Advances in Enzymatic Glycoside Synthesis - ACS Chemical Biology

May 13, 2016 - Copyright © 2016 American Chemical Society. *E-mail: [email protected]. Cite this:ACS Chem. Biol. 2016, 11, 7, 1784-1794. Top of Pag...
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Advances in Enzymatic Glycoside Synthesis Phillip M. Danby, and Stephen G. Withers ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00340 • Publication Date (Web): 13 May 2016 Downloaded from http://pubs.acs.org on May 25, 2016

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Advances in Enzymatic Glycoside Synthesis

Phillip M. Danby and Stephen G. Withers Department of Chemistry University of British Columbia Vancouver, B.C., Canada Abstract: A robust platform for facile defined glycan synthesis does not exist. Yet the need for such technology has never been greater as researchers seek to understand the full scope of carbohydrate function, stretching beyond the classical roles of structure and energy storage to encompass highly nuanced cell signalling events. To comprehensively explore and exploit the full diversity of carbohydrate functions, we must first be able to synthesize them in a controlled manner. Towards this goal, traditional chemical syntheses are inefficient while Nature’s own synthetic enzymes, the glycosyl transferases, can be challenging to express and expensive to employ on scale. Glycoside hydrolases represent a pool of glycan processing enzymes that can be either used in a transglycosylation mode or better, engineered to function as ‘glycosynthases’, mutant enzymes capable of assembling glycosides. Glycosynthases grant access to valuable glycans that act as functional and structural probes or indeed as inhibitors and therapeutics in their own right. The remodelling of glycosylation patterns in therapeutic proteins via glycoside hydrolases and their mutants is an exciting frontier in both basic research and industrial scale processes.

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1. Introduction: Technologies to efficiently and reproducibly construct synthetic nucleotides and peptides have trivialized the production of these important biopolymers. By comparison glycomics remains unsupported by any comparable synthetic platforms to make custom oligosaccharides.1 This is not surprising: while the stereo-and regio-chemistry of peptides and nucleotides is largely invariant, the same cannot be said for oligosaccharides. The neglect experienced by glycans can certainly not be attributed to limitations in their biological function, which range from cell wall structure and energy storage, to nuanced cell signalling. Indeed the features that imbue carbohydrates with the structural complexity necessary to carry out these diverse functions are responsible for complicating their synthesis. The rich diversity of possible stereochemistries and regiochemistries is one such challenge. This is further complicated by the fact that individual glycoside hydroxyl groups are more or less chemically equivalent when functioning as nucleophiles, frustrating efforts to control the regioselectivity of glycosylation. As a consequence, chemical approaches generally require multiple protection and deprotection steps to achieve defined oligosaccharide targets.2,3,4,5 While some methodologies have explored the selective glycosylation of partially6 or completely7 unprotected acceptors to obviate such problems, these are currently limited in scope. Automated systems for glycan assembly8 have received great attention, and these certainly give researchers access to defined glycans with a variety of stereochemistries and glycosidic linkages. However, these approaches are inherently limited by the chemistry available, and the fundamental methodology remains inefficient, relying upon multi-step protections and deprotections. Nature has leveraged millions of years of evolution to yield enzymes capable of the precise control in glycosylation that we seek. Unfortunately broad application of the enzymes responsible for biological production of oligosaccharides (the glycosyl transferases) is currently impractical both due to challenges in expressing the membrane-associated enzymes required and because the nucleotide-phosphate substrates employed by transferases are often too expensive to be used at a larger scale. Nevertheless exploiting the substrate specificity and conformational control of enzymatic synthesis is desirable. Glycoside hydrolases (GHs) are responsible for severing the glycosidic linkages that connect sugars to other sugars or other bio-molecules. Based upon sequence similarity these enzymes have been classified into over 120 families compiled into the Carbohydrate Active Enzymes, CAZy, database (http://www.cazy.org/). In most cases hydrolysis is achieved by either double displacement (retaining) or single displacement (inverting) mechanisms, as originally proposed by Koshland9, Figure 1. Beyond this, families differ primarily in their substrate specificities. Application of the substrate flexibility encompassed by these GH families towards the synthesis of glycosides would therefore provide valuable tools for glycan production. The present review focuses on various enzyme engineering approaches that force these hydrolases to betray their natural function and serve as efficient catalysts for glycan synthesis. Enzymatic glycosylation by 2 ACS Paragon Plus Environment

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glycoside phosphorylases represents an alternative and emerging strategy, but is beyond the scope of this review, especially since several excellent reviews cover this material very well.10,11,12

Figure 1. (A) Inverting glycosidase mechanism. Water is activated as a nucleophile for direct displacement of the anomeric substituent. (B) Retaining glycosidase mechanism, wherein a glycosyl-enzyme intermediate is formed that is subsequently attacked by water to complete hydrolysis. This double displacement leads to net retention of anomeric stereochemistry. Pyranoside sugars are represented without hydroxyl substituents for clarity.

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2. Transglycosylation The oldest uses of glycosidases for enzymatic synthesis of glycosides involve either the “thermodynamic approach” in which high concentrations of the two sugars are incubated with the enzyme to give products via simple reaction reversal, or the “kinetic approach” in which a reactive donor sugar is used to generate a high steady state concentration of the glycosyl enzyme intermediate that can then be intercepted by a suitable acceptor substrate (Figure 2). The former approach is typically not very useful since a complex mixture of products is generally obtained. The latter approach offers some control over products formed, but often results in poor yields since the reaction product is still a substrate for hydrolysis.13,14,15 Examples include Shoda’s preparations of cellulose and xylan, using a cellulase as a catalyst for the self-condensation of βcellobiosyl and β-xylobiosyl fluoride.16,17 Although the target polysaccharides were achieved, in both cases product hydrolysis severely limited conversion. Some GHs are preferential transglycosylases, examples being the cyclodextrin transglycosylases that synthesise cyclodextrins from starch18,19, the trans-sialidases from Trypanosome sp. that hijack sialyl moieties from host conjugates20 or transglycosylases such as the xyloglucan endo-transglycosylases that are involved in plant cell wall expansion.21 These all follow the same basic mechanisms as retaining glycosidases, but form a glycosyl enzyme intermediate that does not undergo hydrolysis: the acceptor subsites must be occupied to provide the binding interactions that lower the activation barrier for turnover.13 Inspired by these transglycosylases, researchers have sought to engineer glycosidases to favor synthesis over hydrolysis. Indeed, enzyme engineering has been successfully implemented to enhance the transglycosylation activity of glycosidases and simultaneously attenuate hydrolysis, largely by the group of Tellier, as described below. Random or site directed mutations combined with directed evolution have significantly enhanced the transglycosylation/hydrolysis (T/H) activity ratio of several enzymes.22,23,24 Notably many mutants developed in this way incorporate mutations at conserved residues in the donor binding site. Although the mechanism underlying this effect is not fully understood, researchers have sought to exploit this observation by targeting conserved subsite residues to produce efficient transglycosylase mutants.25 The success and generality of this approach is evident in its application to glycosidases from different GH families. Efficient “trans-glycosidases” have been derived from β-glycosidases25 (GH 1), α-galactosidases26 (GH 36) as well as α-fucosidases27 (GH 29). In the latter study, mutants of α-L-fucosidase BiAfcB (B. longum subsp. Infantis ATCC 15697) were used to produce precursors to human milk oligosaccharides (HMOs) with defined fucosylation patterns.27 Single products were prepared from individual precursors and although the yields were poor (11–21%), reactions were completed with non-activated, natural donors. Other hybrid engineering approaches, supported by in silico methods, show particular promise, yielding trans-glycosylases with unique activity.28 Specifically, iteratively prepared triple mutants of an α-arabinofuranosidase from 4 ACS Paragon Plus Environment

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Thermobacillus xylanyliticus (TxAbf) were capable of producing a target arabinoxylooligosaccharide (70–80% yields under different conditions). In these examples the dominance of the trans-glycosylation pathway was confirmed but at the expense of overall enzyme activities. The key limitation of the trans-glycosylation strategy persists, in that the products of such reactions are also substrates for hydrolysis. A strategy that completely precludes the hydrolysis reaction is therefore desirable.

Figure 2. Trans-glycosylation by use of a β-glycosyl fluoride donor. Pyranoside sugars are represented without hydroxyl substituents for clarity.

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3. Glycosynthases The hydrolytic activity of retaining glycosidases may be completely abolished by direct mutation of the catalytic nucleophile. These “glycosynthase” mutants can perform neither hydrolysis nor transglycosylation on the native substrates. However, when used in conjunction with an activated donor having the opposite anomeric configuration to that of the native substrate, glycosidic linkages may be formed (Figure 3, A). Donors of this configuration mimic the glycosyl enzyme intermediate and take advantage of the vacant cavity created by mutation of the nucleophile. Without risk of ensuing hydrolysis, glycosynthases are the ideal tool for the efficient production of oligo- and polysaccharides. The glycosynthase strategy was first introduced by the Withers group in 1998 using a mutant of Abg (β-glucosidase from Agrobacterium sp.) with its glutamate nucleophile replaced by a catalytically inert alanine.29 Incubation of the Abg E358A mutant with activated donors, αglucosyl fluoride or α-galactosyl fluoride, gave β-(1,4) glycosidic linkages to a variety of monoand disaccharide acceptors. Yields with the galactosyl donor ranged from 66–92%, producing both di and trisaccharides at a multi-milligram scale. Shortly thereafter Malet et. al. presented a second glycosynthase derived from an endoglycosidase, from Bacillus licheniformus E134A, which used an oligosaccharyl fluoride donor.30 Transfer of the α-laminaribiosyl fluoride donor to a methylumbelliferyl glucoside (MU-Glc) acceptor, proceeded with a 90% yield, far in excess of yields achieved using kinetically controlled transglycosylation.31 While the earliest examples of glycosynthases were limited to retaining glycosidases acting on β-glycosides, the scope of the synthase strategy has since expanded to include GHs acting on α configured substrates as well as inverting GHs. Okuyama et. al. prepared the first glycosidase mutant with glycosynthase-like activity towards alpha substrates, using an α-glycosidase from Schizosaccharomyces pombe (SPG), D481G.32 The D481G mutant produced α-linked disaccharides by coupling β-glucosyl fluoride to a pNP-glucoside acceptor. In fact both α-1,6 linked iso-maltose and the α-1,4 maltose derivatives were manufactured, in 41 and 29% yields respectively. The use of alternative donors such as β-glycosyl azides for α-glycosylation by glycosynthases has also been reported33,34 and may prove useful to circumvent the poor stability of β-glycosyl fluorides. The most recent α-glycosynthase, derived from an α-glycosidase from Thermoplasma acidophilum, Ag1A D408G, operates via a double-displacement mechanism wherein an exogenous formate nucleophile (as has been reported previously35,36) first displaces the donor leaving group. The β-glycosyl-formate thus formed is then attacked by the acceptor molecules, resulting in net retention of the α configuration. Interestingly the use of wild type AglA in trans-glycosylation mode proceeded with poor regioselectivity, since α-(1,6), (1,3) and (1,4) disaccharides were produced, while the synthase mutant gave only the α-(1,4) linked product at 38% yield. The difference in outcomes is due to the fact that glycosynthase action is irreversible, thus gives the kinetically controlled product, while transglycosylation can occur 6 ACS Paragon Plus Environment

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repeatedly thus is thermodynamically controlled. An α-L-fucosynthase derived from Bifidobacterium bifidum. (BbAfcBD703S) has been used in the production of Lewis blood group antigens.37 Fucosylation of lacto-N-biose (LNB) and N-acetyllactosamine (LacNAc) by transfer of fucosyl fluoride was completed in 47% and 55% yields respectively, producing trisaccharides that represent two key Lewis antigen epitopes (Figure 3B). In combination with a previously developed 1,2-α-L-fucosynthase,38 access to the full complement of Lewis antigens (Figure 3B) may be possible. A system to site-selectively fucosylate glycoproteins or glycolipids would give control over the antigen presentation that is linked to various biological processes and allow dissection of these events.39 Importantly the glycosidase from which the 1,2-α-L-fucosynthase is derived is not only an alpha glycosidase but also an inverting glycosidase. The transformation of inverting glycosidases to synthases was first reported by Honda and Kitaoka.40 In these cases mutation of either the catalytic base40 or the residue responsible for orienting water as a nucleophile41,42 have both proven to be successful strategies. Both methods require that the native glycosidase employ a Hehre re-synthesis hydrolysis mechanism43,44 whereby the activated donor is first attacked by an acceptor molecule; only then does hydrolysis of this new glycosidic linkage occur. Glycosynthases interrupt this process such that the addition of the acceptor is completed but active site mutations prevent the subsequent hydrolysis. In the 1,2-α-L-fucosynthase discussed above it was not the basic residue that was mutated, but rather an adjacent residue D766 that is responsible for activating the catalytic base (N423).38 Recent attempts to rationally design a GH9 exo-inverting synthase by similar methods resulted in depletion of both synthetic and hydrolysis activities, yielding only an inefficient synthase.45 To realize their full potential glycosynthases must be generated that are able to act on a range of acceptors and donors, concomitant with access to new GH families. Extending this principle to unnatural acceptors such as pseudo-glycosides allows the facile preparation of inhibitors for polysaccharide hydrolases. A series of xylanase inhibitors was prepared using a xylosynthase derived from Bacillus halodurans, Bhx E334G.46 Oligosaccharides prepared from iminosugar and sulphur-linked acceptors included competitive inhibitors of both GH10 and GH11 xylanases of unrivalled potency,47 with Ki’s as low as 18 nM. Non-glycosidic acceptors are also of interest as they pertain to the glycosylation of important bio-molecules. Glycosylated derivatives of glycosphingolipids48, flavonoids49 and steroids50 have all been reported. The self-condensation of donors can be promoted by glycosynthases to make polysaccharides. Synthase mutant, Cel7B E197A (derived from a Humicola insolens endocellulase) was the first shown to catalyze self-condensation of a cellobiosyl fluoride donor to produce cellulose.51 Mutant Cel7B E197A further tolerated amino, bromo and even sulfur substitution at the 6′ position of the cellobiosyl fluoride donors, efficiently forming new functionalized glycans. Recently the use of Cel7B E197A has been revisited to produce azido functionalized polysaccharides, as footholds for further chemical modification.52 The use of engineered enzymes to synthesize glycopolymers has been recently reviewed.53,54 7 ACS Paragon Plus Environment

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Alternatives to glycosyl fluorides as donors are few and far between since space limitations in the active site exclude most other leaving groups. However, recent conversion of a sialidase to a glycosynthase, MvNA Y370G, highlights that this need not always be the case.55 Since the catalytic nucleophile in neuraminidases (also known as sialidases) is a tyrosine, aryl sialoside donors may be used in place of the traditional fluoride leaving groups. Recently Kim and coworkers reported the first conversion of a GH35 hydrolase from Bacillus circulans (BgaC) to a galactosynthase (BgaC-E233G).56 The galactosynthase was shown to glycosylate five aryl glycoside acceptors, producing disaccharides and a single trisaccharide derivative, with yields ranging from 42 to 98%. Specifically, pNP galacto-N-biose (pNP-αGNB) and pNP lacto-N-biose (pNP-α-LNB) were produced in 98 and 97% yields respectively, at a ~10 mg, preparative scale. These important disaccharides are core components found in a range of bioactive molecules including glycosphingolipids57, HMOs58 and tumor associated antigens.59 In all but one case, the desired regio-isomer was obtained, in contrast to transglycosylation studies using the WT enzyme, which produced mixtures.60 Although neither pNP-β-GalNAc nor pNP-β-GlcNAc functioned as acceptors for the mutant, subsequent studies from a different group, also using BgaC-E233G showed that β-GlcNAc was recognized as an acceptor when linked to non-aromatic aglycones of various types, even UDP-GlcNAc.61 Blending the use of glycosynthases with glycosyl transferases can provide access to oligosaccharides or glycoconjugates that are otherwise unavailable from these enzymes alone. These combined strategies have proven useful in the production of glycosphingolipids,48,62 wherein glycosyl transferases were employed to build an oligosaccharyl fluoride donor for a glycosynthase capable of coupling the glycan to various sphingolipids. Alternatively glycosynthases may be used to prepare acceptors for glycosyl transferases, as was shown for the enzymatic assembly of defined fucosylated xyloglucans63 and more recently in the production of the blood group A antigen.64 In the latter example glycosynthase Abg-2F665 was used to prepare a methylumbelliferyl (MU) disaccharide (Gal-β(1,4)-GlcNAc-β-MU) that was sequentially decorated with fucose and N-acetyl galactosamine units by a fucosyl transferase (WbgL)66 and an N-acetyl galactosaminyl transferase (BgtA)67 respectively. The MU-tetrasaccharide prepared is being used as a substrate for coupled assays to identify glycosidases capable of cleaving type 2A blood group antigens from cell surface glycans as part of a program to produce universal donor blood. Glycosynthase BgaC-E233G can be used to transfer galactose onto β-GlcNAc acceptors, thereby producing β(1,3) linked N-acetyllactosamine: “Type 1” LacNAc.61 Subsequent treatment of the LacNAc product with a glycosyltransferase, β3-GlcNAcT, appends an additional GlcNAc foothold suitable for further derivatization by the synthase.68 Continuing with sequential use of the glycosynthase and the glycosyltransferase therefore allows for the formation of poly-LacNAc oligomers containing up to four contiguous Type 1 units. A similar assembly of “Type 2” LacNAc oligomers (Gal-β(1,4)-GlcNAc) was previously reported using both β3-GlcNAcT and 8 ACS Paragon Plus Environment

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another glycosyltransferase: β4-GalT-1.69 Remarkably, the controlled use of the glycosynthase in concert with both the β3-GlcNAcT and β4-GalT-1 transferases has proven a creative route to LacNAc oligomers with alternating Type 1 and Type 2 units (Figure 3B).68 Briefly, a GlcNAc is first decorated with a galactose moiety by either the synthase giving a β(1,3) linkage (Type 1) or by β4-GalT-1 to yield the β(1,4) linkage (Type 2). As before, transferase β3-GlcNAcT then installs a new GlcNAc residue onto the terminal galactose. Alternating the galactosylation catalysts (β4-GalT-1 followed by BgaC-E233G) yielded oligomers with up to four LacNAc units, in an alternating Type 2 – Type 1 pattern. Unfortunately, isolation of products by HPLC was necessary after each monosaccharide extension. Additionally yields decreased as the acceptor grew, from 99% (2 LacNAc units, tetrasaccharide) to 26% (4 LacNAc units, octasaccharide). Glycosynthases therefore offer an efficient and increasingly versatile platform for defined glycan synthesis. Even where glycosynthases cannot yet usurp existing synthetic strategies they can be incorporated to expand these approaches to new glycans or glycoconjugates.

β3

β3 α4

α2

β4

β4

α4

α3

α2

α3

Figure 3. (A) Glycosynthase mechanism. Pyranoside sugars are represented without hydroxyl substituents for clarity. (B) Structures of Lewis antigens; αFuc. refers to alpha fucose. (C) Oligomers with alternating Type 2 and Type 1 LacNAc residues can be prepared by a combination of glycosyl transferase and glycosynthase catalyzed addition.68

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4. Glycoligases An alternative strategy for glycoside assembly with a mutant glycosidase, that is more limited in scope, is to replace the catalytic acid/base residue of a retaining glycosidase. When presented with activated glycosyl donors that do not need acid-catalytic assistance, such enzymes, called “glycoligases” rapidly form the glycosyl-enzyme intermediate (Figure 4) but only undergo useful transfer to an acceptor (in the absence of base catalytic assistance) if either the acceptor is an exceptional nucleophile, or if the intermediate formed is unusually activated. Useful nucleophiles for this purpose are the thiols of appropriately substituted thiosugars, with the first such thioglycoligases being mutants of the β-glucosidase from Agrobacterium sp. (Abg E171A) and the β-mannosidase from Cellulomonas fimi (Man2A E429A).70 Use of 2,4dinitrophenyl β-glucoside (for Abg) and β-mannoside (for Man2A) in conjunction with the cognate thiosugar acceptors provided good coupling yields of typically around 70%, with transfer to the thiosugar occurring about 100 times faster than to its hydroxy equivalent (Figure 4). The approach was extended to several other β-glycosidases and the optimal choice of acid/base substitution explored via site-saturation mutagenesis at the acid/base position, with small, polar side chains proving best.71,72 Applications of the technology have been in rapid assembly of thio-disaccharide libraries as enzyme inhibitors73 as well as in glycoprotein modification, with the thioglycoligase-catalysed formation of metabolically stable thiogalactoseterminated glycans on a neoglycoprotein.74 The strategy was shown to also be applicable to α-glycosidases with the conversion of YicI (an α-xylosidase from Escherichia coli) to an efficient thioglycoligase, YicI D482.75 Usefully one of the thioglycosides synthesized using the thioglycoligase was found to be a potent inhibitor (Ki = 2 µM) of the parent YicI, allowing structural analysis of the Michaelis complex. Interestingly this same mutant, YicI D482A could also employ hydroxyl acceptors, yielding O-α-glycosidic linkages, thus acting as the first O-glycoligase (Figure 4).76 Reaction of α-xylosyl fluoride as donor sugar with either pNP-β-glucopyranoside or pNP-β-mannopyranoside produced single disaccharide products in yields of over 96%. It is assumed that the greater inherent reactivity of the beta-glycosyl enzyme intermediate of alpha-retaining glycosidases is the essential driving force towards transglycosylation.77 In contrast only a fraction of the glycosynthases developed are capable of forming α-glycosidic linkages.32,33,34,38,78,79 In this way O-glycoligases have the potential to provide important complementary activity to their glycosynthase cousins. Kim and coworkers recently reported the second example of an O-glycoligase.80 A cyclodextrin glucanotransferase (GH13) was transformed to an O-glycoligase by mutation of the acid/base residue to alanine, glycine and serine mutants. Several pNP acceptor sugars (alpha and beta, maltosides and glucopyranosides) were used along with α-maltosyl fluoride as the donor. The glycine modification, CGT-E284G, imbued the best transglycosylation activity with no hydrolysis, whereas the serine mutant retained modest hydrolytic activity. This O-glycoligase 10 ACS Paragon Plus Environment

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was used for the glycosylation of ascorbic acid, with the goal of improving ascorbate stability.81 Reactions with this non-glycosidic acceptor were successful at a preparative scale (14.5 mg), albeit with a modest yield of 29%. The same group also showed that Yic1 D482A when incubated with a range of phenols, along with the glycoligase mutant of another GH31 enzyme, the MalA D416A α-glucosidase, successfully converted the α-xylosyl and α-glucosyl fluorides respectively to a series of aryl glycosides in excellent yields, thereby providing an efficient way of synthesising such a series of glycosides for mechanistic studies.82

Enzyme O O F CH3 Enzyme

Enzyme

O

O O

CH3 Enzyme

Enzyme

O X

O O

O

O X CH3 Enzyme

O

X = OH, S-

Figure 4: Glycoligase mechanism: X = S- for thio-glycoligases and X = OH for O-glycoligases. Activated donors rapidly form the glycosyl-enzyme intermediate, this is then attacked by a nucleophilic acceptor. Mutation of the catalytic base prevents hydrolysis of the intermediate. Pyranoside sugars are represented without hydroxyl substituents for clarity.

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5. Endohexosaminidases Accessing the synthetic power of enzymes from disparate glycoside hydrolase families is an ongoing effort, undertaken to expand the range of acceptors and donors available in glycoside synthesis. However, glycosidases from some GH families operate by mechanisms that are significantly different from those discussed above and thus require new strategies to achieve efficient transglycosylation. Among these non-Koshland families, the hexosaminidases from GH18, GH20, GH25, GH56, GH84 and GH85 rely upon a neighboring group participation mechanism83, 84,85 to accomplish the cleavage of glycosidic linkages. Specifically the enzyme activates the acetamido group at the 2 position, promoting intramolecular nucleophilic attack at the anomeric carbon and severing the glycosidic bond with the formation of an oxazoline intermediate that subsequently undergoes hydrolysis (Figure 5A). The endo-β-Nacetylglucosaminidases (ENGases) found in GH families 18 and 8586 have drawn particular interest due to their ability to cleave the GlcNAc-β(1,4)-GlcNAc linkage found in N-glycans.87

Figure 5: (A) Endo-glucosaminide hydrolysis by neighboring group participation (B) Mutant ENGase synthase catalyzed transglycosylation. Pyranoside sugars are represented without hydroxyl substituents for clarity. In 2001 Shoda and coworkers demonstrated that oxazoline derivatives act as donors for ENGase-catalysed transglycosylation.88 A disaccharide (Man-β(1,4)-GlcNAc) oxazoline was coupled to β-pNP-GlcNAc forming a trisaccharide with yields reaching 54%, albeit at a microgram scale. Since these early studies, facile syntheses of the oxazoline donors required for transglycosylation have been reported.89 Remarkably, in many cases oxazoline groups may be installed regio-selectively at the reducing ends of complex oligosaccharides without the need for 12 ACS Paragon Plus Environment

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protecting groups,90 thereby opening the possibility of harvesting N-glycans from convenient “donor” proteins, activating them as oxazolines and transferring them to desired acceptors. The use of wild type enzymes for tranglycosylation is again hindered by subsequent hydrolysis of the products.91 Using mutant enzymes for which hydrolysis is compromised, alongside activated oxazoline donors, Lai-Xi Wang and coworkers were able to demonstrate the first ENGase based synthases, dramatically improving transglycosylation yields.92 Synthase mutants were prepared by mutating the catalytic residue responsible for orienting the acetamide group (Figure 5B). Alternatively the catalytic acid/base residue may be mutated to attenuate hydrolytic activity.93 Several endohexosaminidases (Endo enzymes) from the GH85 family have thus been transformed into glycosynthases: Endo-A94,95, Endo-M96,92 and Endo-D91 isolated from Arthrobacter protophormiae, Mucor hiemalis and Streptococcus pneumoniae respectively. In all cases mutations improved transglycosylation activity and curtailed hydrolysis. One area where ENGases have garnered significant interest is in the convergent synthesis of glycosylated proteins and peptides.86 Over 70% of therapeutic proteins are glycosylated,97 including therapeutic antibodies, which are universally N-glycosylated through asparagine residues.98 Differential activity and potency between glycoforms (proteins with different glycosylation patterns), even those with only subtle modifications, is well documented.99,100 Protocols to homogenize these glycoforms, for use as therapeutics or in studies to explore glycosylation state-function relationships, are therefore highly desirable. Glycan replacement using ENGases begins with deglycosylation of heterogenous glycans using wild type Endo enzymes, leaving the protein bound to a single GlcNAc residue. Incorporation of new, custom glycans, is then completed using Endo synthases in conjunction with oxazoline donors.101,102 This strategy has been successfully applied to the remodelling of antibodies (e.g. IgG-Fc)91, synthetic peptides (e.g. Saposin C)103 and other glycoproteins such as the classic model glycoprotein ribonuclease B.104 Glycan cleavage is not always trivial, and the diversity of glycoforms present may render a single Endo enzyme insufficient to completely cleave all surface glycans, sometimes necessitating the combined use of multiple Endo enzymes.105 The substrate specificities of the available WT enzymes have been summarized in a recent review.86 In the next step, reglycosylation, the choice of individual Endo enzymes for the remodelling of particular proteins is determined by both their acceptor and their donor specificities. Since these specificities can differ from those of the hydrolytic WT parent, a brief outline of the differential donor specificities observed for the synthase mutants studied to date is provided in Table 1. Not included in this list is a newly isolated ENGase enzyme Endo-CC1,106 which has been advanced as a possible alternative to Endo-M. This has been tested in classical transglycosylation mode and the N180H mutant shown to exhibit a similar transglycosylation profile to Endo-M. However this enzyme has not yet been used with oxazoline donors.

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One important limitation of ENGases from GH85 is an inability to act upon substrates that incorporate a fucose at the 6 position of the protein bound “core” GlcNAc unit.107 Though EndoD cleaves linkages to fucosylated GlcNAc proteins108, transglycosylation activity is limited.91,109 Such “core-fucosylated” glycans are commonly found in mammalian glycoproteins. Only in 2011 were GH18 enzymes shown to exhibit transglycosylation activity on N-glycans and revealed to be tolerant of fucosylated GlcNAc derivatives.110 Quickly thereafter the GH18, EndoS (from Streptococcus pyogenes), was transformed into an efficient synthase and remodelling of a core-fucosylated antibody was demonstrated.111 In this study a model IgG antibody, Rituximab, was subjected to Endo-S catalyzed removal of heterogenous glycoforms, leaving only GlcNAc1,6-α-fucose at the glycosylation sites (or GlcNAc alone if treated with a fucosidase). The deglycosylated antibody was then derivatized with a number of complex type oligosaccharides, using Endo-S synthase mutants (D233A and D233Q) and activated oxazoline donors. Specifically, fully sialylated glycoforms, truncated Man3GlcNAc and azido-tagged glycoforms were installed at a multi-milligram scale. Indeed it had been previously reported that Endo-S was able to complete transglycosylation towards homogenous IgG antibodies.112 However, it appears that the efficiency of transglycosylation is dependent on quenching the hydrolytic activity by significantly reducing the reaction temperature.91 Very recently researchers reported the “optimal synthetic glycosylation of a therapeutic antibody” showcasing the full utility of Endo-S.113 Therapeutic antibody ‘Herceptin’, isolated as a mixture of 7 major glycoforms, was targeted for remodelling. To remove heterogenous glycoforms Herceptin was treated with wild type Endo-S leaving the GlcNAc-6-fucose footholds in place (Figure 6A). Subsequent treatment with the Endo-S D233Q mutant, along with a variety of prepared glycan oxazoline donors, yielded homogenous glycosylated antibodies with purities greater than 90%. Several of these donors were tagged through non-reducing end sialic acids, with functional groups useful for bio-orthogonal coupling. These functionalized substrates still served as donors for Endo-S D233Q, albeit with modest losses in efficiency. The utility of these tagged Herceptin antibodies was further demonstrated by linking them (using azide/alkyne click chemistry) to a fluorescent molecule (rhodamine) and, in a separate experiment, a potential treatment module (cemadotin toxin). Derivatized antibodies were then successfully used in fluorescence-activated cell sorting and cell killing of breast adenocarcinoma cells. Finally the authors report a previously undocumented non-enzymatic coupling of oxazoline donors to surface lysine and histidine residues of the antibody (Figure 6B). This unwanted addition leads to uncontrollable glycosylation patterns. Fortunately modifying reaction conditions through the use of higher enzyme concentrations, and lowering the peak concentrations of the oxazoline donor were sufficient to eliminate this “glycation” pathway. The most recently reported Endo synthase is Endo-F3 (a GH18 from Elizabethkingia meningoseptica) expressed as a fusion protein and with mutations D165A or D165Q.114 Although both Endo-S and Endo-F3 synthases are able to process core-fucosylated glycans, Endo-F3 is unique in accepting tri-antennary complex-type glycan donors for transfer to 14 ACS Paragon Plus Environment

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GlcNAc-1,6-α-fucose acceptors. This activity promises to provide access to highly branched glycoforms that may promote improved target binding114 and anti-inflammatory function.115 This brings the total number of ENGase-derived synthases to six, when Endo-CC1 is included. These strategies may also be applied to produce novel synthetic N-glycan derivatives. In one example N-glycan “clusters” were constructed that incorporate multiple core glycan patterns (e.g. both complex and high mannose cores) onto a biantennary, biotinylated glycan backbone.116 Wild type Endo-A and Endo-M N175A synthase were used in tandem, alongside oxazoline donors to install full N-glycan cores directly onto a biotinylated heptasaccharide acceptor. These synthetic N-glycan clusters were deployed in binding assays against an array of 45 immobilized lectins. Lectins recognized and bound clusters containing the core glycan units for which affinity was expected. However, unexpected recognition motifs were also seen, likely as a consequence of both the high density of the branched glycan cores as well as their unique core configurations. Although one would anticipate that the donor and acceptor tolerances of synthase mutants will always match the native hydrolase specificities, this is not universally true. As indicated above Endo-D is able to hydrolyse linkages to fucosylated GlcNAc acceptors108, but this activity is not reflected in its transglycosylation. Interestingly mutation to N322Q restores substrate recognition, thereby allowing for transglycosylation.91 Specificity differences can also be in the opposite sense also, since attempts to transfer high mannose donors to IgG antibodies failed when mutated Endo-A E173H was used117, despite the documented utility of the wild type enzyme in the hydrolysis of high mannose derivatives. More recently the first ever transfer of phosphorylated high mannose glycans to a peptide was completed using ENGase catalysis.118 The incorporation of phosphorylated glycans has been linked to increased protein transport to the lysosome.119 Synthetically prepared high mannose oxazoline donors, functionalized with terminal mannose-6-phosphate moieties, could be transferred using wild type Endo-A (40% yield) but not with the more promiscuous Endo-M; the Endo-M synthase mutant N175Q also proved inadequate. Interesting commentary on the sensitivity of ENGases to both donor modification and enzyme mutation may be found in a recent review.86 Beyond these frustrations, the growing pool of new ENGase enzymes and their successful conversion to synthases continues to expand the range of glycans and their modified versions that can be attached to proteins.

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Figure 6: (A) Remodelling of a Herceptin antibody by a combination of WT Endo-S and Endo-S D233Q.113 (B) Non-enzymatic “glycation” introduces glycans onto surface lysine (or histidine) residues.

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Table 1: Summary of Endo synthase substrate donor/acceptor specificities Oxazoline Donors

Competent synthase

Acceptor with core fucose?

Example citations

Core Glycan

Endo-M

No

92

Endo-A

No

94

Endo-D

Yes

91

Endo-M

No

92, 96

Endo-A

No

104

High Mannose

Hybrid-type

Endo-M

No

120

Endo-A

No

104

Complex-type biantennary

Endo-M

No

94, 96

Endo-F3

Required

114

Endo-S

Required

111, 113

Endo-F3

Required

114

Triantennary

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6. Future Directions The range of glycosynthases currently available is not sufficient to produce the full complement of oligosaccharides that researchers may wish to explore. One approach to expand the pool of useful glycosynthases is to generate them from a wider range of GHs. Efforts to accomplish this rationally are sometimes hindered by the differential substrate specificity and synthetic potential of individual family members. Indeed over a decade elapsed between initial interest121,88 in GH3 and GH18 derived synthases before these activities were finally reported.122,111 Chances of identifying synthetically competent GHs may by improved by drawing on extensive genetic libraries, such as those developed by large scale gene synthesis123 or from metagenomic sources.124 The other approach would be to engineer existing glycosynthases to broaden their specificity and improve activity.65 Either strategy requires high-throughput screens capable of detecting synthase activity from large mutant libraries. Glycosynthase modification through directed evolution already relies upon screening of large mutant libraries. Coupled assays65,125 and ELISA (enzyme-linked immunosorbent assay) plate based screening approaches126 have both proven effective to evaluate the synthetic competency of mutants. In the latter study 10,000 colonies were screened, identifying 130 hits from which 32 new glycosynthase mutants were characterized. The generality of these assays is limited by the need for enzymes or antibodies that specifically bind the synthase products. The renaissance in transglycosylation development from the Tellier lab has been supported by a novel high-throughput screening methodology. A digital screening approach has allowed transglycosylation activity to be assessed directly from colonies grown on agar plates, avoiding enzyme isolation and enabling the high-throughput analysis of mutants.24,127 As this technique relies upon chromogenic glycosyl donors it is unsuitable for the detection of synthases that require fluoride donors. Thus any assay that detects fluoride release could provide a non-specific indicator of synthase activity. One such assay relies upon pH measurement to detect hydrofluoric acid formation during the glycosylation reaction. Although successful, this procedure does not distinguish between hydrolysis and synthase activity.128 Planas and coworkers recently developed a fluoride chemo-sensor assay for the express purpose of evaluating putative synthase mutants.129 In this assay chemical detection of fluoride is effected indirectly using a protected fluorophore: tert-butylsilyl protected methylumbelliferone (MUTBS). Fluoride catalyzed cleavage of MU-TBS generates fluorescent MU, allowing detection of fluorescence in a high-throughput manner. As proof of concept multiple nucleophile mutants of 1,3-1,4-β-glucanase from Bacillus licheniformis were prepared and reacted with αlaminaribiosyl fluoride. Subsequent reaction with MU-TBS monitored by fluorescence identified six mutants with synthase activity: of these three, E134T, E134D and E134C, had not been previously reported. Expanding this assay to larger more diverse libraries would represent an exciting step in acquiring new, useful synthase enyzmes. Combining these approaches with

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emerging technologies, such as microfluidic droplet based platforms,130 may provide a way forward to rapidly generate glycosynthases from diverse GH families.

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Keywords: Glycosidic Linkage – The bond between two discrete sugars in an oligo- or polysaccharide. Glycosidase – A glycoside hydrolase enzyme which cleaves the bonds between sugars. Glycosyltransferase – Enzymes that catalyze the formation of glycosidic linkages using nucleotide-phospho sugar donors. Glycosynthase – Synthetically useful glycosidase mutated to remove catalytic nucleophile. Transglycosylase – Enzymes of the glycoside hydrolase family that interconverts glycosidic linkages rather than hydrolysing them. Thio glycoligase – Glycosidase with catalytic acid/base mutated, capable of forming thioglycosidic linkages. O-Glycoligase - Glycosidase with catalytic acid/base mutated, capable of forming O-glycosidic linkages. Endo-glucosaminidase – A class of glycosidases that specifically acts upon N-acetylated carbohydrates.

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