Chem Soc Rev REVIEW ARTICLE. Enzymatic glycosylation of multivalent scaffolds. Multivalent structures in glycomics

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1 Chem Soc Rev REVIEW ARTICLE Enzymatic glycosylation of multivalent scaffolds Cite this: Chem. Soc. Rev., 2013, 42, 4774 Received 25th September 2012 DOI: /c2cs35395d Pavla Bojarová,z a Ruben R. Rosencrantz,z b Lothar Elling* b and Vladimír Křen* a The design of glycoclusters, glycodendrimers, glycopolymers and other complex glycostructures that mimic the multivalent carbohydrate display on the cell surface is of immense interest for diagnosis and therapy. This review presents a detailed insight into the exciting possibilities of multiple glycosylation using enzymes, particularly glycosyltransferases (EC 2.4). A representative choice of available scaffolds for the enzyme action is practically infinite and comprises synthetic polymers, carbosilane dendrimers, multiantennary glycans or hyperbranched conjugates. The introduced glyco-patterns range from common sialyl Lewis x and sialyl lacto-chains to chemically functionalized carbohydrate units for detection purposes. The possibilities of in vitro enzymatic production of N- ando-glycans and other natural polymers are also discussed. In harmony with their natural tasks, glycosyltransferases may in vitro complete the imperfect glycosylation pattern of proteins, recombinantly produced in pro- and eukaryotic hosts. What is more, the required enzymatic battery may be directly co-expressed with the protein, in order to elegantly accomplish the production of eukaryotic glycans. Ingenious metabolic labeling enables facile imaging of glycostructures. The boom of glycoarray technology opens vast possibilities in high-throughput screening for novel enzymes and substrate specificities as well as in the synthesis. Though there is still a long way until the Nature s ideal of multivalent glycans is achievable in the laboratory, the sketched pathways to multivalent glycostructures open tremendous possibilities for the future glycobiological research. Multivalent structures in glycomics Most natural proteins are covered in covalently linked carbohydrate moieties, which participate in essential in vivo processes like protein folding, fertilization, hormonal metabolism, immune response, cell signalling and proliferation. 1 3 In the laboratory, this multivalent sugar display may be mimicked by preparing glycopolymers, glycodendrimers and glyco-particles with multiple glycosylation sites. Thus, the weak monovalent interaction is augmented in order to produce glyco-therapeutics and diagnostic tools for antimicrobial therapy, targeted drug delivery or cell imaging. 4 6 Contrary to the routine oligonucleotide and peptide synthesis, 7 the preparation of such complex glycomaterials cannot be approached by any generalized protocols and each target molecule represents an individual, often challenging, research project. A plethora of a Institute of Microbiology, Center for Biocatalysis and Biotransformation, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ , Prague 4, Czech Republic. kren@biomed.cas.cz; Fax: ; Tel: b Laboratory for Biomaterials, Institute for Biotechnology and Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University, Worringer Weg 1, D Aachen, Germany. l.elling@biotec.rwth-aachen.de; Fax: ; Tel: Part of the multivalent scaffolds in glycosciences themed issue. P. Bojarová and R. R. Rosencrantz contributed equally to this work. carrier scaffolds are on choice for multivalent display of carbohydrates, ranging from peptides, 8 polymers, 9 oligonucleotides, 10 fullerenes, 11 and calixarenes 12 to dendrimers 13,14 nanoparticles, 15 and arrays. 16 This review aims to present the most important trends and methods in this field, leading to the production of multivalent glycosylation products. It should be noted that the concept of multivalency as used herein is fulfilled by such substrate scaffolds that bear at least two sites prone to enzymatic glycosylation. Generally, it may either be a linear (polymeric) structure with multiple glycosylation sites in the chain, a surface with attached binding precursors or a multiantennary structure. There are numerous methodical strategies for accomplishing elegant synthesis of multi-glycosylated conjugates. Classical organic synthesis, though probably the most universal approach to the preparation of carbohydrates, 17 is often complicated by the tedious (de-)protection strategies; therefore, more straightforward solutions are sought for, which are closer to the one-pot, one-step ideal. From the purely chemical field, these requirements are well met by three popular strategies. These are (i) click chemistry, typically a Cu + -catalyzed 1,3-dipolar cycloaddition of azides to terminal alkynes yielding 1,4-disubstituted 1,2,3-triazoles, which is widely used for the preparation of glycodendrimers; 18 (ii) Ru-catalyzed olefin metathesis, where two alkylene moieties are combined to generate a new C C 4774 Chem.Soc.Rev.,2013, 42, This journal is c The Royal Society of Chemistry 2013

2 Review Article double bond; 19 and, finally, (iii) multicomponent reactions, such as the Ugi-type four-component condensation. 20 Chemoenzymatic approach to multiple glycosylation finds its use especially in the synthesis of glycosaminoglycans and their analogues, for example in the preparation of bioengineered heparin from microbially produced polymer heparosan material. 21 An alternative chemoenzymatic strategy comprises chemical coupling of enzymatically glycosylated adducts to a polymeric carrier, i.e. in the case of neoglycoconjugates carrying a defined number of trisaccharide antigens of Schistosoma mansoni parasite. 22 Besides minor enzyme classes able to catalyse glycosylations, such as polysaccharide lyases (EC 4.2.2), there are Chem Soc Rev primarily glycoside hydrolases (EC 3.2.1) and glycosyltransferases (EC 2.4) acting as enzymatic glycosylation engines. 23 The possibility of purely enzymatic glycosylation of multivalent scaffolds is somewhat limited by the fact that there has not been any such glycosylation so far accomplished through the catalytic action exo-acting glycoside hydrolases or their sitedirected mutants, glycosynthases. In the attempt to glycosylate a multivalent acceptor, glycoside hydrolases tend to selectively glycosylate just one acceptor site and rather build a linear oligosaccharide by prolonging the non-reducing chain at the newly added unit. 23 On the other hand, this property is directly applicable in one-step enzymatic monoglycosylation of multivalent acceptors, thus avoiding any (de)protection steps. 24 Pavla Bojarová graduated at Charles University in Prague, Faculty of Sciences in 2002, and obtained her PhD in biochemistry in For her postdoctoral study she joined Dr Spencer Williams group at the University of Melbourne, to work on the time-dependent inactivation of sulfatases. Since her undergraduate years she has been working in the Laboratory Pavla Bojarová of Biotransformation with Prof. Vladimír Křen, Academy of Sciences of the Czech Republic, Prague. Her main research interests include (chemo-)enzymatic synthesis of complex glycostructures using glycoside hydrolases, and analysis of these enzymes at the molecular level. She is a (co-)author of 26 papers and 4 book chapters. Ruben R. Rosencrantz received a BS degree in Biology in 2009 from the RWTH Aachen University, working on enzymatic syntheses of glycoconjugates. Additionally he received a BS degree in Chemistry in 2010 for his work on glycan-mediated assembly of metal nanoparticles. In 2011 he obtained his MS degree in Biology, working on combinatorial biocatalysis for Ruben R. Rosencrantz the production of glycoconjugates. Currently he is a PhD candidate under the supervision of Prof. Lothar Elling. His research now involves microgel-based glycoconjugates for biomedical applications as well as novel polymer nanoparticles and glycoarrays. Lothar Elling graduated in Biology and received his PhD from RWTH Aachen University. After a post-doctoral fellowship with Professor Maria-Regina Kula, Institute for Enzyme Technology of the Heinrich- Heine-University Düsseldorf in the Research Center Jülich, he headed since 1990 the research group Enzymes in Oligosaccharide Synthesis at the Lothar Elling same institute. Since October 2001 he has been a full Professor at the Institute for Biotechnology, RWTH Aachen University, and Head of the Laboratory for Biomaterials in the Helmholtz-Institute for Biomedical Engineering. His research interests focus on the area of biocatalysis, glycobiotechnology, and biofunctionalization of biomaterial surfaces. Vladimír Křen graduated in Biotechnology and obtained his PhD in Microbiology from the Czechoslovak Academy of Sciences. He spent postdoctoral fellowship with Prof. Crout, University of Warwick, and Prof. Thiem at University of Hamburg and sabbatical as a visiting professor in Japan (Prof. Suzuki). At present he is a Head of Department of Vladimír Křen Biotechnology of Natural Products, a Head of the Centre of Excellence Biocatalysis & Biotransformation at the Institute of Microbiology, Academy of Sciences of the Czech Republic in Prague, and a full Professor of Medicinal Chemistry at Palacky University, Olomouc. His main interests include biotransformation of natural products by enzymes and microorganisms, glycobiology and glycochemistry. This journal is c The Royal Society of Chemistry 2013 Chem. Soc. Rev., 2013, 42,

3 Chem Soc Rev Otherwise, glycosidases are mostly applied in a selective trimming of glycoconjugates. Better results in the area of multiple glycosylations were reached with endoglycosidases and their glycosynthase mutants. 23 In a typical arrangement, proteins are simultaneously glycosylated at several N-glycosylation sites by branched oxazoline glycans, which are further modified by other enzymes directly on the protein. The catalysing endoglycosidases either display negligible or no hydrolytic activity, which protects the resulting glycoprotein from degradation. This topic was thoroughly explored by Wang and coworkers, such as in the synthesis of diglycosylated HIV-1 N-glycopeptides 25 or Fc N-glycans of IgG antibodies. 26 Despite these promising results, the major enzyme class able to perform routine multiple glycosylation are still glycosyltransferases. Glycosyltransferases and in vitro glycosylations Glycosyltransferases (GTs; EC 2.4) 27 catalyze the regio- and stereospecific transfer of sugar units from activated donors to a variety of acceptors, upon creating a glycosidic bond. They are Nature s tools for the close-to-quantitative synthesis of most cell-surface glycoconjugates. 27 The array of acceptor substrates ranges from glycans, lipids, peptides, and nucleic acids to complex natural or synthetic compounds a variability remarkable in the enzymatic kingdom. In the last ten years, exciting results have been accomplished in the understanding of genomics, catalytic mechanism and structure of glycosyltransferases. 27 Their structural diversity is encompassed in the CAZy (Carbohydrate-Active enzymes) database ( 28 based on amino-acid sequence similarities. Here, the class of glycosyltransferases currently comprises 94 entries, grouped in four clans related in the fold and catalytic mechanism. The structure and evolution of GTs are subjected to constant detailed examination. 29 Two main groups of GTs are distinguished that differ in the type of activated glycosyl donor: Leloir GTs processing nucleotide sugars (CMP-, UDP-, GDP- or dtdp-activated) and non-leloir enzymes utilizing either sugar phosphates (glycoside phosphorylases) or even non-activated oligosaccharides (e.g., glucosylor fructosyltransferases). Though the latter group has a good biotechnological potential thanks to the low price of their donors, the limited choice of catalysts available and the side hydrolytic activity somewhat restrict the full exploitation of this type of enzymes. A couple of non-leloir GTs have been reported to glycosylate multivalent acceptors, for instance cyclodextrin glucanotransferase (EC ) and sucrose phosphorylase (EC ), synthesizing a-di-o-glucosides of resveratrol 30 and epigallocatechin gallate, 31 respectively. Potato phosphorylase (EC ) was able to build up amylose chains (a1,4-glc bond) on multifunctional primers bound to a synthetic matrix. In tandem with glycogen branching enzyme (EC , a1,6-glc bond), they constructed extensive hyperbranched glycoconjugates with several arms 32 (Fig. 1a and b) or a dense polysaccharide brush 33 (Fig. 1c). The branching enzyme was also applied in Review Article the synthesis of branched polysaccharides like amylopectin 34 or glycogen. 35 Choudhury et al. 13 presented successful glucosylations of up to 16-armed cellobiose-coated poly(amidoamine) dendrimers (PAMAM) using cellodextrin phosphorylase (EC ) from Clostridium thermocellum. PAMAMs of higher branching were not processed by the enzyme, apparently due to sterical hindrance. The majority of multiple glycosylations described herein are, however, accomplished through the action of GTs of the Leloir pathway. UDP-glycosyltransferases represent more than a half of all known GTs 36 and the majority of in vivo synthetic reactions are performed by the nucleotide sugar dependent Leloir GTs. These enzymes depend on the nucleotide moiety as on a substrate leaving group. Moreover, its presence in the donor molecule ensures conformational changes needed to bind the acceptor substrate. Since the use of expensive sugar nucleotides is limiting for potential scaled-up industrial applications of GTs, multienzyme reactions, ideally in one pot, were elaborated for the generation and in situ regeneration of sugar nucleotides. The first successful use of this concept was demonstrated in production of N-acetyllactosamine (LacNAc; Galb1,4GlcNAc) on a multigram scale (85% yield) using five enzymes besides b1,4-galt to generate UDP-Gal from UDP, the by-product of UDP-glycosyltransferase catalysis. 37 LacNAc synthesis may even be accomplished with three enzymes only, using sucrose synthase and UDP-GlcNAc 4 0 -epimerase for the synthesis of UDP-Gal from sucrose and UDP, 38 and combined with respective GTs (Scheme 1a). 39 Many more combinatorial biocatalytical systems for (re)generation of nucleotide sugars have been developed In combination with cascade reactions of GTs, the synthesis of glycoconjugate epitopes may be achieved (Scheme 1b). 43 Biotransformation processes utilize metabolically engineered bacteria, either as permeabilized or whole cells. 44 For example, CMP-Neu5Ac was generated either by coupling of permeabilized cells of four different E. coli strains resulting in a large-scale production of sialyllactose from lactose 45 or, alternatively, by creating a recombinant superstrain that simultaneously co-expressed CMP kinase, sialic acid aldolase and CMP-NeuAc synthetase. 46 Though biotransformations are undebatably elegant, the in vitro combinatorial multi-enzyme approach seems to offer more flexibility to GT-catalyzed syntheses. Analogous to glycoside hydrolases, GTs may be functionally classified as either retaining (a - a) or inverting (a - b or b - a) enzymes, 47 according to the stereochemistry of the ejected saccharide product. Both inverting and retaining catalytic mechanisms have been proposed; however, only the inverting mechanism has been well described. 47 The true nature of the retaining process still evades complete mapping due to the lack of thorough studies on the roles of the catalytic nucleophile and the glycosyl intermediate. Inverting GTs act via a single displacement mechanism based on a nucleophilic acceptor attack at the donor anomeric carbon somewhat analogous to inverting glycoside hydrolases (Scheme 2a). The retaining mechanism proceeds either through an S N 2 double displacement mechanism including the formation of 4776 Chem.Soc.Rev.,2013, 42, This journal is c The Royal Society of Chemistry 2013

4 Review Article Chem Soc Rev Fig. 1 Hyperbranched glycoconjugates built through the action of potato phosphorylase and glycogen branching enzyme on matrices of (a) amine functionalized polyethyleneglycol, 32 (b) tris(2-aminoethyl)amine 32 or (c) on aminosilanized surface. 33 a covalent glycosyl-enzyme intermediate (Scheme 2b); or, alternatively, the retaining GTs act via an S N 1 transition state where both the donor and the leaving group approach from the same side of the sugar ring (Scheme 2c). 48 The synthetic reactions catalyzed by GTs are typical of exclusive stereoselectivity and regioselectivity and close-to-quantitative yields, not burdened by parasitic hydrolytic activity. In contrast to glycoside hydrolases, GTs are less robust enzymes with a This journal is c The Royal Society of Chemistry 2013 Chem. Soc. Rev., 2013, 42,

5 Chem Soc Rev Review Article Scheme 1 In situ regeneration of sugar nucleotides in GT-catalysis. (a) Synthesis of the Galili-epitope with in situ regeneration of UDP-Gal. 39 SuSy, sucrose synthase; UGE, UDP-Glc 4 0 -epimerase; b1,4-galt, b1,4-galactosyltransferase; a1,3-galt, a1,3-galactosyltransferase; (b) a series of cascade reactions of GTs. 43 Circles indicate multienzyme systems for the (re)generation of nucleotide sugars. 42 rather narrow specificity for both substrates and the type of reaction catalysed (cf. the one enzyme one linkage concept). There is just one known exception to this rule, i.e., the Lewis blood group a1,3(4)-fuct III that fucosylates the GlcNAc unit of LacNAc at either O-3 or O-4, depending on the type of LacNAc (type 1, Galb1,3GlcNAcb or type 2, Galb1,4GlcNAcb). This limited substrate specificity evokes the need to have a large library of enzymes available, in order to cover specific synthetic needs. The variety of synthetic applications of glycosyltransferases is demonstrated in a number of reviews. 26,41,49 In the following sections, we are going to concentrate on the multivalent aspect of GT-catalyzed glycosylations. Natural multivalent scaffolds Complex N-glycan branches N-Glycans and mucin-type O-glycans on glycoproteins represent the classical Nature s ideals of multivalent scaffolds Chem.Soc.Rev.,2013, 42, This journal is c The Royal Society of Chemistry 2013

6 Review Article Chem Soc Rev Scheme 2 (a) Inverting GTs follow a direct displacement reaction mechanism via a single oxocarbenium ion-like transition state, resulting in inverted anomeric configuration. For retaining GTs, two alternative mechanisms have been proposed: (b) a double-displacement mechanism with the formation of a covalent glycosyl enzyme intermediate or (c) mechanism involving S N 1 transition state, in which both the incoming acceptor and the leaving donor approach the same side of the sugar ring. These scaffolds bear a high intricacy in substrate recognition by glycosyltransferases and reveal the not fully understood coding capacities of glycan structures. Besides O-glycosylation, N-glycosylation is the most abundant glycosylation form of proteins, revealing a high variety in multivalency by tightly controlled and complex branching processes. N-Glycosylation is catalyzed by multi-enzyme apparatus residing in the endoplasmic reticulum and Golgi complex and starts with the biosynthesis of a branched lipid-linked precursor glycan Glc 3 Man 9 GlcNAc 2 -PP-dolichol the carrier of a typical core structure of N-glycans. After transfer onto Asn residues of the target protein, within the Asn-X-Ser/Thr recognition sequence, the precursor glycan is trimmed down to the Man 5 GlcNAc 2 -Asn structure (precisely, Mana1,6[Mana1,3]Mana1,6- [Mana1,3]Manb1,4-GlcNAcb1,4GlcNAc-Asn). This is the starting point of the biosynthesis of branched complex N-glycans by more than six N-acetylglucosaminyltransferases (GnTs) (Scheme 3). Each of them shows distinct specificity for a certain mannose residue of a multivalent substrate, which is mainly determined by mannose branch localization and the action of preceding GnTs. The story of revealing the enzymes catalyzing N-glycosylation was published in a review by H. Schachter. 50 Here, we are going to focus on the specificity of GnTs and on the key controlling mechanisms of branching. The GnT-I links a b1,2-glcnac moiety to the a1,3-man-branch of the core Man 5 GlcNAc 2 -Asn substrate (Scheme 4). Interestingly, the catalytic mechanism of GnT-I was recently investigated by a combined quantum mechanical/molecular mechanical approach, which revealed that this inverting and GT-A-folded enzyme Scheme 3 Biosynthesis of branched complex N-glycan products by GnTs. Each GnT transfers GlcNAc in a branch- and linkage-specific manner, yielding different derivatives of the N-glycan core structure., GlcNAc;, Man. binds the acceptor-substrate in a high-energy tensed state. 51 The trimannosyl-core Mana1,3(3,6-O-Me-Mana1,6)Manb structure was used as a minimal acceptor substrate. The molecular model showed the distance between the nucleophile hydroxyl group and the anomeric carbon of the donor substrate to be 2.97 Å, and all substrate hydroxyl groups to be in a tight interaction with the enzyme. Besides the identity of three amino acids (Tyr181, Tyr184 and Phe289) that participate in acceptor recognition and binding via a hydrophobic interaction between the central mannose and the enzyme, the study This journal is c The Royal Society of Chemistry 2013 Chem. Soc. Rev., 2013, 42,

7 Chem Soc Rev Review Article Scheme 4 Substrates utilized by GnTs., GlcNAc;, Man. revealed flexibility in the orientation of the a1,6-linked mannose and its conformational change during the enzymatic reaction. Similar conclusions could be drawn for other inverting glycosyltransferases. Studies on the catalytic mechanism and especially X-ray-assisted modeling approaches unveiled new aspects in understanding substrate specificity and opened the field for designing specific glycosyltransferase inhibitors. 52,53 Following the path to bi-antennary complex N-glycans, GnT-II, closely related to GnT-I, links b1,2-glcnac to the core a1,6-man branch after trimming the GlcNAcMan 5 GlcNAc 2 Asn-structure by a-mannosidase II to the GlcNAcMan 3 GlcNAc 2 Asn-structure. Recently an important aspect of regulation of glycosylation in insect cells was demonstrated in the concerted action of GnT-I, GnT-II and FDL, a b-n-acetylglucosaminidase which produces paucimanosidic N-glycans in insect cells. 54 These three enzymes contribute to a novel branching pathway of N-glycosylation in insect cells regulated by the abundancy and, therefore, ratio of enzyme activities. Furthermore a co-localization of GnT-I and GnT-II in the Golgi apparatus was found, which is also valid for the human homologues. 55 Each fluctuation in expression of GnTs caused a dramatic change in N-glycan composition in cells, especially as GnT-II specifically acts on the GnT-I product. Interestingly, GnT-I from insect cells was shown to transfer GlcNAc onto GlcNAcMan 3 GlcNAc 2 Asn glycan (Scheme 4). 54 The in vitro substrate spectra of recombinant mammalian GnT-I and GnT-II were extensively studied and revealed a remarkable tolerance to chemically modified acceptor substrates. 50 However, these enzymes were not used for the preparative synthesis due to the problems with their recombinant production in insect cells, mainly caused by low activity yields and difficulties to express active forms. 56,57 While GnT-I and GnT-II lay the cornerstone for bi-antennary and multi-antennary N-glycans, the processing by GnT-III yields bisected hybrid N-glycans. The GnT-III transfers b1,4-glcnac to the central mannose of GlcNAcMan 5 GlcNAc 2 -Asn glycan, the product of GnT-I catalysis (Scheme 4). The activity of GnT-III is crucial for controlling N-glycan pathways since the introduction of bisecting GlcNAc inhibits a-mannosidase II and various glycosyltransferases like GnT-II, -IV and -V as well as core fucosylation. Therefore, GnT-III activity is of a high interest in the matters of activity and substrate specificity. In vitro studies revealed that recombinant GnT-III is capable of reversing the 4780 Chem.Soc.Rev.,2013, 42, This journal is c The Royal Society of Chemistry 2013

8 Review Article reaction in the presence of high UDP concentration. 58 With 100 mm UDP, the bisecting GlcNAc was removed from the glycan and UDP-GlcNAc was formed. The substrate specificity within the reverse reaction stayed the same as with the forward type: GnT-III prefers bi-and tri-antennary oligosaccharides and shows reduced activity with tetra-antennary oligosaccharides. 59 Despite the fact that the required UDP concentration of 100 mm for an efficient reverse reaction is never reached intracellularly or within the Golgi apparatus, these findings are relevant for synthetic applications of GnT-III. In vitro activity of recombinant GnT-III is directly related to its specificity for UDP-GlcNAc; however, other UDP-sugar derivatives are bound with similar affinities without being transferred. 60 GnT-III is related to the inhibition of cancer metastasis, which proves the necessity of understanding and utilizing the involved glycosyltransferases. A GnT-III knockdown leads to decreasing amount of bisecting glycan on the one hand, and increased occurrence of b1,6- linked GlcNAc, the product of GnT-V catalysis, on the other hand. This increase of branched structures is related to decreased cell cell adhesion and, in the next step, it is related to tumor metastasis. 61,62 The finding that, by producing the bisected structure, GnT-III may be helpful in inhibiting tumor metastasis, makes this enzyme an interesting target for carbohydrate-based drug research. Notably, GnT-V and GnT-IV activity leads to a higher amount of branched structures, which again promote tumor metastasis. Therefore, the cross-talk of GnT-III and GnT-V may matter in the search after new cancer therapies. This emphasizes the importance of utilizing glycosylation reactions and their enzymes in vitro GnT-IV is special among the GnT enzymes since it exists in at least two isozymes (GnT-IVa, GnT-IVb) occurring in humans. Both forms transfer b1,4-glcnac to the GlcNAcb1,2Mana1,3- branch of bi- and tri-antennary N-glycans. The originated compounds serve as precursors for tetra-antennary N-glycan and as substrates for GnT-V (Scheme 4). 66 Kinetic studies reveal a broad acceptance of multi-antennary N-glycan substrates by both recombinant isoforms. 67 Both enzymes are thus potential candidates for building of multivalent N-glycan scaffolds by enzymatic synthesis. Understanding the in vivo substrate specificities of both GnT-IV isoforms is important for their role in cancer metastasis and diabetic disease. Recently, it was shown that overexpression of GnT-IVa in the mouse hepatocarcinoma cell line Hepa1 6 (with a generally low metastatic potential) leads to a dramatic increase of metastatic capability via increasing the amount of antennary branches and simultaneously decreasing the amount of bisecting GlcNAc both in vitro and in vivo. 68 In contrast, the down-regulation of GnT-IVa expression in Hca-F cells (a mouse hepatocarcinoma cell line with a high metastatic potential) resulted in a lower amount of tetra-antennary structures and a significantly decreased metastatic capability. A possible glycosylation target is CD147. Both GnT-IVa and GnT-IVb utilize the product of GnT-I as a substrate for ongoing glycosylation. Remarkably, GnT-IVa is localized in the gastrointestinal tract whereas GnT-IVb may be found in most tissues. The GnT-IVa isozyme shows 3 6 times lower K m in vitro than the isozyme b. This may Chem Soc Rev insinuate that GnT-IVb is responsible for basal glycosylation processes while GnT-IVa kicks in under certain circumstances to maintain a higher amount of multiantennary N-glycans. Substrate affinity of both isozymes is increased after the action of GnT-I, -II, and -V. 67 The knockout of GnT-IVb led to an increased expression of GnT-IVa and GnT-V, retaining N-glycan branch complexity. In contrast, the knockout of GnT-IVa did not lead to an increasing expression of GnT-IVb, but rather to a diabetic phenotype in the monitored mice. This leads to the question, whether the isoforms utilize different substrates and whether GnT-IVa substrates may not be accessible for GnT-IVb; GnT-IVa is capable of substituting GnT-IVb at least partially. 69 GnT-V transfers b1,6-glcnac onto the a1,6-man branch of the core of bi- and tri-antennary N-glycans with tremendous impact on various other enzymes and on the glycosylation pattern (Scheme 4). In addition, GnT-V is thought to increase metastatic potential of cancer and therefore is a counter player of GnT-III. There are at least two isoforms of GnT-V reported, known as GnT-Va and -Vb. GnT-Vb exists in two spliceisoforms; the 6 bp is expressed in human brain only, and the GnT-Vb +6 splice-isoform is also referred to as GnT-IX. 70,71 GnT-Vb shows 41% identity and 53% similarity with GnT-Va but it has a different catalytic activity, along with ph-optima and substrate preference. While GnT-Vb needs divalent metal ions for catalysis, GnT-Va is active in the EDTA environment. While both isoforms link b1,6-glcnac to the a1,6-mannose branch of the core of bi- and tri-antennary N-glycans, GnT-Vb also glycosylates the a1,3-mannose branch. This branching specificity was observed in vitro after an 8-hour reaction with a bi-antennary N-glycan substrate, and it was probably induced by accumulation of the first intermediate product. GnT-Vb starts to transfer b1,6-glcnac onto the a1,6-man branch, leading to an intermediate tri-antennary structure, which is then glycosylated onto the a1,3-man branch, yielding a tetraantennary N-glycan. 72 An additional aspect of GnT-V activity is the existence of a truncated, soluble form that is regulated by a protease called g-secretase, and it seems to be involved in a novel angiogenesis pathway. 73 GnT-V exhibits high activity in placental trophoblast cells, corroborating the key role of GnT-V and GnT-III in cell adhesion and migration processes, such as increased cell migration accompanying metastasis. These phenomena open new possibilities in the search after therapeutical targets. 74 The last enzyme of the GnT class to be described is GnT-VI, which transfers b1,4-glcnac onto the a1,6-man arm. In vitro analysis revealed that GnT-VI is only efficient on tetra- and triantennary structures (Scheme 4). 75 This emphasizes the fact that for almost all GnTs, the acceptor substrate is rather specific, and must be provided either by deglycosylation of certain proteins or in a chemical way for in vitro syntheses. 76 This substantially limits the applicability of the GnTs in in vitro syntheses of glycan structures. Furthermore, the recombinant expression of many glycosyltransferases is a complex task. Only few of them may be expressed in an active form in the E. coli host. Other protein expression systems like insect cells or yeast cells give better results but the number of commercially This journal is c The Royal Society of Chemistry 2013 Chem. Soc. Rev., 2013, 42,

9 Chem Soc Rev available glycosyltransferases is still somewhat limited. 77 As soon as substrate limitations and inactive expression are overcome, the in vitro glycosylation and, possibly, the building of diverse artificial multivalent scaffolds for binding and recognition studies will gain depth and complexity. An outstanding example of efficient expression and characterization is GnT-VIII. This recombinant enzyme was expressed in Pichia pastoris and fully characterized. 78 It transfers b1,3- GlcNAc preferably onto tetra- and 2,6-branched tri-antennary structures (Scheme 4). Interestingly, by mixing GnT-VIII with GnT-II, the enzymatic activity was dramatically enhanced; V max /K m increased 9.3- to 160-fold in comparison to individual enzymes. The formation of a hetero-complex was suggested the first case reported for in vitro mixed glycosyltransferases. This work may be a good starting point for optimizing expression of glycosyltransferases for in vitro use. Another enzyme class important for N-glycosylation are galactosyltransferases (GalTs). Branch specificity of bovine colostrum and calf thymus b1,4-galt was investigated as early as in the 1980s, revealing preference ratios from 1.6 : 1 for the a1,3-branch of GlcNAcb1,4(GlcNAcb1,2Mana1,6)(GlcNAcb1,2- Mana1,3)Manb1,4GlcNAc up to 3.7 : 1 for the non-bisecting structure or 1 : 3.5 for the b1,6-branch over the b1,2 branch of GlcNAcb1,6(GlcNAcb1,2)Man. This study revealed a specific order of maturing N-glycosylation. 79 The Ehrlich tumor cell a1,3-galt also showed a certain branch specificity for the a1,6-man branch, which increased to 13-fold for tetra-antennary structures. 80 The incorporation of galactose into a specific branch alters the substrate usability of GnTs or other enzymes involved in N-glycan synthesis. A large study on branch-specific galactosylation of six recombinantly expressed human GalTs demonstrated that b1,4-galt-i prefers a GlcNAcb1,2Man branch to a GlcNAcb1,6Man branch for a1,3-mannosylation. For b1,4-galts-iv, -V, and -VI the opposite result was obtained whereas no branch preferences could be determined for b1,4-galt-ii and -III. 81 These results open the question, how the galactosylation is regulated and what the biological impact of a certain galactosylated structure is. Some GalTs are associated with tumor metastasis but there is still little known about the impact of individual enzymes. They exist in varying amounts within various tissues; however, tissue-specific galactosylation may not be the only reason for this delicate concerto of specific galactosylation. 82 The impact of a variety of galactosylation patterns, specific preferences of the involved enzymes and their abundancy do matter when recombinant production of immunoglobulins is considered. It was shown that the glycosylation pattern is species-specific and depends on the branch specificity of GalTs. As the proper glycosylation is crucial for the bioactivity of IgGs, alterations could substantially affect their applicability. 83 In the case of elongation of N-glycan branches with poly-nacetyllactosamine (poly-lacnac), opposite substrate specificities of the involved enzymes were reported. 84 In the synthesis of linear poly-lacnac (i-antigen), b14-galt-i and the extension enzyme (ignt) favored different branches: poly-lacnac was formed more efficiently on Galb1,4GlcNAcb1,2Mana1,6-R than Review Article on Galb1,4GlcNAcb1,6Mana1,6-R. ignt showed preference for the b1,2-linked substrate while b1,4-galt-i preferred the b1,6- linked acceptor. This is somewhat in contrast to the finding that b1,4-galt-i showed higher turnover of b1,2-linked GlcNAc moieties compared to b1,6-linked ones. 81 Nevertheless, in this case acceptors with a1,6-linked mannose were utilized. 84 Furthermore, the poly-lacnac synthesis was investigated with Gal-terminated acceptor substrates; therefore the step catalyzed by ignt was rate-determining. Pre-galactosylation yielding a partially galactosylated substrate (Galb1,4GlcNAcb1,6(GlcNAcb1,2)- Mana1,6Manb-) resulted in a homogenous formation of poly- LacNAc on both branches. Scheme 5 shows an example of how the outcome of enzymatic reaction may be controlled by varying the ratios of enzymes with different substrate specificities. Thus, homogeneous poly-lacnac formation was achieved on both arms as well as the preference for the b1,6-branch. The differing enzyme preferences are important in the early stage of synthesis. The observed more abundant occurrence of poly-lacnac substitution on the b1,6-branch (GnT-V) is attributed to the branch preference of b1,4-galt-i, which subsequently creates the substrate for ignt. At low enzyme activity of b1,4-galt-i, poly-lacnac is preferentially elongated. Higher b1,4-galt-i activity leads to the galactosylation of the b1,2- GlcNAc branch. This results in balanced poly-lacnac synthesis on both branches, which is initially attributed to the b1,2-branch specificity of ignt. A recent work aided by X-ray crystallography revealed distinct changes in branch specificity of b1,4-galt-1 depending on concentration. 85 At lower acceptor concentrations, GlcNAcb1,2Mana1,6- is preferred to GlcNAcb1,2Mana1,3- while this preference inverts at higher concentrations. This may support the fact that even the mannose linkage has an impact on substrate preference and, therefore, may explain the contrary findings mentioned above. 81,84 Additionally, X-ray analysis and a modeling study demonstrated a new binding mode for b1,6-glcnac-configured oligosaccharide substrates of b1,4- GalT-I. 86 A secondary binding site may lead to a concentration-dependent substrate preference. In summary, we may conclude that the concerted action with other GalTs is necessary to ensure the galactosylation of multi-antennary N-glycans; nevertheless, the branching preference of the involved enzymes is a complex action and is not only dependent on the linkage of the nearest saccharide. Recent years have seen the unveiling of new aspects of how the N-glycosylating enzymes interact with their multivalent substrates and control the glycosylation pattern. The useful examination tools comprise modeling studies together with crystallographic analysis of increasing resolution. Two distinct fields are covered: (i) biological impact of glycosylation and the failure thereof; (ii) understanding of catalytic processes. The combination of both of them will lead, together with better availability of substrates and enzymes, to a high impact of N-glycosylation research on practical applications. Mucin O-glycans The prevalent type of O-glycosylated proteins in mammals are mucin type glycans. They typically comprise a GalNAca1-OR 4782 Chem.Soc.Rev.,2013, 42, This journal is c The Royal Society of Chemistry 2013

10 Review Article Chem Soc Rev Scheme 5 Branch specificity of b1,4-galt-1 and ignt for poly-lacnac synthesis on complex N-glycans. Poly-LacNAc is more readily incorporated onto the b1,6-branch, due to the branch preference of b1,4-galt-i. A homogenous poly-lacnac formation may be reached by elevating activity of b1,4-galt-i (dotted pathway)., GlcNAc;, Man;, Gal. pattern attached to a Ser or Thr residue, which leads to multivalency within a protein scaffold. Detailed reviews summarize the biochemical and physiological characteristics of mucin-type O-glycan biosynthesis O-Glycosylation is closely associated with diverse biological events like embryonic development, cell adhesion processes, cardiovascular risk or even cancer. Thus, studying processes and regulation of this type of glycosylation may result in pathways to carbohydrate-based drugs and vaccines. 92,94 97 Therefore, sophisticated synthesis strategies are needed for enzymatic synthesis of O-glycan multivalent scaffold using glycosyltransferases. The enzymes initiating O-glycosylation are polypeptide GalNAc transferases (ppgalnact). The number of enzymes within this class has been set to over 20. All of them show distinct, sometimes overlapping, substrate specificity, which is crucial for understanding O-glycosylation and predicting glycosylation sites or disease-associated alterations. 88, In vitro glycosylation of a random peptide carrying multiple putative O-glycosylation sites may be separated into two crucial steps: (i) peptide-sequence specific glycosylation; (ii) preglycosylation-dependent follow-up glycosylation (Scheme 6). Understanding peptide-sequence specificities and pre-glycosylation preference of ppgalnacts is mandatory for in vitro glycosylation of peptides and will be described in more detail. Initial peptide glycosylation. Initial glycosylation and peptide specificity of ppgalnacts are mainly associated with the amino acids neighboring to the putative glycosylation site of a Ser/Thr residue. A C-terminal TP(G/A)P-sequence seems to be determining for substrate binding of many ppgalnacts, most probably via interaction with a highly conserved Trp/Phe within the catalytic domain. 102 However, ppgalnact-10 does not contain these conserved residues and, as a result, shows no preference for glycosylation sites flanked by the TP(G/A)P-sequence. It was investigated to which extent certain amino acid residues neighboring to the peptide glycosylation site enhance substrate turnover rates with some ppgalnacts. 102 It turned out that three neighboring residues are highly involved in recognition by the enzymes. N-Terminal Pro at position 1 from the targeted site greatly enhances the affinity for ppgalnact-2, whereas ppgalnact-1 and -3 prefer Val at this position. Additionally, the overall charge of the peptide chain consisting of 20 amino acids influences substrate recognition: while ppgalnact-3/5 utilize more overall cationic sequences, ppgal- NAcT-1/2 favor acidic peptides. This correlates with the IP of the catalytic domains and could be related to a ph-dependent glycosylation control mechanism. These findings lead to a better predictability of an overall glycosylation status of peptides within in vitro glycosylation (Scheme 6). This journal is c The Royal Society of Chemistry 2013 Chem. Soc. Rev., 2013, 42,

11 Chem Soc Rev Review Article Scheme 6 Initial and follow-up glycosylation of a random peptide with multiple glycosylation sites by various ppgalnacts. The asterix indicates the second S/T in a sequence of S/T-S/T-S/T. S, Ser; T, Thr;, GlcNAc;, GalNAc;, Gal. A random peptide substrate library was used to study the impact of the neighboring residues other than the identified consensus motif. 103 Combination of lectin affinity chromatography and Edmann sequencing of fully soluble substrates revealed that ppgalnact-1 activity is enhanced by hydrophobic residues, except for Leu, at position 1, and inhibited by charged residues at this position. Furthermore, Pro/Ala at position +2 show enhancing effects, as well as Pro at position 1, +1 or +3. The optimized peptide sequence for ppgalnact-1 was determined as (Phe/Asp)(Phe/Ala)(Pro/Val)ThrPro(Gly/Ala)- Pro. In contrast, the ppgalnact-2 shows differences in preference at positions 1, +3 and 3. Nevertheless, the optimized peptide sequence ((Pro/Ile)GlyProThrProGlyPro) carries the motif ProThrProGlyPro, which seems to be common for ppgal- NAcTs utilizing nonglycosylated substrates. 104 ppgalnacts show altered activity with pre-glycosylated substrates. 105 The influence of mono- and disaccharide modified glycopeptides was investigated and analyzed in terms of altered site-specific glycosylation. In this study, recombinant ppgal- NAcT-1, -2 and -3 were used. The results showed that the non-glycosylated peptide substrates gave significantly different glycosylation pattern in comparison to the partially glycosylated ones. In addition, the choice of ppgalnact also influenced the glycosylation pattern. Different ppgalnacts addressed different sites and some of them (ppgalnact-4 and -10) showed almost no activity with non-glycosylated peptides, which makes them essential for controlled follow-up glycosylation procedures. 106 Especially ppgalnact-10 has some striking features as it shows very low activity for non-glycosylated peptides; in fact, UDPdonor hydrolysis was faster than the transfer reaction, and great 4784 Chem.Soc.Rev.,2013, 42, This journal is c The Royal Society of Chemistry 2013

12 Review Article activity enhancement by neighboring glycosylation was observed. 107 Interestingly, the activity could not be enhanced by certain amino acids flanking the glycosylation site. Additionally, a binding site for GalNAc-O-Ser/Thr could be found within the catalytic domain, which is unique among the family of ppgalnacts. This finding shows a novel recognition pattern of glycopeptides besides the lectin domain mediated way as discussed below. In the light of these results, ppgalnact-1,-2,-3 and -5 seem to be the most promising tools for initial glycosylation of a peptide scaffold with multiple glycosylation sites. The exact choice depends on the overall charge of the desired peptide (Scheme 6). Follow-up glycosylation. ppgalnacts exhibit an outstanding structural feature. In addition to the N-terminal catalytic domain, mainly involved in the initial, peptide sequence dependent glycosylation, these enzymes have a C-terminal lectin-like domain (similar to ricin) which is involved in glycopeptide specificity, and therefore follow-up glycosylation processes. 108 To obtain a dense O-glycosylation on a random peptide with multiple glycosylation sites in vitro, the lectin domain is one of the key features (Scheme 6). Evidence for the influence of the lectin domain in in vitro glycopeptide specificity was found with ppgalnacts-4 and These enzymes selectively act on glycopeptides and the single amino acid exchange Asp459His in the lectin domain erased not only lectin function of the domain, but also the ability to perform follow-up glycosylations. However, the activity for non-glycosylated peptides was not altered. Moreover, glycopeptide activity could be inhibited by adding high concentrations of free GalNAc sugar and incorporation of core 1 (Galb1,3GalNAca1-O-S/T) structures. This finding supported the hypothesis that the lectin domain specifically recognizes GalNAc transferred by ppgalnacts. In addition, these results also reveal core 1 glycosylation to be a regulative element in O-glycosylation, by competitive action of individual enzymes (Scheme 6). The lectin domain is reported to be important for maintaining dense glycosylation within proteins or peptides carrying multiple glycosylation sites. 110 This cannot be overrated if a heaped in vitro O-glycosylation of a peptide is favored. Interestingly, for ppgalnact-1, not known for high glycopeptide specificity and reported to possess an inactive lectin domain, newer studies reevaluated its status and reported a complete loss of enzyme activity after removal of the lectin domain. 111 This was expanded to the suggestion that ppgalnact-1 may catalyze two distinct reactions: on the one hand, the peptide sequence dependent initial glycosylation; on the other hand, the lectin or previous glycosylation dependent follow-up glycosylation reactions; the latter only within proteins with multiple glycosylation sites. Recently the lectin function of various ppgal- NAcTs was investigated by means of a glycopeptide-array and bead library. 112 A large number of glycopeptides based on sequences from MUC1, MUC2, MUC4, MUC5AC, MUC6 and MUC6 were tested with ppgalnacts-1-4 addressing the lectin domain specificity for certain glycosylation patterns and peptide sequences. For this in vitro approach, recombinant ppgalnacts expressed in insect cells were used. To make sure that the interaction is based on lectin mediated binding, UDP-donor Chem Soc Rev and the necessary divalent metal cation were excluded. This ensured that the catalytic domain was not involved in binding events. Additionally, all interactions were proven to be reversible or inhibitable by the addition of 250 mm GalNAc therefore, the binding was based on the glycan protein interaction mediated by the lectin domain. The combined results suggest that the lectin domain is not only involved in glycan binding but, moreover, it prefers different peptide sequences. Lectin binding was proven to be ph or charge dependent; therefore ph-mediated control of glycosylation is conceivable. These findings are striking because now it becomes clear that substrate specificity is not only related to recognition by the catalytic domain but especially the follow-up glycosylations are mediated by peptide sequence and the glycan specific lectin interaction. This enables ppgalnacts to utilize a glycosylation site immediately adjacent to the pre-glycosylation site. However, this is in contrast to the modeling approaches that suggested a certain distance between recognized glycan and catalytic domain due to the distance between the catalytic and lectin domains. 108 Further investigation is ongoing to reveal the lectin functions by comparing fulllength ppgalnacts with lectin domain deficient ones utilizing unnatural glycopeptide substrates. 113 An interesting study on the in vitro follow-up glycosylation of small glycopeptide fragments derived from MUC2 revealed the delicate concerted actions of ppgalnacts. 114 The peptide fragment ProThrThrThrProLeuLys was used as an acceptor substrate and the Thr-residues were partially glycosylated with GalNAc or core 1 (Galb1,3GalNAca1-O-Ser/Thr). This revealed site specificity of tested ppgalnacts: ppgalnact-4 utilized preferably sites one and three whereas ppgalnact-1 and -2 glycosylated clearly site one, and ppgalnact-3 transferred GalNAc onto site three. Remarkably, glycosylation of one site could enhance activity of ppgalnacts. Incorporation of GalNAc at site three improved dramatically the glycosylation rate in the other sites for ppgal- NAcT-2, -3 and -4. Expanding the glycosylation on site three to core 1 structure, the enhancement effect was totally extinct. Interestingly, the incorporation of GalNAc at site one did not show any enhancing effect. Moreover, ppgalnact-2 transferred GalNAc onto site two only with GalNAc previously added to site three (Scheme 6). These findings suggest a highly active and occurrence-based regulated O-glycosylation process where initial glycosylation has a tremendous impact on follow up glycosylation as well as on site specific glycosylation. To sum up, if a dense glycosylation is required starting from a preglycosylated peptide scaffold, ppgalnact-2, -5, -7 or -10 should be considered, as they exhibit outstanding performance in follow-up glycosylation. (Scheme 6). Additional influence on glycosylation-dependent ppgalnact activity may be found in altered peptide conformation caused by the initial glycosylation. 115 This was found by combining MS-aided identification of preferred glycosylation sites with chemical synthesis of glycopeptides (derived from tandem repeat sequences of human mucin glycoproteins MUC4 and MUC5) and NMR-aided peptide conformation analysis. A conformational change responsible for better or worse binding of certain ppgalnacts to the peptide sequence was suggested. This journal is c The Royal Society of Chemistry 2013 Chem. Soc. Rev., 2013, 42,

13 Chem Soc Rev Previous studies revealed conformational changes in MUC1 tandem repeats that may be related to altered ppgalnact-1/- 2/-4 activity. 116 For these three enzymes a conformation that enhances activity could be defined. These conformational changes are caused by glycosylation and recognized by the catalytic domain independent of interactions between sugar residues and the lectin domain. There are other enzymes besides ppgalnacts involved in mucin type O-glycosylation with striking specificity for glycans, peptide sequences and core structures For example, adjacent Pro, Lys and Arg residues inhibit formation of core 1 structures in a peptide scaffold whereas Glu, Gly and Asp seem to enhance the action of the b1,3-galt core 1 enzyme. Furthermore, a1,2-fucosylation is more prominent on Ser-linked glycans than in Thr-linked ones. These findings are applicable in the design of carbohydrate based therapeutics Nevertheless, the specificities of these enzymes regarding peptidesequence are clearly of a more subtle matter in comparison to sequence specificities of ppgalnacts, which are crucial in peptide glycosylation, especially for in vitro applications. Combining peptide and pre-glycosylation specificities of the ppgalnact family, various glycosylation patterns from spatial to dense glycosylation are accessible. With better understanding of their preferences and regulation mechanisms, these enzymes may ensure successful in vitro glycosylation of multivalent scaffolds like peptides or peptide-like structures. Other natural multivalent acceptors Besides the multiple glycosylation of O- and N-glycans described in the previous sections, there are other natural scaffolds processed by GTs. One of the typical multivalent glycosylation patterns synthesized by GTs is the sialyl Lewis x epitope (sle x ; Neu5Aca2,3Galb1,4(Fuca1,3GlcNAcb1-O-), such as that demonstrated by Furuike et al. 124 Here, a set of seven sle x tetrasaccharides were constructed by enzymatic galactosylation, fucosylation and sialylation of core GlcNAc units chemically attached to b-cyclodextrin. Zou et al. 125 showed a different approach, obtaining multivalent lactoside units by controlled Smith degradation of Streptococcus capsular polysaccharide GBSIa. This is a simple way to defined multivalent oligosaccharidic epitopes, which are otherwise built up from monomer blocks. The obtained bi- to pentavalent lactoside scaffolds were selectively fucosylated, and the resulting sle x/a oligomers were suitable for further potential modifications (sialylation). The sle x pattern was also chemo-enzymatically introduced to the polymeric scaffold of chitosan, 126 on the route to selectinbinding anti-inflammatory agents (Fig. 2a). The impressive 75% overall yield was accomplished over four steps, out of which three were enzymatic (b1,4-galt, a2,3-siat, a1,3-fuct). Incorporation of sialylated structures such as sialyllactose 127 onto the chitosan chain (Fig. 2b) is an important step in the design of inhibitors of influenza virus, based on binding to hemagglutinin, the viral surface protein. 128 Moreover, an insoluble sialyllactose-functionalized chitosan fibre 127 proved to efficiently remove the virus from media (Fig. 2c), which opens immense applications for the construction of virus filters in face mask, air conditioners, air cleaners etc. Chitosan is an especially suitable scaffold due to its biocompatibility, biodegradability, antimicrobial character and easy availability. Advantageously, GTs responsible for the glycosylation of the natural polymer chain may be heterologously expressed in the production host. In the work by Anders et al. 129 wheat and rice glycosyltransferases of family 61 expressed in Arabidopsis thaliana introduced a1,3-arabinosylation of xylan. Since the degree of xylan arabinosylation directly influences the fibre solubility, the tailoring of arabinoxylan biosynthesis is applicable in, e.g., improving the digestibility of lignocellulose for biofuel, feed and other uses. Multiple glycosylation is not necessarily limited to polymeric scaffolds Ahn and coworkers 130 have shown di-o-glucosylation of flavonoids, especially of kaempferol, using recombinant UDP-glycosyltransferase from Bacillus cereus. Naturally or synthetically produced glycopolymers and glycoconjugates may be subsequently modified on the carbohydrate chain by other enzymes than glycosyltransferases, to accomplish changes like epimerization, O-acetylation, O-sulfation, O-methylation, N-deacetylation or N-sulfation. These so-called carbohydrate post-glycosylational modifications (PGMs), as coined by Chen in 2007, 131 are vital for the synthesis of complex glycostructures. The new modifications may complete the existing pattern or they may be incorporated de novo, e.g., after production of the carbohydrate material in microbial strains. This is especially useful in the synthesis of glycosaminoglycans (GAGs), 132 such as chondroitin sulfate, heparin, hyaluronan or keratan sulfate. GAGs are linear heteropolysaccharides composed of repeating disaccharide units, which always include an amino sugar derivative (GlcNAc or GalNAc) and mostly a uronic acid (IdoA or GlcA). The carbohydrate units are often modified, especially sulfated. There are numerous works devoted to, e. g., enzymatic sulfation 133,134 and deacetylation 135 of GAGs, especially of the heparin/heparan sulphate/ heparosan type. Advantageously, all regioselective sulfotransferases concerned may be heterologously co-expressed in one host, such as Kluyveromyces lactis, and used without purification. 136 Linhardt and coworkers 137 designed an artificial analogue of the Golgi organelle, in which D-glucosaminyl 3-Osulfotransferase sulfated a heparan sulfate chain immobilized on magnetic nanoparticles. Functionalization of a carbohydrate chainmayproveveryusefulfordetectionmethods,suchasinthe case of lipase-catalyzed esterification of starch and subsequent fluorescent labeling or biotinylation via click chemistry. 138 Synthetic multivalent scaffolds Review Article Glycosylations on multivalent surfaces This part will cover the use of glycosyltransferases for glycosylation of solid multivalent synthetic scaffolds, mainly arraybased. Although enzymatic glycosylation of solid substrates may suffer from some drawbacks like accessibility and sterical hindrance, there are elegant examples of utilizing glycosyltransferases with arrays of wide biotechnological applicability. 139, Chem.Soc.Rev.,2013, 42, This journal is c The Royal Society of Chemistry 2013

14 Review Article Chem Soc Rev Fig. 2 Functionalized chitosan conjugates: (a) synthesis of sialyl Lewis x -chitosan; 126 (b) sialyllactose-chitosan; 127 (c) inhibition of influenza virus by fibrous sialyllactosechitosan. 127 Adapted with permission from ref. 127 r (2011) American Chemical Society. The enzymatically attached carbohydrate units are marked in color. Two types of this design are distinguished: array-aided substrate or inhibitor screening with GTs, and GT-catalyzed synthesis for lectin ligand preference analysis (Scheme 7). The array technology has greatly contributed to the introduction of high-throughput technology to biological research. While DNA- and peptide arrays are known and used for quite some time, glyco-arrays are rather at the dawn of usage. Well established in this field are the glycoarrays provided by the consortium of functional glycomics (CFC), which delivers arrays for protein carbohydrate interaction screening. Regarding the current mammalian version of the arrays, over 600 glycans are immobilized and are usable for screening of binding specificities The reasons for utilizing immobilized biomolecules on chips are the same for all array-types: fast screening of a molecule library, possible robotics-aided preparation, and easy handling. 144,145 Glyco-arrays provide information about protein glycan binding events, substrate recognition and specificity or they find use in disease-related research. 146,147 This journal is c The Royal Society of Chemistry 2013 Chem. Soc. Rev., 2013, 42,

15 Chem Soc Rev Review Article Scheme 7 Screening and glycosylation of multivalent substrates with GTs on arrays. Glycan immobilisation on the array is controllable from the viewpoints of glycan sequence, glycan density and glycan surface distance. Arrays equipped with multiple types of glycans serve for the screening of GTs and their substrates. Enzymatic modification of array-bound glycans affords a multivalent ligand library for lectin binding studies. Analysis of turnover is carried out by SPR, MS-aided methods or detection of labeled lectins. There are multiple methods reported for designing glycan arrays, mostly based either on covalent ligand binding or on hydrophobic interactions Outstanding is the use of selfassembled monolayers (SAMs) providing a high amount of controllable functionalization for varying ligand density (Scheme 7). 151 Substrate and inhibitor screening for GTs. The ability of screening a vast number of substances in a fast way is among the chief purposes of array technology. Thus, inhibitor or substrate screening may be carried out using carbohydrate arrays. For example, inhibitors of a1,3-fuct were screened on an array with the acceptor substrate (LacNAc) immobilized via non-covalent lipid interaction. 152 The common inhibitor motif was a triazole-group at the O-GDP site. FucT inhibitors are especially sought after because the resulting sle x structure is a known inflammation mediator. Detection was carried out with a fluorescence-labeled fucose-dependent lectin. A simple approach for studying lectin binding, enzymatic glycosylation and inhibition of fucose transfer was carried out in a microtiter plate based array. 153 Eleven oligosaccharides were immobilized with a C14-spacer and attached to the surface through hydrophobic interactions. Lectin binding assays as well as enzymatic synthesis of sle x and sle a were carried out. Additionally, inhibition of a FucT utilizing an anhydro-donor-analogue was investigated. Substrate screening was carried out for multiple enzymes, ranging from GTs involved in plant cell-wall synthesis to recombinantly expressed FucT, able to bis-fucosylate synthetic N-glycans. 154,155 The bis-fucosylation ability was studied with a1,3- and a1,6-fucts and eighteen synthetic N-glycans. The scope of fucosylation was detected with respective 4788 Chem.Soc.Rev.,2013, 42, This journal is c The Royal Society of Chemistry 2013

16 Review Article lectins, revealing a broad substrate specificity of the FucTs tested. More than 10 out of 18 screened substrates were processed by the enzymes. Additionally, the study brought new insights into lectin preferences. Various peptides were screened to define preferred sequences of ppgalnact-2, b1,4-galt-1 and proteases with non- and pre-glycosylated peptides. 156,157 The substrates were immobilized on arrays via SPOT-synthesis and SAMs and analyzed by surface MALDI-TOF. In another study, the substrate promiscuity of SiaTs was revealed in a high-throughput screening on arrays. 158 A modified donor substrate labeled with biotin was transferred by five different mammalian SiaTs onto various acceptors. The product formation was monitored with a fluorophore-labeled streptavidin conjugate. Furthermore, the readily available b1,4-galt-1 was used for enzymatic modifications of arrays carrying immobilized GlcNAc structures. 159,160 b1,4-galt-1 showed 11-times higher activity with b-glcnac than with a-glcnac. The dependence of enzyme activity on the acceptor substrate density followed a bell-like curve, with a dramatically decreased activity at elevated glycan densities. These findings respond to the question of how many glycans may be enzymatically glycosylated using surface bound acceptors. So far glycosyltransferase activity assays have mostly been accompanied by lectin-aided detection of the resulting saccharide, which represents a major drawback in substrate screening with glycan arrays, due to possible alterations of lectin binding. A striking approach for more variability in substrate screening with arrays used self-assembled monolayers for matrix-assisted laser desorption-ionization mass spectrometry analysis (SAMDI-MS), which is mainly a MALDI optimization for analyzing SAMs. 161 This method enables completely label-free activity assays and substrate screening utilizing glycan arrays. In a pioneering study, b1,4-galt-1-catalyzed galactosylation of disaccharide acceptors with b1,2-, b1,3-, b1,4- and b1,6-linkages between Gal, Glc and GlcNAc was shown. Remarkably, the preparation of the array for the screening of 24 disaccharides took as few as 4 hours. This versatile and fast approach was used to perform the largest acceptor-substrate screening on arrays so far. 162 In total, combinations of enzymes, immobilized acceptor substrates and donor substrates in four different buffer systems were screened to identify new glycosyltransferases and their substrate specificity. The resulting reactions were monitored using SAMDI. Besides the identification of novel GTs, some yet unknown donor promiscuity of established GTs was identified and, in addition, the kinetic constants of novel enzymes and new enzyme substrate combinations were determined by this approach. The expression of putative GTs from various bacterial strains was carried out in vitro. Theonly drawback of this method is the necessity to determine the type of glycosidic linkage via NMR; therefore, preparative syntheses in solution are still needed for identifying new GTs. In sum, glycan arrays are a promising platform for substrate and inhibitor screening purposes, especially combined with direct, non-lectin mediated detection. GT-catalyzed synthesis on multivalent surfaces. In addition to substrate and inhibitor screening for glycosyltransferases, Chem Soc Rev the array-approach enables enzymatic synthesis for identifying lectin glycan interactions. Recently, the production of 40 different N-glycan structures using three glycosyltransferases with 13 chemically synthesized acceptor substrates via nanodroplet technology was demonstrated. 163 The acceptors comprising natural and non-natural high mannose, complex and hybridtype N-glycans were immobilized on NHS-activated glass slides. b1,4-galt-1, a2,6-sialt, and a1,3-fuct (core fucosyltransferase) were used for building the N-glycan library. In this assay, enzymes and donor substrates were applied as nanodroplets on the printed array and the reactions were detected using respective lectins. In another study, binding of lectins to ten monosaccharides immobilized through a Diels Alder-type reaction on a SAM revealed the correlation between lectin binding and glycan density. 164 The binding constants of used lectins could be determined via surface plasmon resonance measurement (SPR) on the arrays. Further modification of glycans by b1,4-galt-1 was monitored with the Gal-specific lectin from Erythrina crista-galli. This simple approach led to the idea that array technology may outperform classical ELISA-based studies on protein carbohydrate interactions. However, the immobilization step was crucial with more complex glycans for proving antibody antigen-interactions, and in comparison to ELISA the amount of needed glycans for sufficient functionalization of the surface could not be reduced, which is opposed to one of the major advantages of array usage. 165 Recently, the issue of functionalization density and of its influence on lectin binding was solved by synthesizing a surface with thorough control of glycan density and orientation. 166 Temporary spacers, namely BSA, lysozyme and polyacrylamide, were used as boronic acid ligands for glyco-polymers. After immobilisation through amino-functionalized glass-slides, the spacers were removed to yield a glycan-coated surface with highly controlled interglycopolymer distance. The use of various lectins within SPR measurements revealed a lectin-dependent preference for a certain density of functionalization, which introduced a new parameter in glycan-dependent interactions. New insights into sle x recognition and binding were gained in a high-throughput screening of lectin preferences combined with a chemoenzymatic synthesis of complex saccharides using b1,4-galt-1, a2,3- SialT a1,3-fuct. 167 The maleimide-modified GlcNAc precursor was immobilized on thiol-functionalized glass-plates and the reactions were detected by respective labeled lectins. The formed product could not be quantified in this way; therefore no insights into the efficiency of enzymatic reactions on a surface were gathered and the focus was solely on the investigation of lectin-binding events. Another approach to studying the multivalency in glycan protein interaction combined glycodendrimers and SAMs with array technology. 168 Here, the dendrimer synthesis as well as SAM modification could be monitored using click chemistry. The use of azide-modified monosaccharides (Man, GlcNAc, Gal) greatly enhanced the multivalency of binding of each lectin. Interactions between glycans and cells based on array-technology were also reported. 16 Immobilizing lactose via SAM on chips and subsequent transfer of sialic acid from fetuin by trans-sialidase This journal is c The Royal Society of Chemistry 2013 Chem. Soc. Rev., 2013, 42,

17 Chem Soc Rev from Trypanosoma cruzi showed significant enhancement of binding of CHO-cells in comparison with non-sialylated arrays. Enzymatic sialylation was controlled by MALDI-TOF MS, which revealed that about 50% of glycans were modified. This compares well to turnover rates, utilizing this enzyme with soluble substrates in solution. 169 Indubitably, array technology used in the screening for glycosyltransferases and their substrate specificities and in investigating lectin ligands has a great potential. It will have tremendous impact on future research in glycobiology, making high-throughput glycan assays as common as the established DNA- and peptide arrays. Glycosylations of soluble substrates A diverse array of multivalent synthetic carriers may be enzymatically glycosylated in solution, often with the aim of preparing new carbohydrate-based vaccines 170 or well-defined glycostructures for analysis or detection aims. They range from short synthetic peptides, flexible water-soluble synthetic polymers or polypeptides to carbosilane clusters and dendrimers. Concerning the type of carbohydrate epitopes presented, sialyl N-acetyllactosamine structures (Neu5Aca2,3/6Galb1,4GlcNAcb1-O-), appearing on the surface of glycoproteins and glycolipids in vivo, are of great interest. They may be readily synthesized Review Article by applying the sequence of b1,4-galt and a2,6- or a2,3-siat to a GlcNAc primer chemically attached to a suitable carrier. An example of a sialyl N-acetyllactosamine dendrimer is a tetraantennary carbosilane structure prepared by enzymatic galactosylation and sialylation of O- or S-bound GlcNAc (Fig. 3a). 171 The sialyl N-acetyllactosamine pattern may also be displayed on branched N-linked glycopeptides, 172 and it often forms the tips of antennae of various synthetic glycan blocks as described further in this section. Sialyl lacto-structures type I (containing b1,3-gal) or type II (containing b1,4-gal) are important recognition patterns on the host cell surface for binding of hemagglutinins of influenza A and B viruses. Since the multivalent effect is necessary for successful interaction, the sialyl-lacto epitopes are displayed on a polymeric backbone, such as polyacrylamide. This approach was first used at the onset of 1990s with the simplified pattern of sialic acid moieties directly chemically attached to the polymer. 173 Advantageously, the displayed oligosaccharidic epitopes may be multiantennary for enhanced binding effect (Fig. 3b). 174 The synthetic glycopolymers display a high affinity to the viral hemagglutinin; however, they may suffer from cytotoxicity and immunogenicity. Another alternative is a poly(a-l-glutamic acid) backbone. Ogata et al. prepared such sialoglycopolypeptides with sialyllactose or N-acetyllactosamine Fig. 3 Products of glycosylation of various synthetic scaffolds in solution: (a) tetraantennary sialyl N-acetyllactosamine carbosilane, 171 (b) polyacrylamide with triantennas of sialyl N-acetyllactosamine, 174 (c) sialyl lacto-functionalized poly(a-l-glutamic acid), 175 (d) fluorescently labelled sle x dimer, 179 (e) sialyl-trimeric-lewis x decasaccharide, 181 (f) octavalent synthetic dendrimer, 183 (g) rare triantennary N-glycan, 187 (h) biantennary poly(lacnac), 188 (i) bivalent molecular ruler. 191 The enzymatically attached carbohydrate units are marked in color Chem.Soc.Rev.,2013, 42, This journal is c The Royal Society of Chemistry 2013

18 Review Article epitopes (Fig. 3c) by using recombinant rat a2,3- and a2,6-siats expressed either in Spodoptera frugiperda 175 or, with particular efficiency, in silkworm larval hemolymph injected by Bombyx mori shuttle vector. 176 Totani et al. 177 went even further, preparing a whole bunch of polypeptide sialo-lacto-analogues in a structure activity relationship study with two strains of influenza virus A and B. Another popular carbohydrate epitope is sle x tetrasaccharide and its mimics. In vivo, this structure acts as a minimum ligand for selectin glycoproteins. Its preparation involves sialylation (a2,3-siat) and fucosylation (a1,3-fuct) of an LacNAc type II primer, which is either prepared chemically or enzymatically by galactosylation (b1,4-galt) of GlcNAc attached to the carrier. SLe x and its variants have been displayed in a range of oligosaccharidic structures, such as biantennary sle x dimers with various synthetic linkers 178 or with fluorescent probes (Fig. 3d), 179 attached to the termini of a trimannose core 180 or in the form of linear sialyl-trimeric-lewis x decasaccharide with Fuc branching (Fig. 3e). 181 Wong and coworkers 182 also prepared mercury-modified sialic acid and incorporated it into a bivalent sle x -decorated undecasaccharide and an RNase glycoprotein. This derivatisation should serve for X-ray determination of the carbohydrate structure. Palcic et al. 183 presented enzymatic glycosylation of synthetic dendrimer scaffolds. SLe x epitopes were enzymatically constructed on the GlcNAc moieties chemically attached to the 2,4,8-valent dendrimer antennas, based on L-lysine (Fig. 3f). The peptidic scaffold was already used two years earlier 184 in the construction of di- and trivalent pseudo-glycopeptides. Unverzagt and coworkers prepared a variety of extensive N-glycan building blocks, based on enzymatic galactosylation and sialylation of biantennary, chemically prepared N-glycans; 185,186 also a rare N-glycan type containing the third short branch of a single b1,4-glcnac unit, leading from the core b-mannosyl residue (Fig. 3g). 187 A synthetic O-glycan mimic was constructed using Neisseria meningitidis b1,3-glcnact and human b1,4- GalT (Fig. 3h). 188 Here, a double poly-lacnac chain was built up on a core-2-o-glycan-based trisaccharide. The glycosylating abilities of many GTs are efficiently transferable between the solution and solid phase, where the latter medium is typical of the multivalent effect. Flitsch and coworkers 189 performed total synthesis of a glycopeptide containing O-mannosyltetrasaccharide, using recombinant O-mannose b1,2-n-acetylglucosaminyltransferase 1 (GnT-I), bovine b1,4- GalT, and trans-sialidase from Trypanozoma cruzi in one pot. The enzyme performance was optimized both for the solution and for solid-phase synthesis on a gold platform. In a study of core fucosylation, two recombinant FucTs (a1,3- and a1,6-) were used to fucosylate a series of synthetic N-glycans immobilized on a microarray, and prospective reactions were scaled up in solution. 190 One of the advantages of purely synthetic backbones such as alkyl chains or polymers is their spacious flexibility, which results in a more efficient enzymatic synthesis or enables using the multivalent effect for, e.g., more efficient interactions in biological assays. Křen, Elling and coworkers used mutant Chem Soc Rev b1,4-galt-i Tyr284Leu from human placenta for efficient b-galnac-ylation of bivalent molecular rulers of various lengths (Fig. 3i). 191,192 In the course of this work, Drozdová et al. 192 demonstrated that glycosidases are rather inept catalysts for glycosylation of multivalent acceptors, in contrast to GTs. Nishimura and coworkers presented several cases of multienzyme glycosylation of GlcNAc primers, attached to a flexible polyacrylamide polymer backbone, and yielding sphingoglycolipids or other, non-natural glycolipids. The polymer carrier was elegantly discarded by transfer of the constructed oligosaccharide to ceramide 193 or D-sphingosine 194 using ceramide glycanase. The enzymes employed were various SiaTs, and recombinant b1,4-galt, both optionally immobilized, 195 as well as a1,3-fuct and b1,3-glcnact from Streptococcus agalactiae. 196 This strategy may be applied with other glyco-chains, such as LacNAc glycans. 188 A one-pot approach utilizing recombinantly produced human b1,4-galt-1 and b1,3-glcnact from Helicobacter pylori yielded monovalent poly-lacnac from a chemically modified GlcNAc acceptor. 197 Product-determining preferences of b1,3-glcnact for certain saccharide chain-lengths were found, and, in addition, the synthesis was optimized to gain a broad product distribution ranging from tri- to heptadecasaccharides within 24 hours of reaction time. 198 By coupling of the products to hydrogel coated surfaces, a multivalent mimic of the extracellular matrix was accessed. 199 This synthetic system was also used with a set of modified acceptors, expanding the possibilities of multivalent-glycan coatings on functionalized materials. 200,201 Furthermore, glycans modified at the C6-position were turned over to yield internally labeled and multivalent chemo-enzymatically branched structures. 202 These special non-natural glycans are promising tools for studying the interactions with galectins a class of lectins closely associated with cancer research and general cell adhesion processes. 203 Brilliant examples of multivalent glycosylations In this section we aim to demonstrate a choice of elegant and ingenious applications of complex enzymatic glycosylations that reflect recent trends in glycomics. The first example describes the pathway toward recombinant production of eukaryotic N-glycans in bacterial hosts. Although N-glycosylation was originally considered to be restricted to eukaryotic organisms, at the turn of the millennium this dogma was contradicted by Szymanski and coworkers. 204,205 In a short succession, the pioneering work by Aebi and coworkers proved the possibility of transferring the Campylobacter jejuni N-linked glycosylation pathway into E. coli. 206 They also described the key role of oligosaccharidyltransferase PglB in the process 207 thanks to its relaxed substrate specificity, this enzyme is virtually predestined to mediate in vivo production of tailor-made carbohydrate protein conjugates. 208 These findings formed the platform for further research in this direction. Schäffer and coworkers 209 designed an experimental E. coli production system, capable of integrating two types of tailored N-glycosylation patterns into the S-layer of self-assembled neoglycoproteins on the bacterial surface. Namely, a heptasaccharide from Campylobacter jejuni and the O7 polysaccharide antigen This journal is c The Royal Society of Chemistry 2013 Chem. Soc. Rev., 2013, 42,

19 Chem Soc Rev Review Article Fig. 4 Labeling of cell-surface glycans in vivo. 218 (a) Tagged monosaccharide precursors are metabolized and built up into any surface glycostructures containing the respective monosaccharide or (b) tagged GDP-Fuc is in situ transferred to specifically yield tagged LacNAc. In the second step, tags are coupled with the probe of choice. Adapted with permission from ref. 218 r (2011) John Wiley and Sons. from E. coli were recombinantly transferred onto the engineered N-glycosylation sites of an established S-layer protein self-assembly system of Geobacillus stearothermophilus using oligosaccharyltransferase PglB. This work represented a new concept of in vivo creation of a highly organized, periodic protein system at a nanometer scale; an excellent nanobiotechnological tool. The N-glycosylation battery in E. coli, though functional and versatile, still faced the problem of the vast difference between the bacterial N-glycan structure and the eukaryotic glycopatterns. The E. coli produced N-linked heptasaccharide contains a unique sugar, bacillosamine (2,4-diamino-2,4,6-trideoxy-D-Glc), and is immunogenic, which sadly limits its biotechnological applications. Schwarz et al. 210 addressed this problem by combining bacterial glycoprotein expression with subsequent in vitro chemoenzymatic glycan trimming and extension. The N-glycan was trimmed by a-n-acetylgalactosaminidase to give a GlcNAc-linked glycoprotein and eliminate the bacillosamine moiety. Then, endoglycosynthase-catalyzed transglycosylation with oxazoline donors yielded glycoproteins containing natural complete eukaryotic N-glycans. In recent work, 211 N-linked eukaryotic Man 3 GlcNAc 2 glycans were produced through the action of four co-expressed eukaryotic GTs and the bacterial oligosaccharyltransferase PglB. Although very promising, there are still bottlenecks to be overcome before this technology may be successfully implanted into everyday practice, such as the substrate specificity of oligosaccharidyltransferase and the scale-up potential. Despite these drawbacks, the glycoengineered E. coli is a promising model production system for cracking the glycocode and revealing the secrets of non-template synthesis of complex eukaryotic glycoproteins. The second example of sophisticated use of carbohydrateprocessing enzymes is metabolic labeling. 212 This versatile method of glycan detection makes an alternative to the antibody or lectin assays, which are often stricken with weak affinity, limited specificity and risk of cross-reactivity. In general, metabolic labeling is based on treating cells or organisms with aptly tagged saccharide monosaccharide precursors. When these monosaccharides are taken up by cells, they are transformed into activated nucleotide donors in the cytoplasm and are built up into glycostructures on the cell surface using glycosyltransferases. Then, the bioorthogonal chemical tags may be covalently conjugated with, e.g., fluorescent or affinity probes for imaging, mapping, detection and analysis. Standardly used azido or cyclooctyne probes are detected through Cu + -catalyzed azide alkyne cycloaddition or copper-free click chemistry but there are other possibilities, suchasuvactivateddiazirinetags. 213 A range of glycan types have been analyzed by this method, including mucin O-glycans, 214 sialylated, 215 and fucosylated glycans, 216 and cytosolic O-GlcNAc-ylated proteins Chem.Soc.Rev.,2013, 42, This journal is c The Royal Society of Chemistry 2013