

HO HO HO OH HO a Abg 2F6 OH 42 n=1 63% a HO OO Abg 2F6 HO O O 42 n=1 63% HOHO OpNP HO O O a HO OpNP O O HO OpNPOpNP 43 n=2 35% a OH HH O O n HO 43 n=2 35% OH n OHOH 3737 OH a O Abg 2F6 OH 44 n=1 42% a HO O Abg 2F6 HO O OO 44 n=1 42% HOHO OpNP HO O OpNP a HO OpNP H OO O HOHO OpNP 45 n=2 30% a OH H O n 45 n=2 30% OH n OHOH 38 HO O 38 H O O HO O HO O H O O HO O 46 a HO HO O OpNPOpNP 46n=1n=1 4%4%a HO OH HO O a OH + OH OH 47 n=2 10% a F + OH n OH 47 n=2 10% F n 1414 OH OH OH O OH HO O O AbgAbg 2F62F6 HO O O HOHO HOO OpNP HO O O a HO O HO OpNP O O HO OpNPOpNP 4848 98% a O OH HH O O 2 HO 98% OH 2 OHOH 3939 NR Abg 2F6 OH HO NR Abg 2F6 OH b HO HO NR 40 28% b HOHO HO OO NR 40 28% OH HOHO O HOHO OH O OH 4141 OH SchemeScheme 7.7. SynthesisSynthesisSynthesis ofof of variousvarious various xylosidesxylosides xylosides 404040,, 4242, 42––4848–48 bybyby thethe the glycosynthaseglycosynthase glycosynthase AbgAbg Abg 2F62F6 2F6 derivedderived derived fromfrom from thethe the ββ-- glucosidaseβglucosidase-glucosidase AbgAbg Abg ofof ofA.A.A. tumefacienstumefaciens tumefaciens [44].[44].[44 ]. TheThe The enzymeenzyme enzyme exhibitedexhibited exhibited variablevariable variable selectivityselectivity selectivity dependingdepending depending onon the the a a acceptoracceptor substrate, substrate, producingproducingproducing predominantly predominantlypredominantlyβ ββ-1,4-1,4-1,4 linkages. linkages.linkages. aYields YieldsYields determined determineddetermined by byby HPLC HPLCHPLC analysis analysisanalysis of b b oftheof thethe reaction reactionreaction mixture; mixture;mixture;Yield b YieldYield of of isolated,of isolated,isolated, acetylated acetylatedacetylated product. product.product.

AfterAfter these these first firstfirst syntheses, syntheses, various various groups groups acco accomplishedaccomplishedmplished the the synthesis synthesis of of xylooligomers xylooligomers with with glycosynthasesglycosynthases derivedderived derived fromfrom from actualactual actual xylanases.xylanases. xylanases. TheThe reducing-end Thereducing-end reducing-end xylose-releasingxylose-releasing xylose-releasing exoexo-xylanase-xylanaseexo-xylanase (REX)(REX) mutated(REX)mutated mutated inin thethe acid/baseacid/base in the acid/base catalyticcatalytic catalyticresidueresidue (E236C)(E236C) residue waswas (E236C) utilizedutilized was byby utilized HondaHonda byetet al.al. Honda inin thethe et synthesissynthesis al. in the ofof xylotrimers from xylobiosyl fluoride (X2F, 49) and xylose [45]. This mutant enzyme was also the first synthesisxylotrimers of from xylotrimers xylobiosyl from fluoride xylobiosyl (X2F, 49 fluoride) and xylose (X2F, 49[45].) and This xylose mutant [45 enzyme]. This was mutant also enzymethe first reportwasreport also ofof the aa glycosynthaseglycosynthase first report of aderivedderived glycosynthase fromfrom anan derived invertinginverting from glycosidase.glycosidase. an inverting KimKim glycosidase. etet al.al. alsoalso Kim synthesizedsynthesized et al. also xylooligosaccharidessynthesizedxylooligosaccharides xylooligosaccharides withwith thethe CFXcd-E235CFXcd-E235 with the CFXcd-E235GGG mutantmutant ofof mutant thethe retainingretaining of the retaining xylanasexylanase xylanase ofof C.C. fimifimi of C. [46].[46]. fimi The[The46]. enzyme catalyzed the oligomerization of X2F (49) and Xyl2pNP (39) or benzylthio-β-xylobioside Theenzyme enzyme catalyzed catalyzed the theoligomerization oligomerization of ofX2 XF 2F((4949) and) and Xyl Xyl2pNP2pNP ( (3939)) or or benzylthio-benzylthio-β-xylobioside (Xyl2BT)(Xyl2BT) resulting resulting in in a a mixturemixture ofof oligomersoligomers oligomers rangingranging ranging fromfrom from xylotetrasaccharidesxylotetrasaccharides xylotetrasaccharides toto -dodecasaccharides-dodecasaccharides -dodecasaccharides withwith a a totaltotal yieldyield ofof transfertransfer productsproducts ofofof 61%61%61% andandand 66%,66%,66%, respectively.respectively.respectively. TheThe SugimuraSugimura etet al.al.al. group group expandedexpanded thethe the repertoirerepertoire repertoire ofof of xylanasexylanase xylanase originatingoriginating originating glglycosynthsesycosynthses glycosynthses byby fourfour by examples fourexamples examples ofof thethe ofGH10GH10 the familyfamily GH10 (XylBfamily(XylB TT (XylB maritimamaritimaT; maritima; XynAXynA B.B.; XynAhaloduranshaloduransB. halodurans;; XynBXynB C.C. ; stercorariumstercorarium XynB C. stercorarium;; CexCex C.C. fimifimi;)) Cex enablingenablingC. fimi thethe) enabling synthesissynthesis the ofof xylooligomers from X2F (50) in yields from 29–69% (7.8–18.4 mg) [47]. The products precipitated synthesisxylooligomers of xylooligomers from X2F (50 from) in yields X2F( 50from) in 29–69% yields from (7.8–18.4 29–69% mg (7.8–18.4) [47]. The mg) products [47]. The precipitated products enablingprecipitatedenabling easyeasy enabling isolationisolation easy andand isolation werewere ofof and highhigh were interestinterest of high asas interestunsubstitutedunsubstituted as unsubstituted xylan,xylan, aa homopolymerhomopolymer xylan, a homopolymer ofof ββ-1,4--1,4- xylopyranoseofxylopyranoseβ-1,4-xylopyranose units,units, doesdoes units, notnot existexist does inin not nature.nature. exist ThTh inee nature. productionproduction The ofof production largerlarger xylooligomersxylooligomers of larger xylooligomers waswas achievedachieved bywasby thethe achieved groupgroup ofof by Ben-DavidBen-David the group etet of al.al. Ben-David [48].[48]. TheThe etXynB2XynB2 al. [48 E335GE335G]. The mutantmutant XynB2 ofof E335G thethe retainingretaining mutant ofexoexo the-glycosidase-glycosidase retaining (GH52)exo(GH52)-glycosidase ofof G.G. stearothermophilusstearothermophilus (GH52) of G. stearothermophilus catalyzedcatalyzed thethe synthesissynthesiscatalyzed ofof xylosidesxylosides the synthesis withwith α ofαXylFXylF xylosides ((1414)) andand with pNPGlcpNPGlcαXylF ((37 (3714),),) pNPXylandpNPXyl pNPGlc ((3838),), ( and37and), pNPXylpNPManpNPMan (38 ((66),)) andwithwith pNPMan aa ββ-1,4-1,4 linkagelinkage (6) with inin a βyieldsyields-1,4 linkage ofof 49%,49%, in 42%42% yields andand of 10% 49%,10% (disaccharide(disaccharide 42% and 10% yields),(disaccharideyields), respectively.respectively. yields), respectively.ByBy exploitaexploitationtion By exploitationofof thethe self-condensationself-condensation of the self-condensation ofof ααXylFXylF ((1414 of))α bybyXylF thethe (14 enzymeenzyme) by the toto enzyme XylXyl22FF (49), a combinatorial synthesis with a glycosynthase of the xylanase XT6 (E265G; also of G. to(49 Xyl), a2 F(combinatorial49), a combinatorial synthesis synthesis with a withglycosynthase a glycosynthase of the of xylanase the xylanase XT6 XT6(E265G; (E265G; also alsoof G. of stearothermophilusG.stearothermophilus stearothermophilus)) resultedresulted) resulted inin xylooligomer inxylooligomer xylooligomer comprisingcomprising comprising ofof 6–100 of6–100 6–100 monomersmonomers monomers (Scheme(Scheme (Scheme 8)8) 8 [48,49].[48,49].)[48,49 ].

Scheme 8. Production of xylooligomers comprising 6–100 monomers by exploitation of the self- Scheme 8. ProductionProduction of ofxylooligomers xylooligomers comprising comprising 6–100 6–100 monomers monomers by exploitation by exploitation of the of self- the 2 condensationcondensation ofof ααXylFXylF ((1414)) andand XylXyl2FF ((4949)) catalyzedcatalyzed byby thethe enzymeenzyme combinationcombination ofof XynB2XynB2 E335GE335G andand self-condensation of αXylF (14) and Xyl2F(49) catalyzed by the enzyme combination of XynB2 E335G XT6 E265G [48,49]. andXT6 XT6E265G E265G [48,49]. [48 ,49].

TheThe activityactivity ofof thethe XynB2XynB2 E335GE335G mutantmutant inin productionproduction ofof XylXyl22FF ((4949)) andand XylXyl33FF waswas improvedimproved byby thethe developmentdevelopment ofof aa generalgeneral screeningscreening assayassay utilizingutilizing thethe pHpH indicatorindicator MethylMethyl Red,Red, resultingresulting inin

Molecules 2017, 22, 1434 9 of 20

The activity of the XynB2 E335G mutant in production of Xyl2F(49) and Xyl3F was improved Moleculesby the development2017, 22, 1434 of a general screening assay utilizing the pH indicator Methyl Red, resulting9 of 20 in mutants with much higher kcat values than the original synthase [50]. However, the yields of the mutants with much higher kcat values than the original synthase [50]. However, the yields of the reactions catalyzed by the improved mutants were not reported. More recently Goddard-Borger et al. reactions catalyzed by the improved mutants were not reported. More recently Goddard-Borger et described the effective synthesis of various xylanase inhibitors by the exo-β-xylosidase mutant Bhx al. described the effective synthesis of various xylanase inhibitors by the exo-β-xylosidase mutant Bhx E334G (B. halodurans)[51]. The enzyme accepted different sugar derivatives such as thioglycosides, E334G (B. halodurans) [51]. The enzyme accepted different sugar derivatives such as thioglycosides, iminosugar derived carbamates, and a 2-deoxy-2-fluoroxyloside giving yields of 67–86% of iminosugar derived carbamates, and a 2-deoxy-2-fluoroxyloside giving yields of 67–86% of glycosylated products (acetylated products). A further important type of xyloside are xyloglucans glycosylated products (acetylated products). A further important type of xyloside are xyloglucans (an α-1,4 glucan backbone containing α-1,6-D-xylose branches) which are the principal hemicellulose (an α-1,4 glucan backbone containing α-1,6-D-xylose branches) which are the principal hemicellulose component of the primary cell wall of plant cells and are also an abundant type of storage component of the primary cell wall of plant cells and are also an abundant type of storage polysaccharide in seeds [52]. They often find use in the food, paper, textile, and pharmaceutical polysaccharide in seeds [52]. They often find use in the food, paper, textile, and pharmaceutical industry as for example cellulose crosslinkers or rheology modifiers (in fluid mechanics). The synthesis industry as for example cellulose crosslinkers or rheology modifiers (in fluid mechanics). The of xyloglucans, as custom polysaccharides, was recently carried out by Spaduit et al. with the synthesis of xyloglucans, as custom polysaccharides, was recently carried out by Spaduit et al. with broad-specificity xyloglucan glycosynthase PpXG5 E323G (P. pabuli)[53]. The group synthesized the broad-specificity xyloglucan glycosynthase PpXG5 E323G (P. pabuli) [53]. The group synthesized XXXG- (50) and XLLG-structure (51) type xyloglucans of molecular masses up to 30,000 (n = 29) and XXXG- (50) and XLLG-structure (51) type xyloglucans of molecular masses up to 30,000 (n = 29) and 60,000 (n = 44) by self-condensation of XXXGαF(50) and XLLGαF(51) donor blocks respectively 60,000 (n = 44) by self-condensation of XXXGαF (50) and XLLGαF (51) donor blocks respectively (Scheme9). These were subsequently modified to fucosylated XLFG type glucans with a ratio of 3:1 (Scheme 9). These were subsequently modified to fucosylated XLFG type glucans with a ratio of 3:1 XLFG:XLLG catalyzed by the fucosyltransferase AtFUT1(originating from A. thaliana). XLFG:XLLG catalyzed by the fucosyltransferase AtFUT1(originating from A. thaliana).

SchemeScheme 9. CombinatorialCombinatorial glycosynthase/glycosyltransferase glycosynthase/glycosyltransferase approach approach for for the the production production of of defined, defined, homogenoushomogenous xyloglucans xyloglucans [53]. [53]. Polysaccharide Polysaccharide synthesis synthesis using using compound compound 50 50or 51or was51 was catalyzed catalyzed by PpXG5by PpXG5 E323G E323G resulting resulting in polymers in polymers with witha maximal a maximal molecular molecular weight weight of 30,000 of 30,000(n = 29) (n and= 29) 60,000 and (60,000n = 44), (n =respectively. 44), respectively. Subsequent Subsequent fucosylation fucosylation by AtFUT1 by AtFUT1 reached reached a fucosylation a fucosylation of of 75% 75% of of the the oligosaccharideoligosaccharide repeats. repeats. Nomenclature: Nomenclature: X X= Xyl- = Xyl-α1,6-Glc;α1,6-Glc; L = LGal- = Gal-β1,2-Xyl-β1,2-Xyl-α1,6-Glc;α1,6-Glc; F = Fuc- F =α Fuc-1,2- αGal-1,2- βGal-1,2-Xyl-β1,2-Xyl-α1,6-Glc.α1,6-Glc.

2.4.2.4. Mannosynthases Mannosynthases NextNext to thethe xyloglucans,xyloglucans, mannans mannans are are also also highly highly abundant abundant polysaccharides polysaccharides in plantin plant cell cell walls walls and andmannosyl mannosyl moieties moieties are common are common components components of many of biologicalmany biological structures structures such as suchN-linked as N glycans,-linked glycans,microbial microbial and viral and antigens viral antigens [54,55]. The[54,55]. enzymatic The enzymatic synthesis synthesis of β-mannosyl of β-mannosyl linkages linkages has gained has gainedmuch interest much interest as the chemicalas the chemical synthesis synthesis is complex is complex and demanding and demanding as there as are there no are beneficial no beneficial effects effectspromoting promoting the synthesis the synthesis of this anomeric of this anomeric configuration. configuration. The use of Theβ-mannosynthases use of β-mannosynthases is therefore is a thereforesimple and a practicalsimple and solution practical for the solution synthesis for ofthe this sy typenthesis of bond.of this Nevertheless, type of bond. only Nevertheless, little research only has littlebeen research carried out has toward been carried the creation out toward of mannosynthases. the creation of Nashirumannosynthases. et al. created Nashiru a β-mannosynthase et al. created a βby-mannosynthase the glycosynthase by the method glycosynthase with a retaining method βwith-mannosidase a retaining mutant β-mannosidase of Man2a mu (E519S,tant of GH2) Man2a of (E519S,C. fimi [ 54GH2)]. The of enzymeC. fimi [54]. catalyzed The enzyme the formation catalyzed of βthe-1,4 formation (predominant of β-1,4 linkage) (predominant and β-1,3 linkage) mannosides and βwith-1,3 mannosidesα-mannosyl with fluoride α-mannosyl (15, αManF) fluoride and (15 various, αManF) acceptors and various (pNPMan acceptors (6), -Xyl(pNPMan (38), -Glc(6), -Xyl (37), -cellobioside(38), -Glc (37), ( 8-cellobioside), -gentiobioside, (8), -gentiobioside, and 2,4-dinitrophenyl-2-deoxy-2-fluoro- and 2,4-dinitrophenyl-2-deoxy-2-fluoro-β-mannoside)β in-mannoside) yields from in yields from 70–99% comprising di-, tri-, and tetrasaccharides. The yield was mostly hampered due 70–99% comprising di-, tri-, and tetrasaccharides. The yield was mostly hampered due to the instability to the instability of αManF (15), which can easily undergo hydrolysis by epoxide-formation. The of αManF (15), which can easily undergo hydrolysis by epoxide-formation. The enzyme also exhibited enzyme also exhibited high activity in chemical rescue experiments using 2,4-dinitrophenyl-β-D- high activity in chemical rescue experiments using 2,4-dinitrophenyl-β-D-mannosylpyranoside (52) mannosylpyranoside (52) and external nucleophiles such as azide, formate or acetate, but also with fluoride ions allowing an in situ production of the αManF donor (15, Scheme 10).

Molecules 2017, 22, 1434 10 of 20

and external nucleophiles such as azide, formate or acetate, but also with fluoride ions allowing an in Moleculessitu production 2017, 22, 1434 of the αManF donor (15, Scheme 10). 10 of 20

MoleculesMolecules 2017 2017, ,22 22, ,1434 1434 1010 of of 20 20 MoleculesMoleculesMolecules 2017 2017 2017, ,22 22, 22, ,1434 1434, 1434 101010 of of of20 20 20 MoleculesMolecules 2017 2017, ,22 22, ,1434 1434 1010 of of 20 20 MoleculesMoleculesMolecules 2017 2017 2017, ,22 22, 22, ,1434 1434, 1434 101010 of of of20 20 20

SchemeScheme 10. InIn situsitu production production of of the the glycosylation glycosylation donor donorαManF αManF (15) by(15 exploiting) by exploiting the chemical the chemical rescue rescueof hydrolytic of hydrolytic activity ofactivity Man2a of E519S Man2a in the E519S presence in th ofe sodiumpresence fluoride of sodium as an externalfluoride nucleophileas an external [54]. nucleophile [54].

Larger mannooligosaccharides were produced by Jahn et al. by biocatalysis using the glycosynthase

Larger mannooligosaccharidesScheme 10. In situ production ofwere the glycosylation produced donor by α ManFJahn ( 15et) by al.exploiting by biocatalysis the chemical using the derived fromSchemeSchemeScheme the β 10.10. -mannosidase10. InIn In situsitu situ productionproduction production of ofof of thethe C.the glycosylationglycosylation japonicusglycosylation donordonor Man26Adonor αα ManFαManFManF ([(15 5615(15)) ]. by)by by exploitingexploiting Theexploiting glycosynthase thethe the chemicalchemical chemical produced SchemerescueScheme of 10. 10.hydrolytic In In situ situ production productionactivity of of ofMan2a the the glycosylation glycosylation E519S in th edonor donorpresence α αManFManF of sodium( 15(15) )by by exploiting fluorideexploiting as the thean chemical externalchemical glycosynthasemannooligosaccharidesrescueSchemerescue SchemerescueSchemederived ofof 10.of 10. hydrolytic10.hydrolytic hydrolytic In In fromIn using situsitu situ production production activityproductiontheactivity activityαMan β -mannosidaseofof of of ofMan2a FMan2a of Man2athetheas the glycosylation aglycosylation glycosylationE519SE519S donorE519S inin in of th andth the donor edonor C. epresencedonorpresence presence pNPGlc japonicus αα ManFαManFManF ofof of sodium (sodium (15 sodium1537(15 )) Man26A by)by by as exploitingfluorideexploitingfluoride fluorideexploiting the acceptor as[56].as asthe the ananthe an chemical chemicalexternal external Thechemicalexternal in glycosynthase a total yield of rescuenucleophilerescue ofof hydrolytichydrolytic [54]. activityactivity of2of Man2aMan2a E519SE519S inin ththee presencepresence ofof sodiumsodium fluoridefluoride asas anan externalexternal nucleophilerescuenucleophilerescuenucleophilerescue ofof of hydrolytichydrolytic [54]. hydrolytic[54]. [54]. activityactivity activity ofof of Man2aMan2a Man2a E519SE519S E519S inin in thth thee epresencepresence presence ofof of sodiumsodium sodium fluoridefluoride fluoride asas as anan an externalexternal external produced59%. The mannooligosaccharides tri- (36%),nucleophilenucleophile penta- [54]. [54]. (18%), using and heptasaccharidesαMan2F as a donor (5%) and containedpNPGlc (37 exclusively) as the acceptorβ-1,4 in linkages. a total nucleophilenucleophilenucleophile [54]. [54]. [54]. yield of 59%. TheLarger tri- mannooligosaccharides (36%), penta- (18%), were and produced heptasaccharides by Jahn et al. (5%) by biocatalysiscontained usingexclusively the β-1,4 The acceptorLarger pNPManLargerLarger mannooligosaccharides mannooligosaccharidesmannooligosaccharides (6) on the other handwere werewere resultedproduced producedproduced inby byby a Jahn Jahn trisaccharideJahn et etet al. al.al. by byby biocatalysis biocatalysis inbiocatalysis a 35% yield. using usingusing Inthe thethe contrast to glycosynthaseLargerLarger mannooligosaccharidesmannooligosaccharides derived from the β-mannosidase werewere producedproduced of C. byjaponicusby JahnJahn etMan26Aet al.al. byby [56]. biocatalysisbiocatalysis The glycosynthase usingusing thethe linkages. Theglycosynthaseglycosynthase LargeracceptorLargerLarger mannooligosaccharides mannooligosaccharides mannooligosaccharidesderived pNPManderived from from (the6 )the onβ -mannosidaseβ -mannosidase thewere werewere other produced producedproduced handof of C. C. by byjaponicusresulted byjaponicus Jahn JahnJahn et Man26Aet et Man26Ain al. al. al. a by bytrisaccharideby [56]. biocatalysis [56].biocatalysisbiocatalysis The The glycosynthase glycosynthase using usinginusing a 35%the thethe yield. In these synthesesglycosynthaseglycosynthaseglycosynthase in which derived derivedderived oligomerization from fromfrom the thethe β ββ-mannosidase-mannosidase-mannosidase of the product of ofof C. C.C. japonicus japonicusjaponicus is observed, Man26A Man26AMan26A Yamamoto [56]. [56].[56]. The TheThe glycosynthase glycosynthaseglycosynthase et al. described the producedproducedglycosynthase mannooligosaccharides mannooligosaccharides derived from the using βusing-mannosidase α αManMan2F2F as as ofa a donor donorC. japonicus and and pNPGlc pNPGlc Man26A ( (3737) ) as[56]. as the the The acceptor acceptor glycosynthase in in a a total total contrast toproducedglycosynthaseproducedglycosynthase producedglycosynthasethese synthesesmannooligosaccharides mannooligosaccharides mannooligosaccharides derivedderived derived in fromfrom fromwhich thethe the β usingβ usingoligomerizat-mannosidase βusing-mannosidase-mannosidase α α ManαManMan22FF2 Fas as ofas ofion a aof donor aC.donorC. donor C. ofjaponicusjaponicus japonicus and theand and pNPGlc pNPGlcproduct pNPGlc Man26AMan26A Man26A ( (37 37( 37 ) is[56].) [56].as )as[56]. as observed,the the the TheThe acceptor Theacceptor acceptor glycosynthaseglycosynthase glycosynthase in Yamamotoin in a a totala total total et al. possibilityyieldproducedproduced of mono-glycosylating of 59%. mannooligosaccharides mannooligosaccharides The tri- (36%), penta- acceptors using using (18%), α αManMan with and2F2F asheptasaccharides aas aglycosynthase a donor donor and and pNPGlc pNPGlc (5%) derived (contained 37(37) )as as the the from acceptor exclusivelyacceptor a broad in in a a βtotal total-1,4 glycosidase yieldproducedyieldproducedyieldproduced ofof of 59%.59%. mannooligosaccharides59%. mannooligosaccharides mannooligosaccharides TheThe The tri-tri- tri- (36%),(36%), (36%), penta-penta- penta- using using using (18%),(18%), (18%), α α ManαMan Man andand and22FF2 asFheptasaccharidesasheptasaccharides asheptasaccharides a a donor adonor donor and and and pNPGlc pNPGlc pNPGlc (5%)(5%) (5%) (contained(37contained 37(contained37)) as )as as the the the acceptor exclusivelyacceptorexclusively acceptorexclusively in in in a a β total βatotal-1,4β -1,4total-1,4 describedscaffold [55 theyieldlinkages.yield]. Thepossibility of of 59%. αThe59%.-mannosynthase acceptorThe The of tri- tri- mono-g (36%), pNPMan(36%), penta- lycosylatingpenta- was (6) on(18%), created(18%), the otherand and acceptors by heptasaccharideshandheptasaccharides mutation resulted with of in a an a(5%) glycosynthase(5%) trisaccharideα-glucosidase contained contained inexclusively exclusively aderived 35% of S. yield. solfataricus β fromβ-1,4 -1,4In a broadwith linkages.yieldlinkages.yieldlinkages.yield ofof of 59%. 59%. TheThe 59%.The acceptorTheacceptorThe acceptorThe tri-tri- tri- (36%), pNPMan(36%), pNPMan (36%),pNPMan penta-penta- penta- ((6 6()6) on) on(18%), (18%),on (18%), thethe the other otherand andother and heptasaccharides handheptasaccharides hand heptasaccharideshand resultedresulted resulted inin in a(5%)a(5%) a trisaccharide(5%)trisaccharide trisaccharide containedcontained contained inin exclusively exclusivelyin exclusivelyaa a 35%35% 35% yield.yield. yield. ββ -1,4β-1,4 In-1,4In In glycosidaselinkages.contrastlinkages. scaffold to The The these [55]. acceptor acceptor syntheses The pNPMan pNPManα-mannosynthase in which ( 6(6) )on onoligomerizat the the other other was hand handion createof resulted resulted the productd byin in a a mutationtrisaccharide trisaccharideis observed, of in inYamamotoan a a 35% 35%α-glucosidase yield. yield. et al.In In of S. a preferredcontrastlinkages.contrastlinkages.contrastlinkages. hydrolysis to to TheTheto The thesethese these acceptoracceptor ofacceptor synthesessyntheses glucosidessyntheses pNPManpNPMan pNPMan inin in whichwhich over which ((6 6())6 on)on oligomerizaton mannosides.oligomerizat oligomerizat thethe the otherother other handhandion ionhandion Theof of resulted resultedof resulted thethe the synthase productproduct product inin in aa atrisaccharidetrisaccharide istrisaccharideis variant is observed,observed, observed, MalA in in Yamamoto Yamamoto in Yamamoto aa a35%35% D320G35% yield.yield. yield. etet et catalyzed al. Inal. In al.In the describedcontrastcontrast to tothe these these possibility syntheses syntheses of mono-g in in which whichlycosylating oligomerizat oligomerizat acceptorsionion of of the withthe product producta glycosynthase is is observed, observed, derived Yamamoto Yamamoto from a broad et et al. al. solfataricusformationdescribedcontrastdescribedcontrast ofdescribedwithcontrast oligosaccharides a toto the topreferredthe thesethethese thesepossibility possibility possibility synthesessyntheses syntheses hydrolysis of ifof of βmono-g mono-g in mono-ginGlcF in whichwhich whichlycosylating waslycosylatinglycosylating of oligomerizatoligomerizat oligomerizat used,glucosides acceptors butacceptors acceptorsionionion only ofoverof of thewith thewith mono-glycosylation withthe productmannosides.product a aproduct glycosynthasea glycosynthase glycosynthase isis is observed,observed, observed, The derived derived derived Yamamoto inYamamotosynthase Yamamoto thefrom from from case a a broada broad et etbroadvariant ofet al.al. al. the β MalAManF describedglycosidasedescribed the the scaffold possibility possibility [55]. of ofThe mono-g mono-g α-mannosynthaselycosylatinglycosylating acceptors acceptorswas create with withd by a a glycosynthase mutationglycosynthase of an derived derived α-glucosidase from from a a broad broadof S. D320G catalyzedglycosidasedescribedglycosidasedescribedglycosidasedescribed thethe the the scaffoldscaffold possibilityscaffold possibilityformation possibility [55].[55]. [55]. of of TheofThe mono-g Theofmono-g mono-g αoligosaccharidesα -mannosynthaseα-mannosynthase-mannosynthaselycosylatinglycosylatinglycosylating acceptors acceptors wasacceptorswas was if create create βcreateGlcF with with withdd d by bya awasby glycosynthase aglycosynthase mutation mutation glycosynthasemutation used, ofof butof anan derived anderived derivedααonly -glucosidaseα-glucosidase-glucosidase from frommono-glycosylation from a a broad a broad ofbroadof of S.S. S. donor. Thesolfataricusglycosidaseglycosidase enzyme with wasscaffold scaffold a tested preferred [55]. [55]. on The The ahydrolysis α varietyα-mannosynthase-mannosynthase of glucosides pNP-glycosides was was createover create mannosides.dd by resultingby mutation mutation The inof of synthase yieldsan an α α-glucosidase-glucosidase up variant to 77% MalA of of (S. αS. -1,4 being solfataricusglycosidasesolfataricusglycosidasesolfataricusglycosidase with with scaffoldwithscaffold scaffold aa a preferredpreferred preferred [55].[55]. [55]. TheThe The hydrolysishydrolysis hydrolysis αα -mannosynthaseα-mannosynthase-mannosynthase ofof of glucosidesglucosides glucosides waswas was createovercreateover overcreate mannosides.mannosides.d dmannosides. d byby by mutationmutation mutation TheThe The ofof of synthasesynthasean ansynthase an αα -glucosidaseα-glucosidase-glucosidase variantvariant variant MalA MalA ofMalAof of S.S. S. inthe the predominant casesolfataricusD320G ofsolfataricus the catalyzed β typeManF with with of a athe preferreddonor. linkage,preferred formation The hydrolysis αhydrolysis-1,3 of enzyme oligosaccharides and of of 1,2glucosides glucosideswas were tested ifalso βover overGlcF on observed)mannosides. mannosides.was a variety used, but andThe Theof only pNP-glycosidessynthase thesynthase mono-glycosylation preparation variant variant MalA MalA resulting of naturally in D320GsolfataricusD320GsolfataricusD320Gsolfataricus catalyzedcatalyzed catalyzed withwith with a a thethe apreferred preferredthe preferred formationformation formation hydrolysishydrolysis hydrolysis ofof of oligosaccharidesoligosaccharides oligosaccharides ofof of glucosidesglucosides glucosides ifif if βoverβover GlcFβoverGlcFGlcF mannosides.mannosides. wasmannosides.was was used,used, used, butbut ThebutThe The onlyonly only synthasesynthase synthase mono-glycosylationmono-glycosylation mono-glycosylation variantvariant variant MalAMalA MalA yields up toinD320GD320G 77%the case catalyzed (catalyzedα -1,4of the being βthe theManF formation formation the donor. predominant of Theof oligosaccharides oligosaccharides enzyme typewas tested ofif if β linkage,βGlcFGlcF on awas wasvariety αused, used,-1,3 of but butandpNP-glycosides only only 1,2 mono-glycosylation mono-glycosylationwere alsoresulting observed) in and occurringinD320GinD320GαinD320G the-mannosylthe the case case catalyzedcasecatalyzed catalyzed ofof of thethe the motifs, βtheβthe ManFβtheManFManF formationformation formation donor.donor. suchdonor. of Theof asThe of The oligosaccharidesoligosaccharides oligosaccharidesMan- enzymeenzyme enzymeα (1,4)-Glc, waswas was testedtested testedifif if ββ GlcFβGlcF onGlcF Man-on on a wasa wasa varietywasvariety varietyα used,used,(1,3)- used, ofof of but but pNP-glycosidesLpNP-glycosidesbut pNP-glycosides-Rha onlyonly only mono-glycosylation (mono-glycosylation 54mono-glycosylation), and resultingresulting resulting Man- ininα in (1,2)-Man yieldsinin the the caseup case to of of77% the the ( βα βManF-1,4ManF being donor. donor. the The predominantThe enzyme enzyme was wastype tested testedof linkage, on on a a variety αvariety-1,3 and of of pNP-glycosides 1,2pNP-glycosides were also observed) resulting resulting and in in the preparationyieldsininyieldsin thethe the caseup case ofupcase to naturally ofto of77% of the77%the the ( βα β( ManF-1,4αβManFManF-1,4 occurringbeing being donor.donor. donor. the the ThepredominantThe Thepredominant α enzyme-mannosylenzyme enzyme was wastype was type tested tested motifs, oftested of linkage, linkage, onon on asucha varietya varietyα variety -1,3α-1,3 as and of andofMan- of pNP-glycosides1,2pNP-glycosides pNP-glycosides1,2 wereα were(1,4)-Glc, also also observed) observed) resultingresulting resultingMan- and αand inin(1,3)- in L-Rha (55, Table2yieldsyields).yields upup up toto to 77%77% 77% (( αα(α-1,4-1,4-1,4 beingbeing being thethe the predominantpredominant predominant typetype type ofof of linkage,linkage, linkage, αα α-1,3-1,3-1,3 andand and 1,21,2 1,2 werewere were alsoalso also observed)observed) observed) andand and thetheyields preparation preparation up to 77% of of ( naturallyα naturally-1,4 being occurring occurring the predominant α α-mannosyl-mannosyl type motifs, motifs, of linkage, such such asα as-1,3 Man- Man- andαα(1,4)-Glc, 1,2(1,4)-Glc, were alsoMan- Man- observed)αα(1,3)-(1,3)-LL-Rha-Rha and (54), and Man-theyieldstheyieldstheyields preparation preparation preparationα up up (1,2)-Manup to to to 77% 77% 77% of of( (ofα αnaturally (naturally-1,4α -1,4 naturally(-1,455 being being, being Table occurring occurring the occurringthe the predominant 2).predominant predominant α α -mannosylα-mannosyl-mannosyl type type type motifs, motifs,of ofmotifs, of linkage, linkage, linkage, such such such α αas as-1,3α -1,3as -1,3Man- Man- Man-and and andαα 1,2 (1,4)-Glc,α1,2(1,4)-Glc, 1,2(1,4)-Glc, were were were also alsoMan- Man-also Man- observed) observed) observed)αα(1,3)-α(1,3)-(1,3)-LL-Rha L-Rhaand and-Rha and (the54the), preparation preparationand Man-α of(1,2)-Man of naturally naturally (55 occurring occurring, Table 2). α α-mannosyl-mannosyl motifs, motifs, such such as as Man- Man-αα(1,4)-Glc,(1,4)-Glc, Man- Man-αα(1,3)-(1,3)-LL-Rha-Rha Table(the(the54 2.54(the54), ),preparationSynthetic preparation ),and preparationand and Man- Man- Man- productsαα of(1,2)-Man αof(1,2)-Man (1,2)-Manof naturally naturally naturally of ( (55 55α(occurring 55occurring, -mannosylation ,occurringTable ,Table Table 2). 2). 2).α α -mannosylα-mannosyl -mannosyl catalyzed motifs, motifs, motifs, such bysuch such the as as as Man- MalAMan- Man-αα(1,4)-Glc,α D320G(1,4)-Glc,(1,4)-Glc, [ 55Man- Man- Man-]. Allαα(1,3)-α(1,3)-(1,3)- reactionsLL-Rha-RhaL-Rha were (54(54),), and and Man- Man-αα(1,2)-Man(1,2)-Man ( 55(55, ,Table Table 2). 2). ((5454(54),), ),and and and Man- Man- Man-αα(1,2)-Manα(1,2)-Man(1,2)-Man ( (55 55(55, ,Table Table, Table 2). 2). 2). Tablecarried 2. outSyntheticTable with 2. Syntheticβ productsManF products as of the α-mannosylationdonor. of α-mannosylation Different catalyzed catalyzed regioselectivities by by the the Ma MalA D320G werelA D320G observed[55]. All [55]. reactions All depending reactions were on were the TableTableTable 2. 2. 2.Synthetic Synthetic Synthetic products products products of of of α α -mannosylationα-mannosylation-mannosylation catalyzed catalyzed catalyzed by by by the the the Ma Ma MalAlAlA D320G D320G D320G [55]. [55]. [55]. All All All reactions reactions reactions were were were acceptor structureTablecarriedTable 2. 2.out Synthetic Synthetic and with anomeric β productsManF products as of configuration.the of α α -mannosylationdonor.-mannosylation Different Yields catalyzed catalyzedregioselecti were by by vitiesthe determinedthe Ma Ma werelAlA D320G D320G observed after [55]. [55]. dependingAll purification All reactions reactions on were were the or isolation. carried outcarriedTablecarriedTable carriedTablewith 2. 2. out 2.outSynthetic Syntheticβout SyntheticManF withwith with β βproducts ManFproductsβasManF productsManF the asas as ofdonor. theofthe of theα α -mannosylation donor.α-mannosylationdonor. -mannosylationdonor. Different DifferentDifferent Different catalyzed catalyzed regioselecticatalyzedregioselecti regioselecti by by by vitiesthe vitiesthe vitiesthevities Ma Ma Ma werewerelA lAwerelA D320G wereD320G D320Gobservedobserved observed [55]. observed[55]. [55]. dependingAll dependingAll dependingAll reactions reactions reactions depending onon were onwere thewerethe the on the acceptorcarriedcarried out outstructure with with β βandManFManF anomeric as as the the donor. configuration.donor. Different Different Yields regioselecti regioselecti were determinedvitiesvities were were after observed observed purification depending depending or isolation. on on the the acceptor structureacceptorcarriedacceptorcarriedacceptorcarried out outstructure structureout andstructure withwith with anomeric β βand ManFandβManF andManF anomeric anomeric anomeric asas as theconfiguration.the the donor.configuration. donor.configuration. donor.configuration. DifferentDifferent Different Yields Yields Yields Yields regioselectiregioselecti regioselecti were werewere were determined determinedvities determined vitiesdeterminedvities werewere were after after observed observedafter observed purification afterpurification purification depending dependingpurification depending or or or isolation. isolation. isolation. onon on the theor the isolation. acceptoracceptor structure structure and and anomeric anomeric configuration. configuration. Yields Yields were were determined determined after after purification purification or or isolation. isolation. acceptoracceptoracceptorAcceptorAcceptor structure structure structure and and and anomeric anomeric anomeric configuration. configuration. configuration.Product Yields Yields Yields were were were determined determined determined after after after purification purification purification Yield (Selectivity) (Selectivity) or or or isolation. isolation. isolation. AcceptorAcceptorAcceptor ProductProductProduct Yield Yield Yield (Selectivity) (Selectivity) (Selectivity) AcceptorAcceptor Acceptor ProductProductProduct Yield Yield Yield(Selectivity) (Selectivity) (Selectivity) AcceptorAcceptorAcceptor ProductProductProduct Yield Yield Yield (Selectivity) (Selectivity) (Selectivity) 26% a 26% a a (>95%,26%26%26%α -1,3)a a a 26% a a (>95%,(>95%,26% α aαa-1,3) -1,3)a a (>95%,(>95%,(>95%,26%26%26% α α -1,3)α -1,3) -1,3) 26% 3737 (>95%,(>95%, α α-1,3)-1,3) 373737 6060 (>95%,(>95%,(>95%, α α -1,3)α-1,3)-1,3) 37 37 606060 (>95%, α-1,3) 373737 6060 37 606060 60 55%55% 55%55%55% (60:40(60:40α -1,3/α55%55%-1,3/α -1,2)α -1,2) (60:40(60:40 α55% 55%-1,3/α55%-1,3/ α -1,2)α -1,2) 6 (60:40(60:40(60:40 αα α-1,3/-1,3/-1,3/ααα-1,2)-1,2)-1,2) 6 6 (60:40 α-1,3/α-1,2) 666 6161 (60:40(60:40(60:40 α α -1,3/α-1,3/-1,3/αα-1,2)α-1,2)-1,2) 6 616161 6262 55% 66 6 6161 62 6262 616161 6262 626262 (60:40 α-1,3/α-1,2) 6 44% a a 61 44%44%a aa a 44%44%44% a a 62 (92%,44%44% α -1,3)a (92%,(92%,(92%,(92%,44%44%44% αα α-1,3) -1,3)αa -1,3) -1,3) a 5858 (92%,(92%, α α-1,3)-1,3) 585858 (92%,(92%,(92%, α α -1,3)α-1,3)-1,3) 5858 6363 585858 636363 58 6363 636363 44% a 41% a a 41%41%41% a a a 41%a(92%, a a α-1,3) (3:2 41%α-1,5/41%aaα -1,1) 5959 (3:2(3:2(3:2 α α41% -1,5/41%α-1,5/41%-1,5/ α α -1,1)αa-1,1) -1,1) 58 595959 57 (3:2(3:2(3:2α α-1,5/ α-1,5/-1,5/αα-1,1)α-1,1)-1,1) 5959 5656 575757 (3:2(3:2(3:2 α α -1,5/α-1,5/-1,5/αα-1,1)α-1,1)-1,1) 595959 63 565656 5757 5656 a aIsolated as peracetate.57 5757 595656 56 a a Isolated aIsolated Isolated as as as peracetate. peracetate. peracetate. 57 a aIsolated as peracetate. aa Isolated as peracetate. a Isolated Isolated Isolateda Isolated as asas as peracetate. peracetate. peracetate. 41% a

(3:2 α-1,5/α-1,1) 59

56 57 a Isolated as peracetate.

Molecules 2017, 22, 1434 11 of 20

Molecules 2017, 22, 1434 11 of 20 The method also allowed the production of mannosylated myo-inositol (56/57) which is a component The method also allowed the production of mannosylated myo-inositol (56/57) which is a of the cell wall glycolipids of Mycobacterium tuberculosis. The mono-mannosylated inositols were component of the cell wall glycolipids of Mycobacterium tuberculosis. The mono-mannosylated isolated in a yield of 41% (mixture of Man-α(1,5)- and Man-α(1,1)-myo-inositol 56 and 57 in a 3:1 ratio). inositols were isolated in a yield of 41% (mixture of Man-α(1,5)- and Man-α(1,1)-myo-inositol 56 and 2.5.57 Fucosynthases in a 3:1 ratio).

2.5.A Fucosynthases further important group of compounds relevant in many biological processes are fucosyl residues. This deoxy-aldohexose exists in nature, in comparison to most natural carbohydrates, A further important group of compounds relevant in many biological processes are fucosyl α asresidues. a L-configured This deoxy-aldohexose glycoside and exists linked in predominantly nature, in comparison in -configuration to most natural at carbohydrates, the anomeric as center. a ManyL-configured biological glycoside processes and involvelinked predominantly fucosylated compounds in α-configuration such as,at the cell-to-cell anomeric communication,center. Many cellbiological development, processes recognition involve structuresfucosylated for compounds pathogens, andsuch antigenic as, cell-to-cell structures. communication, cell development,The first synthesis recognition of fucosylatedstructures for structures pathogens, using and antigenic the glycosynthase structures. method was reported by Wada etThe al. first with synthesis a mutated ofα fucosylated-fucosidase ofstructuresB. bifidum usBbAfcAing the glycosynthase D766G [57]. The method enzyme was was reported successfully by 0 utilizedWada inet the al. synthesiswith a mutated of a Fuc- α(1,2)-Gal-fucosidase linkage of B. inbifidum 2 -fucosyllactose BbAfcA D766G by combining [57]. Theβ -fucosylenzyme fluoridewas (βFucF)successfully with lactose. utilized However, in the synthesis the low of stabilitya Fuc-α(1,2)-Gal of βFucF linkage led to yieldsin 2′-fucosyllactose of 6%. The yields by combining caused by β- the instabilityfucosyl fluoride of the β (-fluorideβFucF) with donor lactose. were Howeve overcomer, the by low Cobucci-Ponzano stability of βFucF et al.led byto theyields implementation of 6%. The ofyields more caused stable byβ-azide the instability fucosylpyranoside of the β-fluoride (βFucN donor3) aswere donors overcome for two by Cobucci-Ponzano new α-fucosynthases et al. by [58 ]. Thetheα implementation-fucosynthase SsD242S of more exhibitedstable β-azide a wide fucosylpyranoside acceptor range (β (shownFucN3) foras donors various for aryl-glycosides) two new α- thoughfucosynthases also catalyzing [58]. The theα-fucosynthase self-condensation SsD242S of exhibitedβFucN3 ain wide all acceptor cases. In range comparison, (shown for the various second fucosynthasearyl-glycosides) TmD224G though demonstratedalso catalyzing athe more self-condensation restricted acceptor of βFucN range,3 in all but cases. the self-condensationIn comparison, reactionthe second was notfucosynthase observed. TmD224G The reactions demonstrated were also a carried more restricted out with SsD242Sacceptor range, in a preparative but the self- scale withcondensation total transfucosylation reaction was efficiencies not observed. ranging The fromreactions 29–86% were (for also the carried acceptors out pNPXylwith SsD242S (38), pNPGal, in a preparative scale with total transfucosylation efficiencies ranging from 29–86% (for the acceptors and pNPGlcNAc (7)) providing mostly α-1,4 and 1,3 linkages and additionally α-1,6 linkages in the pNPXyl (38), pNPGal, and pNPGlcNAc (7)) providing mostly α-1,4 and 1,3 linkages and additionally case of the galactoside acceptor. The mutant TmD224G could even reach a transfucosylation efficiency α-1,6 linkages in the case of the galactoside acceptor. The mutant TmD224G could even reach a of 91% producing a mixture of Fuc-α(1,3)- and Fuc-α(1,4)-β-XylpNP in a near 1:1 ratio. The application transfucosylation efficiency of 91% producing a mixture of Fuc-α(1,3)- and Fuc-α(1,4)-β-XylpNP in a of α-L-fucosynthases was more recently transferred to relevant biological structures such as epitopes near 1:1 ratio. The application of α-L-fucosynthases was more recently transferred to relevant a ofbiological the antigens structures Lewis andsuch ABO. as epit Sakuramaopes of the et antigens al. reported Lewis the and synthesis ABO. Sakurama of the lewis et al. antigens reported Le theand x β α a/x α Lesynthesis(Gal- 1,3/4-(Fuc- of the lewis 1,4/3)-GlcNAc:antigens Lea andLe Lex (Gal-64/β651,3/4-(Fuc-) catalyzedα1,4/3)-GlcNAc: by the 1,3-1,4- Le-a/xL -fucosynthase64/65) catalyzed BbAfcB by β D703Sthe 1,3-1,4- [59]. Theα-L-fucosynthase antigen structures BbAfcB were D703S created [59]. The by the antigen reaction structures of FucF were and created lacto- byN-biose the reaction I (66, LNB,of Gal-βFucFβ(1,3)-GlcNAc) and lacto-N-biose and N I-acetyllactosamine (66, LNB, Gal-β(1,3)-GlcNAc) (67, LacNAc, and Gal- N-acetyllactosamineβ(1,4)-GlcNAc) resulting(67, LacNAc, in yields Gal- of a x 47%β(1,4)-GlcNAc) and 55% for resulting Le (64) andin yields Le (of65 47%), respectively and 55% for (Scheme Lea (64) and11). Lex (65), respectively (Scheme 11).

Scheme 11. α-Fucosylation of LNB 66 or LacNAc 67 for the production of the Lewis antigens Lea (64) Scheme 11. α-Fucosylation of LNB 66 or LacNAc 67 for the production of the Lewis antigens Lea (64) and Lex (65), respectively, catalyzed by the α-1,3-1,4-L-fucosynthase BbAfcB D703S [59]. and Lex (65), respectively, catalyzed by the α-1,3-1,4-L-fucosynthase BbAfcB D703S [59].

An increase in yield using the more stable donor βFucN3 as demonstrated by Cobucci-Ponzano et al.An could increase not inbe yieldachieved using as the no moretransfucosylation stable donor productsβFucN3 as could demonstrated be observed by with Cobucci-Ponzano this donor. The et al. couldenzyme not bealso achieved interestingly as no transfucosylation only acted on productsdi- or trisaccharide could be observed acceptors with unable this donor. to fucosylate The enzyme alsomonosaccharides interestingly only such acted as, Glc, on di- Ga orl, trisaccharideGlcNAc, and acceptorsGalNAc. unableThe specific to fucosylate production monosaccharides of lacto-N-

Molecules 2017, 22, 1434 12 of 20 such as, Glc, Gal, GlcNAc, and GalNAc. The specific production of lacto-N-fucopentaose II Molecules 2017, 22, 1434 12 of 20 (LNFP II: Gal-β1,3-(Fuc-α1,4)-GlcNAc-β1,3-Gal-β1,4-Glc) was also achieved in a yield of 41% with lacto-fucopentaoseN-tetraose II (LNT: (LNFP Gal- II: Gal-β1,3-GlcNAc-β1,3-(Fuc-αβ1,4)-GlcNAc-1,3-Gal-β1,4-Glc)β1,3-Gal- asβ the1,4-Glc) acceptor. was also achieved in a yield of 41%The with repertoire lacto-N-tetraose of synthesized (LNT: Gal- antigenβ1,3-GlcNAc- epitopeβ1,3-Gal- structuresβ1,4-Glc) was as expanded the acceptor. by the work of SugiyamaThe repertoire et al. who of synthesized introduced antigen the 1,2- epitopeα-L-fucosynthase structures was BbAfcA expanded N423H by the [ 60work]. Inof Sugiyama addition to fucosylatinget al. who introduced many types the 1,2- ofα mono--L-fucosynthase and disaccharides, BbAfcA N423H producing [60]. In addition for example to fucosylating H type-1 many or H type-2types chainsof mono- from and LNBdisaccharides, (66) or LacNAcproducing (67 for) respectively,example H type-1 the or antigens H type-2 Le chainsb and from Ley LNB(Fuc- (66α)1,2- Gal-orβ LacNAc1,3/4-(Fuc- (67)α respectively,1,4/3)-GlcNAc: the antigens Leb/y) were Leb and produced Ley (Fuc- inα yields1,2-Gal- ofβ1,3/4-(Fuc- 43% and 62%α1,4/3)-GlcNAc: from the Le a andLe Leb/y)x weretrisaccharides produced (64in yieldsand 65 of). In43% comparison and 62% from to the the BbAfcB Lea and D703S Lex trisaccharides fucosynthase, (64 BbAfcA and 65 N423H). In producedcomparison LNFP to Ithe (Fuc- BbAfcBα1,2-Gal- D703Sβ1,3-GlcNAc- fucosynthase,β1,3-Gal- BbAfcAβ N423H1,4-Glc) produced rather than LNFP LFNP I (Fuc- II withα1,2-Gal- an efficiencyβ1,3- ofGlcNAc- 75%. Subsequently,β1,3-Gal-β1,4-Glc) the transfer rather than of fucosyl LFNP II residues with an toeffi theciency non-reducing of 75%. Subsequently, end of O-linked the transfer glycans wasof demonstratedfucosyl residues by to the the successful non-reducing reintroduction end of O-linked of H-antigen glycans was structures demonstrated to pretreated by the glycopeptidesuccessful porcinereintroduction gastric mucin. of H-antigen The application structures was to even pret furtherreated extended glycopeptide by the porcine group introducinggastric mucin. H-antigens The toapplication the non-reducing was even ends further of N extended- and O-glycans by the group in fetuin introducing glycoproteins, H-antigens GM1 to the ganglioside non-reducing (68 ),ends and a xyloglucanof N- and nonasaccharideO-glycans in fetuin [61 glycoproteins,]. It was demonstrated GM1 ganglioside that BbAfcA (68), and N423H a xyloglucan could fucosylate nonasaccharide asialo-bi-, [61]. It was demonstrated that BbAfcA N423H could fucosylate asialo-bi-, asialo-tri-, and monosialo- asialo-tri-, and monosialo-tri-antennary N-glycan of the asialo fetuin glycopeptide with transfer tri-antennary N-glycan of the asialo fetuin glycopeptide with transfer efficiencies of 9%, 26%, and efficiencies of 9%, 26%, and 20% respectively. It was also possible to fucosylate the sialo form of the 20% respectively. It was also possible to fucosylate the sialo form of the fetuin peptide containing di- fetuin peptide containing di-sialo-bi-, di-sialo-tri, tri-sialo-tri, and tetra-sialo-tri-antennary glycans, yet sialo-bi-, di-sialo-tri, tri-sialo-tri, and tetra-sialo-tri-antennary glycans, yet with lower transfer with lower transfer efficiencies. The fucosylation of the xyloglucan nonasaccharide XLLG (51, Structure efficiencies. The fucosylation of the xyloglucan nonasaccharide XLLG (51, Structure shown in Scheme α shown9) occurred in Scheme with9 )an occurred efficiency with of 57% an efficiency resulting in of a 57% mixture resulting of mono- in a mixture(α-1,2-Gal), of mono- di- (α-1,2-Gal), ( -1,2-Gal), and di- α α ( -1,2-Gal),trifucosylated and ( trifucosylatedα-1,3-Glc) XLLG ( -1,3-Glc)glycosides. XLLG The enzyme glycosides. also The fucosylated enzyme glycolipids, also fucosylated transferring glycolipids, α- transferring1,2-fucosylα residue-1,2-fucosyl to the residueglycoside to of the the glycoside GM1 ganglioside of the GM1 (68, ganglioside Scheme 12).( 68, Scheme 12).

SchemeScheme 12. 12.Selective Selective introduction introduction ofof an α-fucosyl residue residue to to the the glycoside glycoside of of the the GM1 GM1 ganglioside ganglioside (68 ()68 ) demonstrateddemonstrated by by Sugiyama Sugiyama etet al.al. [[61].61]. TheThe enzyme BbAfcA N423H N423H showed showed a abroad broad acceptor acceptor range range exhibitingexhibiting fucosylation fucosylation activityactivity also towards towards NN- -and and OO-glycans-glycans and and a nonasaccharide a nonasaccharide xyloglucan. xyloglucan.

2.6. Glycosynthase Variants of endo-β-N-Acetylglucosaminidases 2.6. Glycosynthase Variants of endo-β-N-Acetylglucosaminidases Different from the exo- and endo-glycosidases described above, the hydrolytic mechanism of Different from the exo- and endo-glycosidases described above, the hydrolytic mechanism of endo-β-N-acetylglucosaminidases follows a substrate-assisted pathway in which the anomeric center β endoof -the-N N-acetylglucosaminidases-acetylglucosamine located follows in the a chitobiose substrate-assisted moiety undergoes pathway a in nucleophilic which the anomeric attack by centerthe ofN the-linkedN-acetylglucosamine acetyl group creating located a inoxazoline the chitobiose structure. moiety This undergoes kind of activated a nucleophilic structure attack can by be the N-linkedemployed acetyl by groupmutant creating forms aof oxazoline the endo- structure.β-N-acetylglucosaminidases, This kind of activated which structure are deficient can be employed in the bypromotion mutant forms of the of oxazoline the endo -βformation,-N-acetylglucosaminidases, as glycan donors in which glycosynthetic are deficient reactions. in the The promotion first ofglycosynthase the oxazoline like formation, form of an as glycanendo-β-N donors-acetylglucosaminidase in glycosynthetic was reactions. reported Theby Umekawa first glycosynthase et al. as likethe form N175A of anvariantendo -ofβ- NEndo-M-acetylglucosaminidase (originating from wasM. hiemalis reported) [62]. by UmekawaThe enzyme et could al. as utilize the N175A the variantoxazoline of Endo-M donor (originatingMan9GlcNAc-oxazoline from M. hiemalisin the)[ synthesis62]. The of enzyme the HIV-1 could gp41 utilize glycopeptide the oxazoline donorMan9 ManGlcNAc9GlcNAc-oxazoline2C34, producing the in the glycopeptide synthesis ofin thea yield HIV-1 of gp41 72%. glycopeptideA large improvement Man9GlcNAc for the2C34, producingglycosynthase the glycopeptide method using in endo a yield-β-N-acetylglucosaminidases of 72%. A large improvement was the simplified for the glycosynthase donor synthesis method by Noguchi et al. [63,64]. The synthesis of the oxazoline structure catalyzed by 2-chloro-1,3-dimethyl- imidazolium chloride (DMC) and later 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride

Molecules 2017, 22, 1434 13 of 20 using endo-β-N-acetylglucosaminidases was the simplified donor synthesis by Noguchi et al. [63,64]. TheMolecules synthesis 2017, 22 of, 1434 the oxazoline structure catalyzed by 2-chloro-1,3-dimethyl-imidazolium chloride13 of 20 (DMC) and later 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI) allowed the production(CDMBI) inallowed aqueous thesolution production in unprotected in aqueous form,solution therefore in unprotected ideal for form, subsequent therefore conversion ideal for by enzymaticsubsequent glycosylation. conversion by In enzymatic 2010, Umekawa glycosylation. et al. describedIn 2010, Umekawa a new variant et al. described Endo-M aN175Q, new variant which exhibitedEndo-M a N175Q, much higherwhich synthaseexhibited activitya much higher and transglycosidase synthase activity activity and transglycosidase [65]. This enabled activity the [65]. use of naturalThis enabled glycan donorsthe use suchof natural as the glycan sialoglycopeptide donors such as (SGP), the sialoglycopeptide though leading (SGP), to a lower though yield leading compared to a lower yield compared to the use of oxazoline donors. The variant enabled the synthesis of a high- to the use of oxazoline donors. The variant enabled the synthesis of a high-mannose (84%) and mannose (84%) and complex type glycoform (76%) of the sperm antigen CD52 (70a,b) using the complex type glycoform (76%) of the sperm antigen CD52 (70a,b) using the GlcNAc-CD52 peptide (71) GlcNAc-CD52 peptide (71) as the acceptor (Scheme 13). The group also described the successful as the acceptor (Scheme 13). The group also described the successful synthesis of sialo-complex-type synthesis of sialo-complex-type glycoforms of the bioactive peptides PAMP12 and Substance P in glycoforms of the bioactive peptides PAMP12 and Substance P in yields of 95 and 98%, respectively. The yields of 95 and 98%, respectively. The glycosylation reaction was also shown for the protein RNaseB glycosylation reaction was also shown for the protein RNaseB bearing a single GlcNAc moiety therefore bearing a single GlcNAc moiety therefore allowing glycan modification of proteins/enzymes by this allowingmethod glycan [66]. Amin modification et al. also of proteins/enzymes described the production by this method of monoglycoforms [66]. Amin et al.of alsoRNaseB described by the the productionglycosynthase of monoglycoforms Endo-A N171A of(A. RNaseB protophormiae by the) glycosynthasein yields up to Endo-A 80% with N171A chemically (A. protophormiae synthesized) in yieldsoxazoline up to glycans 80% with [67]. chemically synthesized oxazoline glycans [67].

SchemeScheme 13. 13.Synthesis Synthesis ofof twotwo glycoformsglycoforms of the sperm antigen antigen CD52 CD52 (70a (70a/b/)b by) by the the endoendo-β--Nβ--N- acetylglucosaminidase derived glycosynthase Endo-M N175Q [65]. The high-mannose and complex acetylglucosaminidase derived glycosynthase Endo-M N175Q [65]. The high-mannose and complex type glycoforms were produced under utilization of oxazoline donors 72, 73 and the GlcNAc-CD52 type glycoforms were produced under utilization of oxazoline donors 72, 73 and the GlcNAc-CD52 peptide (71). peptide (71). The Endo-M and Endo-A glycosynthase variants were additionally employed in the synthesis of Thefour Endo-Mglycoforms and (yields Endo-A varying glycosynthase from 59–96%) variants of the glycopeptide were additionally pramlint employedide by Tomabechi in the synthesis et al. ofin four an glycoformsattempt to improve (yields varying the low from circulatory 59–96%) half-life of the and glycopeptide poor solubility pramlintide of the pharmaceutical by Tomabechi et al.compound in an attempt [68].to The improve produced the glycoforms low circulatory were half-lifetested in and vitro poor and solubilityin vivo and of exhibited the pharmaceutical varying compoundproperties [ 68in ].dependency The produced of the glycoforms type of transferre wered tested glycanin and vitro itsand positionin vivo in theand glycopeptide. exhibited varying The propertiesglycoform in library dependency was subsequently of the type expanded of transferred with the glycan same enzymes and its positionby Kowalczyk in the et glycopeptide.al. to 18 N- Theglycosylated glycoform pramlintide library was analogues subsequently bearing expanded either a GlcNAc-, with the pentasaccharide-, same enzymes or by undecasaccharide Kowalczyk et al. toresidue 18 N-glycosylated at different positions pramlintide of the analogues peptide [6 bearing9]. In comparison either a GlcNAc-, to the Endo-A pentasaccharide-, and Endo-M or undecasaccharideglycosynthases, Fan residue et al. at differentdeveloped positions a glycos ofynthase the peptide variant [ 69of]. Endo-D In comparison originating to the from Endo-A S. andpneumoniae Endo-M which glycosynthases, could glycosylate Fan et fucosylated al. developed GlcNAc a glycosynthase residues [70]. This variant unique of Endo-D property originating allowed the remodeling of the glycans of the IgG Fc-domain. However, the strict substrate specificity for Man3GlcNAc oxazoline and not complex type N-glycan oxazolines limits the use of the enzyme greatly. A more variable glycosylation of α-1,6-fucosylated GlcNAc-polypeptides was demonstrated

Molecules 2017, 22, 1434 14 of 20 from S. pneumoniae which could glycosylate fucosylated GlcNAc residues [70]. This unique property allowed the remodeling of the glycans of the IgG Fc-domain. However, the strict substrate specificity for Man3GlcNAc oxazoline and not complex type N-glycan oxazolines limits the use of the enzyme greatly. A more variable glycosylation of α-1,6-fucosylated GlcNAc-polypeptides was demonstrated by Giddens et al. with the Endo-F3 mutants D165A/Q [71]. The mutant was capable of synthesizing asialo biantennary and complex triantennary core-fucosylated glycoforms of rituximab (intact antibody) in yields over 95%. Further applications, indicating the high potential of the method for the synthesis of pharmaceutical relevant compounds were the chemoenzymatic production of vaccine candidates [72]; the site-selective glycosylation of HIV-1 polypeptide antigen bearing two different glycans (yields up to 95%) [73]; the glycan remodeling of human erythropoietin (EPO) [74]; and the synthesis of mannose-6-phosphate-containing glycoproteins [75]. Tang et al. impressively demonstrated a one-pot N-glycan remodeling of IgG proteins by combining the wild type (wt) Endo-M glycosidase with the synthase variant Endo-S D322S [76]. This synthesis comprised the donor production by Endo-M catalyzed SGP hydrolysis, subsequent conversion to the oxazoline with DMC, and donor transfer to the protein by Endo-S D322S with near complete conversion within 30 min. Further yield improvement might be achieved by the new glycosynthase of Endo-CC (C. cinereal) recently introduced by Eshima et al. [77]. The enzyme exhibited high activity at a neutral pH of 7.5 in comparison to the acidic pH between 4–6, which most other endo-β-N-acetylglucosaminidases require for activity. This could be a great advantage for yield improvement due to the increased stability of the oxazoline donors at this pH [78]. A further use of the oxazoline type donor for glycoside synthesis has been demonstrated by the production of glycosaminoglycans such as derivatives of chondroitin sulfate and hyaluronan [79–82]. However, the polymerization of the oxazolines derived from, for example N-acetylchondrosine, was carried out with the natural forms of ovine or bovine testes hyaluronidase, therefore not utilizing the glycosynthase method and not further discussed in this review. Nevertheless, the first step to glycosynthase mediated synthesis of glycosaminoglycans such as heparan, chondroitin sulfate, and hyaluronan was demonstrated by Müllegger et al. by the synthesis of uronic acid-containing glycoconjugates catalyzed by the thermostable glycosynthase derived from T. maritima β-glucuronidase [83].

3. Conclusions The enzymatic synthesis of glycosidic structures utilizing the glycosynthase approach has made many advances and is nowadays a promising alternative to classical chemical synthesis. The direct comparison of enzymatic glycosylation reactions compared to the chemical paths is difficult due to the additional protection and deprotection as well as possible modification steps needed after chemical glycosylation. When solely comparing the glycosylation reaction, the chemical pathway also in many cases produces glycosides in similar high yields (e.g., the synthesis of disaccharide 61 (Section 2.4, Table2), enzymatic glycosylation 55% yield; chemical glycosylation 61%). However, when considering the steps required for synthesis of the acceptor and donor molecule and also the subsequent deprotection steps for the chemical pathway, the advantage of the glycosynthases becomes obvious, especially when thinking of large oligosaccharides (Table3). The repertoire of glycosynthase variants is successfully increasing though it is still insufficient for creating the high diversity of glycosides present throughout Nature. Great advances were acheived with the introduction of synthases derived from inverting enzymes and the recent glycosynthases derived from endo-β-N-acetylglucosaminidases that allow the production of peptides with defined glycosylation. A larger drawback, however, is still the lack of glycosynthase variants capable of glycosylating non-glycosidic structures and the formation of oligomerized side products. MoleculesMolecules 2017 2017, ,22 22, ,1434 1434 1414 of of 20 20 Molecules 2017, 22, 1434 14 of 20 byby GiddensGiddens etet al.al. withwith thethe Endo-F3Endo-F3 mutantsmutants D165A/D165A/QQ [71].[71]. TheThe mutantmutant waswas capablecapable ofof synthesizingsynthesizing by Giddensasialoasialo biantennarybiantennary et al. with the andand Endo-F3 complexcomplex mutants triantennarytriantennary D165A/ Qcore-fucosylatedcore-fucosylated [71]. The mutant glycoformsglycoforms was capable ofof of rituximab rituximabsynthesizing (intact(intact asialoantibody)antibody) biantennary inin yieldsyields and over overcomplex 95%.95%. FurtherFurthertriantennary applicationsapplications core-fucosylated, , indicatingindicating glycoforms thethe highhigh potentialpotential of rituximab ofof thethe methodmethod(intact forfor antibody)thethe synthesissynthesis in yields of ofover pharmaceuticalpharmaceutical 95%. Further relevantapplicationsrelevant compcomp, indicatingoundsounds werewere the thehighthe chemoenzymatic chemoenzymaticpotential of the method productionproduction for ofof the vaccinesynthesisvaccine candidatescandidates of pharmaceutical [72];[72]; thethe site-selectivesite-selective relevant comp glycosylationglycosylationounds were ofof theHIV-1HIV-1 chemoenzymatic polypeptidepolypeptide antigenantigen production bearingbearing of two two vaccinedifferentdifferent candidates glycans glycans [72]; (yields (yields the up site-selectiveup to to 95%) 95%) [73]; [73]; glycosylation the the glyc glycanan remodeling remodelingof HIV-1 polypeptide of of human human er er antigenythropoietinythropoietin bearing (EPO) (EPO) two [74]; [74]; differentandand the theglycans synthesissynthesis (yields ofof up mannose-6-phosphate-containingmannose-6-phosphate-containing to 95%) [73]; the glycan remodeling glycoproteinsglycoproteins of human er[75].[75].ythropoietin TangTang etet al.al. (EPO) impressivelyimpressively [74]; anddemonstrated demonstratedthe synthesis a ofa one-pot one-pot mannose-6-phosphate-containing N N-glycan-glycan remodeling remodeling of of IgG IgG glycoproteins proteins proteins by by combining combining [75]. Tang the the et wild wildal. impressively type type ( (wtwt)) Endo- Endo- demonstratedMM glycosidaseglycosidase a one-pot withwith N thethe-glycan synthasesynthase remodeling variantvariant of Endo-SEndo-S IgG proteins D322SD322S by[76].[76]. combining ThisThis synthesissynthesis the wild comprisedcomprised type (wt) theEndo-the donordonor M glycosidaseproductionproduction bywith by Endo-M Endo-M the synthase catalyzed catalyzed variant SGP SGP hydrolysis, hydrolysis,Endo-S D322S su subsequentbsequent [76]. This conversion conversion synthesis to to comprised the the oxazoline oxazoline the with withdonor DMC, DMC, productionandand donordonor by Endo-M transfertransfer catalyzed toto thethe proteinprotein SGP hydrolysis, byby Endo-SEndo-S suD322SD322Sbsequent withwith conversion nearnear completecomplete to the conversionconversion oxazoline withinwithwithin DMC, 3030 min.min. andFurther Furtherdonor transfer yieldyield improvementimprovement to the protein mightmight by Endo-S bebe achievedachieved D322S by bywith thethe near newnew complete glycosynthaseglycosynthase conversion ofof Endo-CCEndo-CC within (30(C.C. min. cinerealcinereal )) Furtherrecentlyrecently yield introducedintroduced improvement byby Eshima Eshimamight beetet al.al.achieved [77].[77]. TheThe by enzyme enzymethe new exhibitedexhibited glycosynthase highhigh activityactivity of Endo-CC atat aa neutralneutral (C. cinereal pHpH ofof) 7.57.5 recentlyinin comparisoncomparison introduced to toby thethe Eshima acidicacidic et pH pHal. [77].betweenbetween The 4–6,enzyme4–6, whichwhich exhibited mostmost other otherhigh activityendoendo--ββ--N Nat-acetylglucosaminidases-acetylglucosaminidases a neutral pH of 7.5 in comparisonrequirerequire forfor toactivity.activity. the acidic ThisThis pH couldcould between bebe aa great great4–6, advawhichadvantagentage most forfor other yieldyield endo improvementimprovement-β-N-acetylglucosaminidases duedue toto thethe increasedincreased requirestabilitystability for activity. ofof thethe oxazolineoxazolineThis could donorsdonors be a great atat thisthis adva pHpHntage [78].[78]. for AA furtheryieldfurther improvement useuse ofof thethe oxazoline oxazolinedue to the typetype increased donordonor forfor stabilityglycosideglycoside of the synthesissynthesis oxazoline hashas donors beenbeen at demonstrateddemonstrated this pH [78]. by byA thfurtherthee productionproduction use of the ofof oxazolineglycosaminoglycansglycosaminoglycans type donor such suchfor asas glycosidederivativesderivatives synthesis ofof chondroitinchondroitin has been demonstratedsulfatesulfate andand hyaluronanhyaluronan by the production [79–82].[79–82]. However, However,of glycosaminoglycans thethe polymerizationpolymerization such ofasof thethe derivativesoxazolinesoxazolines of derivedchondroitinderived from,from, sulfate forfor exampleexample and hyaluronan NN-acetylchondrosine,-acetylchondrosine, [79–82]. However, waswas carriedcarried the outpolymerizationout withwith thethe naturalnatural of the formsforms oxazolinesofof ovineovine derived oror bovinebovine from, testestestes for example hyaluronidase,hyaluronidase, N-acetylchondrosine, therefthereforeore notnot utilizingutilizing was carried thethe glycosynthaseglycosynthaseout with the natural methodmethod forms andand notnot of ovinefurtherfurther or discussedbovinediscussed testes inin thisthis hyaluronidase, review.review. Nevertheless,Nevertheless, therefore thenotthe firstfirstutilizing stepstep totheto glycosynthaseglycosynthase glycosynthase mediatedmediated method synthesisandsynthesis not ofof furtherglycosaminoglycansglycosaminoglycans discussed in this suchreview.such asas Nevertheless, heparan,heparan, chondroitinchondroitin the first stepsulfate,sulfate, to glycosynthase andand hyaluronanhyaluronan mediated waswas demonstrateddemonstrated synthesis of byby glycosaminoglycansMülleggerMüllegger etet al.al. such byby thetheas heparan,synthesissynthesis chondroitin ofof uronicuronic acid-contasulfate,acid-conta andiningining hyaluronan glycoconjugatesglycoconjugates was demonstrated catalyzedcatalyzed byby thethe Mülleggerthermostablethermostable et al. glycosynthase glycosynthaseby the synthesis derived derived of uronicfrom from T. T. acid-contamaritima maritima β βining-glucuronidase-glucuronidase glycoconjugates [83]. [83]. catalyzed by the thermostable glycosynthase derived from T. maritima β-glucuronidase [83]. 3.3. Conclusions Conclusions 3. Conclusions TheThe enzymatic enzymatic synthesis synthesis of of glycosidic glycosidic structures structures utilizing utilizing the the glycosynthase glycosynthase approach approach has has made made manymanyThe enzymatic advancesadvances synthesis andand isis nowadaysnowadays of glycosidic aa promisingpromising structures alternativealternative utilizing totheto classicalclassical glycosynthase chemicalchemical approach synthesis.synthesis. has The Themade directdirect manycomparisoncomparison advances ofandof enzymaticenzymatic is nowadays glycosylationglycosylation a promising reactionsreactions alternative compcomp toaredared classical toto thethe chemical chemicalchemical synthesis. pathspaths isis difficult difficultThe direct duedue toto comparisonthethe additionaladditional of enzymatic protectionprotection glycosylation andand deprotectiondeprotection reactions ascompas wewearedllll asas to possiblepossible the chemical modificationmodification paths is stepsdifficultsteps neededneeded due to afterafter the chemicaladditionalchemical glycosylation.glycosylation. protection and WhenWhen deprotection solelysolely comparingcomparing as well thetheas glycosylationpossibleglycosylation modification reaction,reaction, steps thethe chemical chemicalneeded pathwayafterpathway chemicalalsoalso in inglycosylation. manymany casescases produces producesWhen solely glycosidesglycosides comparing inin similasimila the rrglycosylation highhigh yieldsyields (e.g.,(e.g., reaction, thethe synthesissynthesis the chemical ofof disaccharidedisaccharide pathway 6161 also(Section (Sectionin many 2.4, 2.4,cases TableTable produces 2),2), enzymaticenzymatic glycosides glycosylationglycosylation in similar high 5555%% yields yield;yield; (e.g., chemicalchemical the synthesis glycosylationglycosylation of disaccharide 61%).61%). However,However, 61 (Sectionwhenwhen 2.4, consideringconsidering Table 2), theenzymaticthe stepssteps requiredrequired glycosylation forfor synthesissynthesis 55% yield; ofof thethe chemical acceptoracceptor glycosylation andand donordonor moleculemolecule 61%). However, andand alsoalso thethe whensubsequentsubsequent considering deprotectiondeprotection the steps required stepssteps for forfor the thesynthesis chemicalchemical of the pathway,pathway, acceptor thethe and advantageadvantage donor molecule ofof thethe and glycosynthasesglycosynthases also the Molecules 2017, 22, 1434 15 of 20 subsequentbecomesbecomes deprotectionobvious, obvious, especially especially steps when forwhen the thinking thinking chemical of of large largepathway, oligosaccharides oligosaccharides the advantage (Table (Table of 3). 3).the glycosynthases becomes obvious, especially when thinking of large oligosaccharides (Table 3). TableTableTable 3. Comparison 3. 3. Comparison Comparison of the of of required the the required required steps steps andsteps yieldsand and yields yields of the of of chemical the the chemical chemical and enzymaticand and enzymatic enzymatic synthesis synthesis synthesis of the of of the the disaccharideTabledisaccharidedisaccharide 3. Comparison Man- Man- αMan-(1,3)- ofαα (1,3)-theD(1,3)--α required-pNPManDD--αα-pNPMan-pNPMan steps (61)[ and( 55(6161,)84 ) yields[55,84–87]. [55,84–87].–87]. of the chemical and enzymatic synthesis of the disaccharide Man-α(1,3)-D-α-pNPMan (61) [55,84–87]. ProductProduct TypeType DonorDonor Acceptor Acceptor Product ReferencesReferences Type Donor Acceptor (Yield,(Yield,Product Selectivity) Selectivity) References Type Donor Acceptor (Yield, Selectivity) References (Yield, Selectivity)

Chemical [84–86]

Chemical [84–86] Chemical 7676 [84–86] 7474 Chemical 7575 76 [84–86] 74 GlycosylationGlycosylation76 61% 61% 22 Steps Steps 83% 83% 275 Steps 50%aa 74 2 Steps75 50% GlycosylationGlycosylationDeprotectionDeprotection 61%61% of of 76 76 81% 81% 2 Steps 83% 2 Steps 50%a MoleculesMolecules 2017 2017, ,22 22, ,1434 21434 Steps 83% 2 Steps 50% a Deprotection(2 Steps ofof 7676 49%) 81%81% 1515 of of 20 20 Molecules 2017,, 22,, 14341434 (2 Steps 49%) 15 of 20 (2(2 StepsSteps 49%)49%)

Enzymatic [55,87]

EnzymaticEnzymatic [55,87][55,87] Enzymatic 1515 [55,87] 15 b b 66 22 Steps Steps 21%b 21% 6 2 Steps 21% b commerciallycommercially available available commercially available

6161 62 62 61 62 15 6 55%55% 55% 2 Steps 21% b commercially available (60:40(60:40 α α-1,3/-1,3/αα-1,2)-1,2) (60:40(60:40 αα-1,3/-1,3/αα-1,2)-1,2) a a bb a StartingStarting fromfrom commerciallycommercially availableavailable αα-pNPMan-pNPMan b (6(6);); StartingStarting fromfrom 2,3,4,6-tetra-2,3,4,6-tetra-OO-acetyl--acetyl-ββ-D-D-- aa StartingStarting from from commercially commercially available availableα-pNPMan α-pNPMan (6); b Starting (6); fromb Starting 2,3,4,6-tetra- fromO -acetyl-2,3,4,6-tetra-β-D-mannopyranose.O-acetyl-β-D- mannopyranose.mannopyranose. mannopyranose. TheThe repertoire repertoire of of glycosynthase glycosynthase variants variants is is successfully successfully increasing increasing though though it it is is still still insufficient insufficient FurtherThe repertoire production of glycosynthase of glycosynthase variants variants is successfully of glycosidases increasing with known though substrates it is still willinsufficient be vital forfor creatingcreating thethe highhigh diversitydiversity ofof glycosidesglycosides presentpresent throughoutthroughout Nature.Nature. GreatGreat advancesadvances werewere forfor thecreating progress the ofhigh this diversity method. of Also glycosides progress present in methods throughout for identification Nature. Great and characterizationadvances were acheivedacheived withwith thethe introductionintroduction ofof synthasessynthases derivedderived fromfrom invertinginverting enzymesenzymes andand thethe recentrecent ofacheived glycosynthases with the from introduction mutant libraries of synthases will be derived critical tofrom produce inverting enzymes enzymes with newand acceptorthe recent or glycosynthasesglycosynthases derived derived from from endo endo-β-β-N-N-acetylglucosaminidases-acetylglucosaminidases that that allow allow the the production production of of peptides peptides donorglycosynthases scopes. Many derived advances from endo have-β-N already-acetylglucosaminidases been made such asthat biological allow the and production biochemical of peptides assays withwith defined defined glycosylation. glycosylation. A A larger larger drawback, drawback, howe however,ver, is is still still the the lack lack of of glycosynthase glycosynthase variants variants utilizingwith defined chemical glycosylation. complementation, A larger fluorescencedrawback, howe andver, photometric is still the methods lack of [glycosynthase50,88–90]. Subsequent variants capablecapable of of glycosylating glycosylating non-glycosidic non-glycosidic structures structures and and the the formation formation of of oligomerized oligomerized side side products. products. analysiscapable of glycosylating structural and non-glycosidic mechanistic details structures will help and tothe identify formation structural of oligomerized requirements, side whichproducts. can FurtherFurther production production of of glycosynthase glycosynthase variants variants of of gl glycosidasesycosidases with with known known su substratesbstrates will will be be vital vital be transferredFurther production to new glycosynthases of glycosynthase by genetic variants engineering of glycosidases and rational with known design substrates [28,91]. will be vital forfor the the progress progress of of this this method. method. Al Alsoso progress progress in in methods methods for for identification identification and and characterization characterization of of for the progress of this method. Also progress in methods for identification and characterization of Acknowledgments:glycosynthasesglycosynthasesWe from from gratefully mutant mutant acknowledge libraries libraries will will the be Jürgenbe critical critical Manchot to to produce produce Stiftung enzymes enzymes for a scholarship with with new new (MH). acceptor acceptor This workor or donor donor wasglycosynthases supported by from the DFGmutant within libraries the IRTG will ‘SeleCa-be critical Selectivity to produce in Chemo- enzymes and with Biocatalysis’ new acceptor (grant or number donor scopes.scopes. Many Many advances advances have have already already been been made made such such as as biological biological and and biochemical biochemical assays assays utilizing utilizing IRTG1628).scopes. Many advances have already been made such as biological and biochemical assays utilizing chemicalchemical complementation, complementation, fluorescence fluorescence and and phot photometricometric methods methods [50,88–90]. [50,88–90]. Subsequent Subsequent analysis analysis Authorchemical Contributions: complementation,M.R.H. andfluorescence J.P. wrote theand paper. photometric methods [50,88–90]. Subsequent analysis ofof structuralstructural andand mechanisticmechanistic detailsdetails willwill helphelp toto identifyidentify structuralstructural requirements,requirements, whichwhich cancan bebe of structural and mechanistic details will help to identify structural requirements, which can be Conflictstransferredtransferred of Interest: to to new newThe authorsglycosynthases glycosynthases declare no by by conflict genetic genetic of interest.engineering engineering The founding and and rational rational sponsors design design had no[28,91]. [28,91]. role in the design oftransferred the study; to in new the collection, glycosynthases analyses, by or genetic interpretation engineering of data; and in therational writing design of the [28,91]. manuscript, and in the decision to publish the results. Acknowledgments:Acknowledgments: We We gratefully gratefully acknowledge acknowledge the the Jürgen Jürgen Mancho Manchot tStiftung Stiftung for for a a scholarship scholarship (MH). (MH). This This work work Acknowledgments: We gratefully acknowledge the Jürgen Manchot Stiftung for a scholarship (MH). This work waswas supported supported by by the the DFG DFG within within the the IRTG IRTG ‘SeleCa- ‘SeleCa- Selectivity Selectivity in in Chemo- Chemo- and and Biocatalysis’ Biocatalysis’ (grant (grant number number Referenceswas supported by the DFG within the IRTG ‘SeleCa- Selectivity in Chemo- and Biocatalysis’ (grant number IRTG1628).IRTG1628). IRTG1628). 1. Stevens, J.; Blixt, O.; Glaser, L.; Taubenberger, J.K.; Palese, P.; Paulson, J.C.; Wilson, I.A. Glycan microarray AuthorAuthor Contributions: Contributions: M.R.H. M.R.H. and and J.P. J.P. wrote wrote the the paper. paper. Authoranalysis Contributions: of the hemagglutinins M.R.H. and J.P. from wrote modern the paper. and pandemic influenza viruses reveals different receptor ConflictsConflicts of of Interest: Interest: The The authors authors declare declare no no conflict conflict of of interest. interest. Th Thee founding founding sponsors sponsors had had no no role role in in the the design design Conflictsspecificities. of Interest:J. Mol. The Biol. authors2006 declare, 355, 1143–1155. no conflict [ CrossRefof interest.][ PubMedThe founding] sponsors had no role in the design ofof the the study; study; in in the the collection, collection, analys analyses,es, or or interpretation interpretation of of data; data; in in the the writing writing of of the the manuscript, manuscript, and and in in the the 2.of theBode, study; L. in Human the collection, milk oligosaccharides: analyses, or interpretation Every baby needs of data; a sugar in the mama. writingGlycobiology of the manuscript,2012, 22, 1147–1162.and in the decisiondecision to to publish publish the the results. results. decision[CrossRef to publish][PubMed the results.] 3. Nicolaou, K.C.; Mitchell, H.J. Adventures in carbohydrate chemistry: New synthetic technologies, chemical ReferencesReferences Referencessynthesis, molecular design, and chemical biology. Angew. Chem. Int. Ed. 2001, 40, 1576–1624. [CrossRef] 4. 1.Douglas,1. 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