Toward Automated Enzymatic Synthesis of Oligosaccharides

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Cite This: Chem. Rev. XXXX, XXX, XXX−XXX

Toward Automated Enzymatic Synthesis of Oligosaccharides Liuqing Wen,*,†,‡ Garrett Edmunds,†,‡ Christopher Gibbons,†,‡ Jiabin Zhang,†,‡ Madhusudhan Reddy Gadi,† Hailiang Zhu,† Junqiang Fang,§ Xianwei Liu,§ Yun Kong,§ and Peng George Wang*,†,§ †

Department of Chemistry, Georgia State University, Atlanta, Georgia 30303, United States National Glycoengineering Research Center and State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China

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ABSTRACT: Oligosaccharides together with oligonucleotides and oligopeptides comprise the three major classes of natural biopolymers. Automated systems for oligonucleotide and oligopeptide synthesis have significantly advanced developments in biological science by allowing nonspecialists to rapidly and easily access these biopolymers. Researchers have endeavored for decades to develop a comparable general automated system to synthesize oligosaccharides. Such a system would have a revolutionary impact on the understanding of the roles of glycans in biological systems. The main challenge to achieving automated synthesis is the lack of general synthetic methods for routine synthesis of glycans. Currently, the two main methods to access homogeneous glycans and glycoconjugates are chemical synthesis and enzymatic synthesis. Enzymatic glycosylation can proceed stereo- and regiospecifically without protecting group manipulations. Moreover, the reaction conditions of enzyme-catalyzed glycosylations are extremely mild when compared to chemical glycosylations. Over the past few years methodology toward the automated chemical synthesis of oligosaccharides has been developed. Conversely, while automated enzymatic synthesis is conceptually possible, it is not as well developed. The goal of this survey is to provide a foundation on which continued technological advancements can be made to promote the automated enzymatic synthesis of oligosaccharides.

CONTENTS 1. Introduction 2. Overview of Oligosaccharide Synthesis 2.1. Chemical Synthesis of Oligosaccharides 2.2. Enzymatic and Chemoenzymatic Synthesis of Oligosaccharides 3. Overview of Automated Solid-Phase Synthesis of Oligosaccharides 4. Toward Automated Enzymatic Synthesis of Oligosaccharides 4.1. Enzymes Used in the Automated Enzymatic Synthesis of Oligosaccharides 4.1.1. Glycosyltransferases 4.1.2. Glycosidases and Glycosynthases 4.1.3. Enzymes Involved in Oligosaccharide Postmodification 4.2. Glycosylation Donors 4.3. Supports Used in Enzymatic Glycosylation 4.3.1. Solid-Phase Supports 4.3.2. Solution-Phase Supports 4.4. Linker and Spacer 4.4.1. Protease-Cleaved Linkers 4.4.2. Disulfide Linkers 4.4.3. Photocleavable Linkers 4.4.4. Acid- or Hydrazine-Sensitive Linkers 4.4.5. Safety-Catch Linkers as a Potential Option

© XXXX American Chemical Society

4.5. Automation Instruments for the Automated Synthesis 4.5.1. Peptide Synthesizer 4.5.2. Early Progress toward Automated Oligosaccharide Synthesis 4.5.3. Seeberger’s Synthesizer 4.5.4. Other Synthesizers 5. Automated Enzymatic Synthesis of Oligosaccharides 5.1. Artificial Golgi Apparatus 5.2. Combination of Automated Solid-Phase and Enzymatic Oligosaccharide Synthesis 5.3. Novel Automated System for Oligosaccharide Synthesis 6. Summary and Outlook Author Information Corresponding Authors ORCID Author Contributions Notes Biographies Acknowledgments References

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Special Issue: Carbohydrate Chemistry

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Received: January 31, 2018

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DOI: 10.1021/acs.chemrev.8b00066 Chem. Rev. XXXX, XXX, XXX−XXX

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Figure 1. Common monosaccharides that occur as building blocks in oligosaccharides and their symbols.

1. INTRODUCTION Biopolymers produced in living systems include three main classes: oligonucleotides, oligopeptides, and oligosaccharides.1 Saccharide polymers, in which monosaccharides are covalently joined together through glycosidic linkages, are called oligosaccharides or polysaccharides.2 While strict definitions of both oligosaccharide and polysaccharide are not established, it is generally agreed that a carbohydrate consisting of 2−10 monosaccharide residues with a defined structure is an “oligosaccharide” and a carbohydrate consisting of more than 10 monosaccharide residues is a “polysaccharide”.3,4 In living cells, oligosaccharides often occur as glycoconjugates attached to other macromolecules such as lipids (called glycolipids) or proteins (called glycoproteins) to express their function.5 Of the more than 300 known protein modifications, glycosylation events (N-linked, O-linked, and C-linked glycosylation) are the most abundant and complex post-translational modifications.6 It has been suggested that more than 50% of human proteins are glycosylated, but this number is likely an underestimation.7 More than 40 different types of linkages between sugars and amino acids have been identified, involving at least 13 different proximal sugar residues and 8 different amino acid residues.8 Glycolipids are found on the surface of all eukaryotic cells, where they extend from the phospholipid bilayer (lipid portion) into the extracellular environment (glycan portion).9 Beyond their traditionally accepted roles as energy sources for living organisms, it is now well established that oligosaccharides play

important roles in a variety of physiological and pathological processes; this includes but is not limited to cell growth and proliferation, immune responses, angiogenesis and tumor cell metastasis, toxin interaction, protein folding and degradation, cell−cell communications, and cell−pathogen interactions.10−25 Additionally, it has also been shown that aberrant glycosylations have a strong relationship with cancer and multiple other human diseases.26−36 For example, the truncated mucin-type O-glycans including Tn-antigen, sialyl-Tn antigen, T-antigen, and sialyl-T antigen have low or no expression on normal cells but are highly expressed in many human cancer cell lines.37−44 These tumorassociated carbohydrate antigens are targets for application in either clinical diagnosis or immunotherapy.45−53 In biosynthetic pathways, oligosaccharides are built stepwise by enzyme-catalyzed reactions. However, unlike oligonucleotides and proteins, the biosynthesis of oligosaccharides is not a template-guided process. It remains unclear whether oligosaccharide biosynthesis in living cells is involved in the transfer of biological information, but the structural diversity and complexity of oligosaccharides is far beyond nucleic acids and proteins. This diversity and complexity arises from the peculiar properties of monosaccharide residues and linkages.54,55 A hexose has 4 chiral centers which results in 16 possible isomers, while a pentose can have up to 8 possible isomers. Additionally, monosaccharides experience modifications to native hydroxyl or amino groups including acylation, sulfation, methylation, and phosphorylation,56−59 which significantly increase overall B


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effective methods for the synthesis of oligosaccharides especially automated synthesisis essential for advancing our understanding of glycobiology and the development of glycanrelated pharmaceuticals.

diversity. The most common monosaccharide building blocks found in oligosaccharides as well as their symbols as suggested by glycobiology community60,61 are shown in Figure 1. Moreover, monosaccharides can form α or β glycosidic linkages with other sugar residues at any hydroxyl group, with one monosaccharide having the possibility to engage in more than two glycosidic linkages. This structural diversity and complexity make the characterization of oligosaccharides extremely complicated. As a glycobiology research tool, the availability of pure glycans is prerequisite to determining the molecular details of a glycan’s function and to produce other homogeneous glycoconjugates such as glycoproteins for functional studies and biomedical applications. One example is the use of oligosaccharides to produce glycan microarrays for high-throughput screening.62−64 With standard glycans in hand, the Consortium for Functional Glycomics (CFG) has produced the largest mammalian-type glycan microarray (∼611 glycans)65 and a microbial glycan microarray with over 300 targets.66 Feizi and co-workers produced the largest neoglycolipid-based oligosaccharide microarray (∼600 glycans).67 The development of glycan microarrays has greatly advanced glycobiological studies.68,69 They facilitate high-throughput experiments exploiting mammalian and microbial glycan-binding proteins,70−76 help with screening for substrate specificity of glycosyltransferases,77−80 and make easier the discovery of glycan-specific antibodies.81−84 In addition, pure glycan standards are also important to develop chemoenzymatic bioorthogonal labeling tools to investigate cell surface glycans.85−88 In pharmaceutical chemistry, carbohydrate-based vaccines have widely been used against bacteria.89−93 To date, most commercial carbohydrate-based vaccines in use are derived from pathogens via the process of large-scale bacterial fermentation, digestion, hydrolysis, and chromatographic purification. Nevertheless, although extraction and purification processes have been improved, unwanted contaminations are still difficult to avoid completely, which may cause serious side effects. Structuredefined oligosaccharides obtained via artificial synthesis have the potential to simplify the procedures for quality control, which could facilitate the creation of a new generation of carbohydratebased vaccines. However, the development of a new generation of carbohydrate-based vaccines also requires the increased availability of homogeneous oligosaccharides.94−98 The vaccine currently used to prevent Haemophilus influenzae serotype b (Hib) infection is an example of a fully synthetic carbohydrate conjugate vaccine.99 The Globo H hexasaccharide, which was first isolated from breast cancer cell line MCF-7 by Hakamori and co-workers100,101 and was later found on a range of other cancer types,102,103 has attracted significant attention. It was found that Globo H, when conjugated with carrier proteins, can elicit strong immune responses in cancer patients, indicating the potential of the Globo H hexasaccharide in the development of a cancer vaccine.104,105 Over the past few decades, many powerful methodologies including one-pot glycosylation, metal-catalyzed synthesis, automated solid-phase synthesis, one-pot multienzyme synthesis, and chemoenzymatic synthesis have been developed to produce structure-defined glycans.106−112 However, there is still no general synthetic method for the routine synthesis of glycans due to the inherent chemical properties of oligosaccharides. Although progress has been made, the ability to form a desired glycosidic linkage on command is a skill that has yet to be acquired. As we review here, the development of simple and

2. OVERVIEW OF OLIGOSACCHARIDE SYNTHESIS 2.1. Chemical Synthesis of Oligosaccharides

Among all biopolymers, the chemical synthesis of oligosaccharides is the most perplexing as they are often highly branched with many different linkages. A general approach to oligosaccharide assembly consists of a glycosyl donor, a glycosyl acceptor, and an activator. The glycosylation process generates a new stereocenter and is characterized by the manipulation of various protecting groups that mask the hydroxyl groups and prevent them from reacting with activated sugar donors and other reagents. The selective exposure of a single hydroxyl affords the ability to control the location of glycosylation on an acceptor. Most often, the donor is activated with a Lewis acid and this group is replaced by a free hydroxyl from the acceptor, creating a glycosidic linkage. For example, one of the most famous glycosylation reactions still in common use is the Koenigs− Knorr reaction, a substitution reaction of a glycosyl halide with an alcohol to generate a glycoside.113 Indeed, the most challenging task in chemical synthesis is the choice of suitable orthogonal protecting groups and their selective manipulation to achieve efficient synthesis. Commonly used protecting groups include esters, benzyl or silyl ethers and derivatives, as well as acid- or base-sensitive protecting groups.114−116 These protecting groups can be divided into permanent and temporary protecting groups. Temporary protecting groups are removed after each glycosylation step to make the molecule able to serve as the glycosyl acceptor for the next glycosylation, with this process continuing until the target oligosaccharide is assembled. Permanent protecting groups are left on the molecule until the end of the synthesis. They prevent glycosylation or other side reactions in places on the molecule where the need for glycosylation is not foreseen. These groups must be orthogonal with respect to the deprotection conditions of the temporary groups but not necessarily the conditions of the removal of the linker. Each glycosylation and deprotection step requires purification as the new stereocenter generated in the glycosylation may not be fully diastereoselective. This process increases the number of purifications required in the synthesis of an oligosaccharide, leading to a labor-intensive and timeconsuming effort. Several convergent glycosylation strategies have taken over the stepwise glycosylation approach owing to the economy, time, and efficiency. These strategies mainly include one-pot, solid-phase, and chemoenzymatic glycosylation. One-pot glycosylation is done in a single flask where glycosyl donors are added sequentially to the reaction vessel in order to obtain the required oligosaccharide. Such an approach requires the ready availability of well-designed donors and acceptors. The lack of efficient methods for mainly stereoselective α-sialyation and β-mannosylation mostly has hindered development of broadly applicable one-pot glycosylation strategies. The introduction of the 4-O,5-N-oxazolidinone protecting group by Takahashi117 and the N-acetyl variant by Crich118 and coworkers has helped to overcome the development hurdle of stereoselective α-sialyation. However, the less active sialic acid donors have hindered their use in one-pot glycosylation. Interestingly, the use of adamantanyl donors along with NC


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Figure 2. Programmable one-pot synthesis of Globo H oligosaccharide through the use of the OptiMer computer program. Reproduced with permission from ref 140. Copyright 2011 John Wiley and Sons.

obtained donor is activated with the promotor again and successively the bifunctional donor−acceptor is added, and the process continues in the same reaction flask until the desired oligosaccharide is obtained. This strategy is highly advantageous over the other one-pot glycosylation strategies as fine tuning of the glycosyl donors is not required. However, for a successful synthesis, a stoichiometric amount of promoter has to be used and the donor has to be completely consumed to give a stable yet reactive intermediate to eliminate the formation of side products. A recent report from Ye and co-workers used this strategy to synthesize the mycobacterial arabinogalactan with 92 monosaccharide units.133 To overcome the labor-intensive process to access complex oligosaccharides, Wong and co-workers introduced a programmable one-pot synthetic strategy to generate oligosaccharides.134 The programmable one-pot synthesis strategy takes advantage of the differences in reactivities between different glycosylation donors and uses a computer program to direct oligosaccharide synthesis. One-pot glycosylation allows multiple glycosylation reactions to take place successively in the same reaction chamber. The most popular one-pot glycosylation approach used in automated synthesis sequentially uses the reactivity differences of a large set of diverse thioglycoside building blocks to form glycosidic bonds. The thioglycoside possesses an −SR group at the anomeric center instead of the natural −OR group. These derivatives are suitable for all monosaccharides and can be easily prepared. Moreover, thioglycoside building blocks are stable under ambient conditions.135 Currently, more than 400 thioglycoside building blocks with a wide spectrum of protecting groups have been prepared and characterized with respect to their relative reactivities. Each has a measured relative reactivity value (RRV) that can be stored in a computer database. A computer program, called “OptiMer”, was created to store the RRVs of many donors and donor−acceptors. Once a user has input a target oligosaccharide structure, OptiMer will list the best combination of building blocks for its preparation. The program will also dictate the stereochemistry as α- or β-directing. With the OptiMer database, oligosaccharides containing 3−6 monosaccharides can be rapidly assembled starting from the most reactive and ending with the least reactive in minutes or hours without intermediate workup or purification procedures. In addition, regioselective and combinatorial one-pot protec-

acyloxazolidinone protection has showed great promise in employing sialic acid donors in the one-pot glycosylation.119 Another landmark development in stereoselective glycosylation is the successful β-mannosylation in one-pot strategies. Crich and co-workers reported an interesting strategy of using 4,6benzylidene protection to obtain α-triflate leading to stereoselective β-mannosylation in an SN2 fashion.120 A recent report from Sasaki and co-workers employed 2,6-lactones to obtain βmannosides.121 One-pot glycosylation strategies are done in one of three ways: an anomeric reactivity-based strategy, an orthogonal protection strategy, and a preactivation strategy. In the anomeric reactivity-based strategy, the anomeric reactivity of the glycosyl donors is altered by arming/disarming the glycosyl donor through the use of protecting groups mainly at the C-2 position. The armed/disarmed concept pioneered by Fraser-Reid122 has been used to produce complex oligosaccharides.123−125 According to this concept, in general glycosyl donors protected with ether protecting groups are armed, whereas ester protection leads to a disarmed donor. Armed donors react faster in the presence of a bifunctional disarmed donor−acceptor. On the basis of this reactivity, glycosyl donors are added successively in the synthesis of oligosaccharides.126 In the orthogonal protection strategy, two or more different types of glycosyl donors are used in the synthesis of oligosaccharides. The leaving group at the anomeric position is selected such that its activation does not affect the leaving group on the other glycosyl donor along with any other protective groups. For example, a fluoride donor can be activated in the presence of a thio donor and vice versa.127 In a report from Ogawa and co-workers,128 A NIS-TfOH/AgOTf promoter system was used to activate a thiophenyl glycosyl donor in the presence of a fluoride donor, whereas Cp2HfCl2−AgClO4 was used to activate a fluoride donor in the presence of a thio donor. Such a strategy is highly advantageous as the glycosyl donors can be selectively activated irrespective of their reactivities and has been used in the synthesis of several complex oligosaccharides.129,130 Finally, in the preactivation strategy, a glycosyl donor is activated by a promotor prior to the addition of an acceptor having the same type of leaving group at the anomeric center.131,132 The acceptor is added to this preactivated donor or the active intermediate for a successful glycosylation. The D


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Scheme 1. Chemical Assembly of Globo H Hexasaccharide

Scheme 2. Enzymatic Synthesis of Globo H Hexasaccharide

blocks (7−12) need to be prepared first for construction. The longest linear sequence of the synthesis was 11 steps and gave an overall yield of 2.6% (Scheme 1).143 Meanwhile, the enzymatic synthesis of Globo H hexasaccharide 14 starting from compound 15 employs only three glycosyltransferases (four enzymatic glycosylation steps) and gives a total yield of 57% (Scheme 2).144 This yield can be increased to 94% by the inclusion of sugar nucleotide regeneration systems.145 In addition, enzyme-catalyzed reactions can be carried out in one pot (one-pot multienzyme strategy) because of the substrate specificity of enzymes.112,146−150 Chemical synthetic methods allow for the preparation of diverse natural and unnatural glycan structures, while donor and acceptor specificity require an enzymatic synthesis to follow natural biosynthetic pathways. Yet many enzymes are unable to tolerate even minor variations in donor or acceptor structure. This distinguishing feature is desirable when using enzymatic methods to produce homogeneous glycans, but it also frustrates efforts to produce diverse glycan structures by enzymatic methods. For example, many glycosyltransferases involved in E. coli O-antigen synthesis require acceptors with a lipid tail.151−155 The human mannosyltransferases involved in N-glycan synthesis use dolichol phosphate mannose as an activated sugar donor,156 which is insoluble in water and has proven difficult to prepare. To address this issue, chemoenzymatic approaches that combine the flexibility of chemical synthesis and high

tion, which has potential for application in automated synthesis of monosaccharides, has also been reported.136−138 An example that used OptiMer for one-pot synthesis of Globo H hexasaccharide is shown in Figure 2.139,140 When the target Globo H hexasaccharide 1 was input into the computer, the OptiMer program calculated a synthetic route in which building blocks 2, 3, and 4 were need for construction. After the successful construction of intermediate 5, a deprotection reaction was required to produce Globo H hexasaccharide 1. 2.2. Enzymatic and Chemoenzymatic Synthesis of Oligosaccharides

As an alternative to chemical synthesis, there is growing interest in using enzymatic methods to synthesize oligosaccharides and glycoconjugates. Enzymatic glycosylation is able to proceed stereo- and regioselectively without the need to undergo tedious protecting group manipulations. Even for very sterically demanding couplings such as those involving sialic acid, enzymatic glycosylation can be performed selectively.141,142 Enzymatic reactions occur in aqueous solutions and only require limited control over reaction conditions such as temperature, system pH, and the presence or absence of metal ions. Also, there are no toxic byproducts produced in glycosylation process, making the enzymatic synthesis of oligosaccharides an environmentally friendly approach. For example, a typical chemical strategy (3 + 2+1) for the synthesis of Globo H hexasaccharide 14 requires multiple glycosylation steps. Multiple building E


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Figure 3. Chemoenzymatic synthesis of oligosaccharides. (A) Chemical synthesis of a primer oligosaccharide with enzymatic extension (chemoenzymatic synthesis of asymmetric N-glycans). Reproduced with permission from ref 160. Copyright 2017 American Chemical Society. (B) Enzymatic synthesis of oligosaccharides followed by chemical modification (chemical modification of oligosaccharides for immobilization on microarray). Reproduced with permission from ref 163. Copyright 2017 Proceedings of the National Academy of Sciences. (C) Enzymatic incorporation of sugar residue modified with unnatural groups (chemoenzymatic synthesis of sialosides containing sialic acid modifications). Reproduced with permission from ref 164. Copyright 2006 John Wiley and Sons.

derivatives.142,164,165 Such oligosaccharides with modified sugar residues can be used as probes to investigate protein− carbohydrate interactions166,167 or used to develop carbohydrate-based vaccines.168

regioselectivity and stereoselectivity of enzyme-catalyzed reactions were developed to achieve highly efficient syntheses of complex oligosaccharides. In general, there are three types of chemoenzymatic synthesis (Figure 3): the chemical synthesis of primer oligosaccharides with enzymatic extension, the enzymatic synthesis of oligosaccharides followed by chemical modification, and the enzymatic incorporation of sugar residues modified with unnatural groups. The first strategy is widely used to construct complicated glycans with large molecular weights.157−161 The second strategy is normally used to modify glycans for further use such as the ligation of an oligosaccharide to a protein or printing oligosaccharides on a microarray.162,163 The third strategy is generally used to produce oligosaccharide

3. OVERVIEW OF AUTOMATED SOLID-PHASE SYNTHESIS OF OLIGOSACCHARIDES Reliable and rapid access to oligonucleotides and oligopeptides by commercial automated synthesizers has fundamentally altered biological research.169−171 However, a general commercial system for the synthesis of oligosaccharides has yet to be implemented, mainly due to the lack of general synthetic methods. With a goal of achieving automated synthesis of F


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overall process. The acquisition of final product requires cleavage of the fully protected material from the solid support and removal of the protecting group. One of the main advantages of the use of a solid-phase support in oligosaccharide synthesis is that considerable excess of the glycosyl donor and other regents can be used to drive the reaction to completion without the need to separate out the final product from these reagents. Side products and other unwanted materials can be removed by a single wash, while the desired glycan remains covalently attached to the solid support. In recent years, many oligosaccharides have been successfully prepared by automated chemical synthesis.176−185 However, the application of automated chemical synthesis of oligosaccharides has been hindered by some limitations. Automated chemical synthesis relies on chemical synthetic methods, which suffer from tedious protection/deprotection manipulations. The control of regio- and stereospecificity is still problematic in many chemical glycosylation reactions. The lack of a general chemical glycosylation method for routine synthesis of oligosaccharides means that a special reaction strategy or reaction cycle needs to be carefully designed for each target structure.

complex oligosaccharides, many methodologies are currently being developed by synthetic chemists to address the challenges in carbohydrate chemistry that have prohibited automation. A major breakthrough in the synthesis of oligonucleotides and oligopeptides was the use of solid-phase synthetic methodologies, Scheme 3.172,173 This development enabled the Scheme 3. Principle of Automated Solid-Phase Synthesis of Oligosaccharides

4. TOWARD AUTOMATED ENZYMATIC SYNTHESIS OF OLIGOSACCHARIDES Enzymatic glycosylation has numerous advantages when compared to chemical glycosylation. In nature, enzymes make complex oligosaccharides with high acceptor and donor specificity in a linear fashion from the reducing end. Decoding such a technology from nature is challenging and can be automated only on a proper platform with inherent purification procedures that can separate out oligosaccharides from a mixture of enzymes and sugar nucleotides. In 2001, Wong and co-workers suggested two ways to achieve automated enzymatic synthesis of oligosaccharides: immobilized enzymes and immobilized substrates (Figure 4).186 Enzyme immobilization is a mature technology that is widely used for enzymatic synthesis.187,188 However, only immobilized enzymes can be removed using this method, while the oligosaccharide product is still mixed with other reaction regents such as metal ions, nucleotide sugars, and byproducts. The remaining reaction regents may interfere with the next reaction step. Therefore,

subsequent creation and eventual widespread use of automated synthesizers for these biopolymers. The use of solid-phase synthesis for the construction of oligosaccharides was initiated with Frechet and co-workers in 1971.174 However, it was not until 2001 that Seeberger and co-workers reported the first automated synthesis of oligosaccharides based on a solid-phase synthetic method.175 The first automated glycan synthesizer was constructed by modifying a peptide synthesizer, and the synthetic process closely resembles protocols established for the generation of peptides. To achieve automated synthesis, the first monosaccharide residue is attached through its reducing end to a solid support via a linker. The linker must be cleavable to release the final product upon the completion of the synthesis. Once the hydroxyl group to be modified in the coupling reaction is exposed by selective deprotection, addition of a glycosyl donor and activator will initiate the glycosylation reaction. Typically, the activator is a reagent that can generate a leaving group at the anomeric position. Upon the completion of one reaction cycle, subsequent sugar residues can be added by repeating the same

Figure 4. Comparison of immobilized enzymes vs immobilized substrates for automated enzymatic synthesis of oligosaccharides. Reproduced with permission from ref 186. Copyright 2001 The American Association for the Advancement of Science. G


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Figure 5. (A) Principle of solid-phase synthesis. (B) Principle of solution-phase synthesis.

enzymes that establish natural glycosidic bonds with regio- and stereoselectivity by catalyzing the transfer of an activated saccharide moiety to a nucleophilic glycosyl acceptor, which can be a specific carbohydrate hydroxyl group in a sugar, amino acid, or lipid. Although the mechanistic details of many GTases are unknown, they are generally categorized as either retaining or inverting to indicate the relative anomeric configuration between donor and product on the basis of analogies with better-studied glycosidases.197 In a popular classification system, glycosyltransferases can be divided into two superfamilies GT-A and GT-B as their structural folds.198 GT-A displays a single Rossmann fold and a conserved “DXD” metal-binding motif.199,200 GT-B does not require metal ions for its activity and possesses twin Rossmann folds that face each other.201 Although most of the known glycosyltransferases can be classified into these two families, many newly identified glycosyltransferases do not fit these two classifications well. Therefore, new superfamilies GT-C and GT-D are also suggested.202−204 GT-C superfamily contains diverse glycosyltransferases that consist of large hydrophobic proteins located in the ER or on the plasma membrane. Recently, it was reported that a β-glucosyltransferase from Streptococcus parasanguinis (DUF1792), which catalyzes the third step of Fap1 (Fimbriaeassociated protein) glycosylation, is structurally distinct from all known GT folds of glycosyltransferases and contains a new metal-binding site.204 Sequence analysis and structural prediction reveal that DUF1792 does not share any homology with known glycosyltransferases; it was suggested to be classified as GT-D. Nevertheless, both GT-C and GT-D classifications have not been well accepted by glycobiology community. Although more than 33 000 glycosyltransferases from both prokaryote and eukaryote have been identified,204 the common glycosyltransferases used in enzymatic synthesis mainly includes 8 classes (Table 1): galactosyltransferases (GalTs), glucosyltransferases (GlcTs), glucouronyltransferases (GlcATs), N-acetylgalactosaminyltransferase (GalNAcT), N-acetylglucosaminyltransferase (GlcNAcT), fucosyltransferases (FucTs), mannosyltransferases (ManTs), and sialyltransferases (SiaTs).

immobilizing enzymes is not an ideal strategy for automated enzymatic synthesis of oligosaccharides. We therefore will focus on the second strategy: immobilized substrates. Currently, there are two prominent approaches to automated enzyme-mediated saccharide synthesis using immobilizing substrates on an appropriate support or resin. In the first approach, the substrate is linked to a solid support and the enzymatic reaction proceeds on the solid phase, which is similar to solid-phase peptide synthesis (SPPS) (Figure 5A). In contrast to the first approach, enzymatic reactions proceed in a solution phase and the resulting product can be captured and released by specialized technologies (Figure 5B). In order to fully realize automation, the fundamental requirement for both strategies is the high-to-perfect conversion rate of the enzymatic reactions of each step, which significantly affects the design of the overall plan. Then the feasibility of linker and spacer, the availability of enzyme and glycosylation donor, and the possibility of automation are the subsequent considerations built upon the fundamental requirement. Thus, there are four key factors that need to be carefully considered in pursuing an automated enzymatic synthesis of oligosaccharides: (1) enzymes, (2) glycosylation donors, (3) supports (resin/polymer-support/ tag), (4) linkers and spacers, (5) automation instruments. 4.1. Enzymes Used in the Automated Enzymatic Synthesis of Oligosaccharides

Considering the advantages of enzymatic synthesis when compared with chemical synthesis as mentioned above, many enzymes have been explored for the assembly of complex oligosaccharides and glycoconjugates.189−194 The three most important categories of enzymes used in oligosaccharide synthesis are glycosyltransferases, glycosidases/glycosynthases, and the enzymes involved in oligosaccharide modification.5,112,195,196 The availability of enzymes is the major prerequisite for automation enzymatic synthesis of oligosaccharides. 4.1.1. Glycosyltransferases. Among the three classes of enzymes used for glycan synthesis, glycosyltransferases are the most widely used.195 Glycosyltransferases (GTFs, EC 2.4) are H


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a wide variety of useful enzymes. This requires future efforts to be made in identifying more enzymes for use in glycan synthesis. In addition, enzyme activity parameters such as reaction temperature, reaction pH, effect of metal ions, and preference of glycosylation donor and acceptor also need to be carefully investigated as these factors will affect glycosylation yield and reaction time. It is worth mentioning that most human glycosyltransferases have been successfully overexpressed using mammalian and insect cell systems by the Moremen and Jarvis groups recently.206 They removed transmembrane domains located at the N-terminal or C-terminal part of human glycosyltransferases to facilitate overexpression in both mammalian and insect cell expression systems. Using this strategy, most human glycosyltransferases can be expressed with a high expression level and high activity. This effort will definitely advance the use of glycosyltransferases for the synthesis of complex oligosaccharides.163 Moreover, an increasing number of enzymes are available from commercial sources, which allows nonspecialists to take advantage of enzymatic methods to synthesize oligosaccharides. 4.1.2. Glycosidases and Glycosynthases. Glycosidases (also called glycoside hydrolases or glycosyl hydrolases, EC 3.2) are enzymes that hydrolytically cleave glycosidic bonds in complex oligosaccharides.207 Since glycosidases catalyze the reversible cleavage of glycosidic bonds in complex oligosaccharides, they can also be used for synthetic purposes in the presence of excess glycosides to drive the reaction to favor product formation. As shown in Scheme 4, GalNAcα-Ser 31 can be prepared from GalNAcα-PNP 29 and N-protected L-serine methyl ester by a α-D-galactosaminidase from Aspergillus oryzae in which up to 50% yield can be obtained.210 Similarly,

Table 1. Eight Common Classes of Glycosyltransferase That Are Used in Glycan Synthesis no.

class

glycosylation donor

1 2 3 4 5 6 7

galactosyltransferases (GalTs) glucosyltransferases (GlcTs) glucouronyltransferases (GlcATs) N-acetylgalactosaminyltransferase (GalNAcT) N-acetylglucosaminyltransferase (GlcNAcT) fucosyltransferases (FucTs) mannosyltransferases (ManTs)

8

sialyltransferases (SiaTs)

UDP-Gal UDP-Glc UDP-GlcA UDP-GalNAc UDP-GlcNAc GDP-Fuc GDP-Man Dol-P-Man CMP-Neu5Ac

The advantages of employing glycosyltransferases for oligosaccharide synthesis is obvious. Reaction condition is simple and mild, without tedious protection/deprotection manipulations. Most glycosyltransferase-catalyzed reactions can finish in 15 min to 48 h. As shown in Figure 6, starting from a single oligosaccharide, a sugar library that contains a large number of oligosaccharides can be obtained by employing only a few robust glycosyltransferases.158,159,205 In recent years, an increasing number of glycosyltransferases have been characterized. Despite this, the number of identified enzymes is still limited when compared to the high diversity of glycan structures. In addition, an ideal glycosyltransferase used in automated enzymatic synthesis should be stable at its favorable glycosylation temperature, with high specific activity, and have tolerance over a wide range of pH values. Currently, the number of such ideal enzymes is very limited. Nevertheless, nature has already addressed the challenges of glycan synthesis by evolving

Figure 6. Enzymatic synthesis of an oligosaccharide library. PmST1, Pasteurella multocida α2,3sialyltransferase; Pd26T, Photobacterium damselae α2,6sialyltransferase; CstII, Campylobacter jejuni α2,8sialyltransferase; α1,3/4FucT, Helicobacter pylori α1,3/4-fucosyltransferase; α1,2FucT, H. pylori α1,2-fucosyltransferase; CgtA, β1,4-N-acetylgalactosaminyltransferase from C. jejuni; CgtB, β1,3-galactosyltransferase from C. jejuni; GalT, α1,3galactosyltransferase; GTA, human α1,3-N-acetylgalactosaminyltransferase; GTB, human α1,3- galactosyltransferase; LgtA, β1,3-N-acetylglucosaminyltransferase from Neisseria meningitidis; LgtB, β1,4-galactosyltransferase from N. meningitidis. I


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Scheme 4. Glycosidase-Catalyzed Synthesis of GalNAcα-Ser and GlcNAcβ-Ser

Scheme 5. Chemoenzymatic Glycoengineering of Human IgG with “Glycosynthase”a

a

Reproduced with permission from ref 222. Copyright 2017 American Chemical Society.

to measure with many enzymes displaying properties that appear to be intermediate between exo and endo.209 In 1998, Withers and co-workers created a “glycosynthase” by mutating a β-glycosidase (E358A) from Agrobacterium sp.211 When the nucleophile residue E358 of this β-glycosidase was replaced by a nonnucleophilic amino acid residue alanine, the glycosidase lost its hydrolytic activity but still ketp high activity in the synthetic reaction direction. After this breakthrough, a number of glycosidases have been successfully converted into glycosynthases for the efficient synthesis of oligosaccharides, glycolipids, and glycoproteins.212−221 As shown in Scheme 5 for the synthesis of homogeneous glycoproteins, heterogeneous glycans were removed by wild-type glycosidases first, and then structure-defined glycans can be installed back by a “glycosynthase” (mutant glycosidases) to produce glycoproteins with homogeneous glycans in high yield.222 Therefore, glycosidases

GlcNAcβ-Ser 34 can be prepared from GlcNAcβ-PNP 32 and N-protected L-serine methyl ester by a β-D-glucosaminidase from A. oryzae. However, only 7.5% yield can be obtained. Most glycosidases perform hydrolytic reactions through either inverting or retaining mechanisms.195 In addition, certain Nacetylhexosaminidases perform hydrolytic reactions through a substrate-assisted mechanism.208 These glycosidases have an acetamido group capable of neighboring group participation to form oxazolinium ion intermediates, which subsequently undergoes hydrolysis. Glycosidases can be classified as exoglycosidases and endo-glycosidases dependent upon whether they act at the end or in the middle of an oligosaccharide chain. Exo-glycosidases hydrolyze the nonreducing glycosidic bond to remove a single terminal sugar residue, whereas endoglycosidases hydrolyze an internal bond to remove more than one sugar residue. However, this classification can prove difficult J


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Figure 7. Representative examples of carbohydrate modifications in nature. Reproduced with permission from ref 223. Copyright 2012 American Chemical Society.

Figure 8. Chemoenzymatic synthetic schemes of ultralow molecular weight heparin construct 42 and 46. Synthesis started from disaccharide 39, which was then elongated to tetrasaccharide 40. Eight additional steps transformed 40 to construct 42 (left column). Steps d−h were combined in a sequential one-pot reaction format. Ten additional steps transformed 40 to construct 46 (right column). Recovery yield at each purification step was determined by parallel synthesis of the corresponding radioactively labeled oligosaccharide. Reproduced with permission from ref 234. Copyright 2011 The American Association for the Advancement of Science.

K


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Figure 9. Structures of the nine most common sugar nucleotides.

and built the target structure by employing glycosyltransferases, epimerase, and sulfotransferases, resulting in 37% and 45% yields of heparin 42 and 46. They also demonstrated that the synthesized compounds display excellent anticoagulant activity and comparable pharmacokinetic properties to commercial Arixtra in a rabbit model. A similar strategy was also used to produce homogeneous chondroitin with sulfate modifications.235 However, other enzymes involved in acylation, methylation, and phosphorylation have not been widely used for producing glycans with modified groups. In future studies, more efforts should be made to synthesize such modified oligosaccharides to understand their important roles in living cells.

and glycosynthases also have significant potential for application in automated enzymatic synthesis of complex oligosaccharides and glycopeptides in future studies. 4.1.3. Enzymes Involved in Oligosaccharide Postmodification. Oligosaccharide postmodifications such as acylation, sulfation, methylation, and phosphorylation are also very common in living cells (Figure 7, 35−38).223 These postmodifications can occur at various positions within a glycan chain to modulate a glycan’s overall biological function.223 Employing glycosyltransferases and glycosidases in oligosaccharide synthesis is widely studied and used by chemists and biologists. However, the use of enzymes involved in postmodification of oligosaccharides for glycan synthesis has been ignored for a long time. In recent years, with the availability of new tools to investigate the postglycosylational modifications of oligosaccharides, more details about the functions of these postmodifications in a variety of physiological and pathological processes have been clarified. For example, the sulfation of heparan sulfate determines its affinity for growth factors, which further influences its diffusion and retention within the extracellular matrix.224 Genetic disruption of carbohydrate sulfotransferases illustrates that sulfated glycosaminoglycans are especially important for development of connective tissue.225,226 As another example, more than 50% of sialic acids present in human colonic mucins are O-acetylated, and a reduced expression level of O-acetylated sialic acids has a strong relationship with colorectal cancer.227,228 In addition, Oacetylated sialic acids are overexpressed in many tumorassociated carbohydrate antigens such as sialyl Lewis X, GD3, and GM3.229,230 Currently, sulfotransferases have been successfully used in the chemoenzymatic synthesis of sulfated glycans.231−233 The commercial drug Fondaparinux (trade name Arixtra) is a structurally homogeneous ultralow molecular weight heparin pentasaccharide with multiple sulfated modifications. It is an anticoagulant and is clinically used for the prevention of deep vein thrombosis in patients who have had orthopedic surgery as well as for the treatment of deep vein thrombosis and pulmonary embolism. The commercial pentasaccharide is synthesized via chemical means through a lengthy process with very low yield. In 2011, Liu and co-workers developed a chemoenzymatic strategy to produce ultralow molecular weight heparin 42 and 46 (Figure 8).234 They started from chemically synthesized disaccharide 39

4.2. Glycosylation Donors

In addition to the enzymes, the availability of glycosylation donors is another important factor to consider in order to achieve automated enzymatic synthesis of oligosaccharides in large scale. Most glycosyltransferases are of the Leloir type that use activated sugar nucleotides as glycosylation donors. The most common sugar nucleotides in eukaryotes include UDPGlc, UDP-GlcNAc, UDP-GlcA, UDP-Gal, UDP-GalNAc, UDPGalA, UDP-Xyl, GDP-Man, GDP-Fuc, and CMP-Neu5Ac (Figure 9). Some other sugar nucleotides such as CMP-KDO, ADP-Hep, dTDP-Rha, and UDP-FucNAc are very common in bacteria.236 In addition, a few glycosyltransferases use sugar phosphates or polyprenol sugar phosphates as their glycosyl donors. For example, human mannosyltransferases are a wellstudied example which use dolichol phosphate mannose as an activated sugar donor.156 Glycosylation donors for glycosidases/ glycosynthases are normally monosaccharide analogues, such as phosphorylated sugars and fluoride sugars,222,237,238 which can be obtained through either commercial sources or prepared via synthetic methods. Conversely, nucleotide sugar substrates the donors utilized by glycosyltransferasesare often commercially expensive (with the exception of UDP-Glc). Over the past few years the large-scale preparation of sugar nucleotides has been explored by many groups.239−261 A convenient method called silver nitrate precipitation was recently developed for quick purification of sugar phosphates,262,263 which are intermediate for preparation of nucleotide sugars. Nevertheless, some sugar nucleotides, such as CMP-Neu5Ac and CMP-KDO, are highly unstable. To address this issue, one-pot multienzyme strategies and in situ sugar nucleotide regeneration systems L


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Figure 10. (A) Sequential one-pot multienzyme (OPME) synthesis of ganglioside oligosaccharides GD1b. Reproduced with permission from ref 149. Copyright 2016 American Chemical Society. (B) Several sugar nucleotide regeneration systems for GalT (galactosyltransferase), GalNAcT (Nacetylgalactosaminyltransferase), SiaT (sialyltransferase), and FucT (fucosyltransferase).

Scheme 6. (A) Enzymatic Elongation of GalNAc on a PEGA Resin Bead with Gal via a GalT; (B) Glycosylation of Glucose with Galactose on a PEGA Resin via a Glycosynthase

4.3. Supports Used in Enzymatic Glycosylation

(Figure 10) have been employed to synthesize complex glycans.112,145,149,264−268 For example, the combination of sialyltransferases with one-pot synthesis of CMP-Neu5Ac from CTP and ManNAc or Neu5Ac analogues using sialic acid aldolase and CMP-sialic acid synthetase is widely used in the large-scale synthesis of sialylated oligosaccharides.142,164,165 The coupling of nucleotide sugar generation systems with enzymatic glycosylation in a one-pot system does not require pure sugar nucleotides as starting materials and, therefore, not only saves time but also makes large-scale preparation of oligosaccharides a viable strategy. In addition to the regeneration of sugar nucleotides, the regeneration of other enzyme cofactors such as NADP+ and NAD+ can also be used in the synthesis of glycans that contain rare sugar residues.269 These strategies can be employed in automated synthetic systems to achieve large-scale synthesis.

Finding a suitable support systembe it a resin, polymer support, tag, or something else entirelyis a key factor in bridging enzymatic reactions and automation and is the first roadblock to overcome in achieving that goal. Different supports have diverse impacts on enzymatic reactions and ultimately require tailor-made designs for the automation machine. Hence, the conversion rate of enzymatic reactions and the suitability toward automation conditions are the two main aspects for the selection of supports. 4.3.1. Solid-Phase Supports. Encouraged by the progress researchers have made in SPPS, solid supports were the first choice when selecting a vector for glycosylation reactions. Conventional resins, such as those used in solid-phase peptide synthesis, are low swelling in an aqueous environment, producing limited or congested cavities and thus providing a limited surface area for reactions to take place. Therefore, the swelling ability and swelled porous structure of resins largely M


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Scheme 7. Enzymatic Solid-Phase Synthesis of Lewis X Tetrasaccharide on a Sepharose Bead

Scheme 8. (A) Solid-Phase Synthesis of Glycopeptide on an Aminopropyl Silica Solid Support; (B) Solid-Phase Enzymatic Synthesis of a Tetrasaccharide with a Controlled Pore Glass Solid Support

Both PEGA and sepharose have good swelling properties in aqueous solutions. PEGA is also widely used in peptide synthesis with its favorable swelling property in organic solvents, which can enable peptide synthesis and enzymatic sugar synthesis on the same resin without cleaving the glycopeptide substrate off before enzymatic extension. Sepharose is not very suitable for peptide synthesis because of the large amount of free hydroxy groups which lead to poor solubility in organic solvents. However, these hydroxy groups provide sepharose with enough hydrophilicity suitable for enzymatic reactions. In 1994, Meldel and co-workers reported the development of a PEGA copolymer that swells 10−20 fold in aqueous solutions,270 enough of an increase to allow large biomolecules access to linked substrates. The resin was made by copolymerizing a mixture of mono- and

affects the productivity of the enzymatic reactions. For the solid supports with hard cores which allow the enzymatic reactions to happen on the outside surface of the solid support, almost all need a suitable spacer to reduce the hindrance from the large surface of the solid support and ensure the enzymes have adequate space to smoothly approach the substrates. As a result, choosing a proper solid support is significant to reaching high conversion rates. Any solid support chosen must have an appropriate structure to provide adequate swelling or sufficient surface area to allow access for large biomolecules and a proper substrate loading density. 4.3.1.1. Swelling Resins. Swelling resins such as poly(ethylene glycol) polyacrylamide (PEGA) and sepharose have been employed in the enzymatic synthesis of oligosaccharides. N


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Figure 11. (A) SAM on a gold surface displaying GalNAc residues. (B) Enzymatic oligosaccharide synthesis of a glycopeptide on a gold surface.

diacryloylated polyethylene glycol with acrylamide or N,Ndimethylacrylamide which swells highly in both organic and aqueous phases. The swelling in aqueous solutions was good enough to allow access to glycopeptides by glycosyltransferases of molecular weight ranging to 50 000, which is adequate to enable some enzymes to perform glycosylation. To demonstrate this new resin, the researchers performed a single glycosylation of a terminal GlcNAc in compound 48 with Gal using UDP-Gal and β1,4-GalT, achieving a near quantitative conversion within 72 h (Scheme 6A) to obtain 49. The resin can be cleaved off by treating with concentrated aqueous acids like trifluoroacetic acid, which in this case resulted in 50. In 2002, Withers and coworkers reported the application of PEGA resin with amide backbone in the galactosylation of a glycopeptide using glycosyl fluorides as donors via a glycosynthase (Scheme 6B).271 In 1997 and 1998, Blixt and Norberg and their co-workers reported a pair of solid-phase oligosaccharide syntheses on a sepharose bead solid support.272,273 In 1997, a glycosyl acceptor with a thio group was synthesized using thibutyrolactone. Treating this with thiopropyl sepharose gave the glycosyl acceptor bound to sepharose via a disulfide bond. Fucosylation was done on such a sepharose-supported disaccharide using α1,3-FucT to obtain a disaccharide in good yield. The resin was cleaved off by treating with mercaptoethanol or DTT to dissociate the disulfide bond. Later in 1998 the same group reported the use of thipyridyl sepharoses with different arm lengths as supports. They noted that the long length of the linker was particularly important, suggesting that sepharose may interfere with the glycosyltransferases they were using. They successfully synthesized the well-known tumor-associated carbohydrate antigen sialyl Lewis X tetrasaccharide 56. The oligosaccharide was retrieved from the resin by treating with dithiothreitol (DTT) (Scheme 7). 4.3.1.2. Nonswelling Resins. The hard core of nonswelling resins when used with an appropriate spacer provides a good support for enzymatic reactions; however, nonswelling resins are less compatible with certain enzymes compared to other techniques. In 1994, Wong and co-workers first reported the enzymatic solid-phase rapid iterative synthesis of oligosacchar-

ides on an aminosilica-based solid support that is compatible with both organic and, importantly, aqueous solvents.274 Among several solid supports tested, aminopropyl silica with an appropriate density of functional groups (1.5 mmol/g) and good hydrophilicity was found to be the best support for the synthesis of sialyl Lewis X glycopeptide 60 which was prepared in good yield (Scheme 8A). Later in the same year275 Wong an co-workers demonstrated a solid-phase enzymatic synthesis of a tetrasaccharide with controlled pore glass (CPG) as the solid support (Scheme 8B). CPG was selected as the support as it is incompressible in aqueous environments and does not sequester enzymes during enzymatic reactions. A disaccharide was initially coupled to CPG via a spacer group containing an ester bond (61), and Gal and Neu5Ac were later coupled to the disaccharide using glycosyltransferases to obtain tetrasaccharide 63. The conversion for each step averaged 98%. 4.3.1.3. Nonwater-Soluble Gold and Polymer Surface Supports. For automated enzymatic microsynthesis of oligosaccharides, nonwater-soluble gold or polymer surface support is a good option that has been successfully applied in sugar microarrays. Substrate density control and proper spacer selection are potential challenges for this type of system, as they do have an effect on overall efficiency enzymatic reactions. Moreover, it seems that it is not possible to use this sort of surface support for large-scale synthesis for now, because substrate density needs to be limited to a certain level to achieve good conversion rates. In this context, self-assembled monolayers (SAMs) on a gold surface have commonly been used to model the surfaces of cells to study biointerfacial interactions. The relative ease with which the density, nature, and environment of the functional groups displayed to enzymes can be controlled is an asset in this application. The use of SAMs eliminates the issue of nonspecific adsorption of proteins. Using this solid support technology, Houseman and Mrksich276 attempted to model glycans and the glycan modification process on cell surfaces through the use of SAMs of alkanethiolates on a gold surface (Figure 11A). They prepared substrates where some alkylthiols were terminated with GlcNAc and others with O


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Figure 12. (A) Generating a dextran brush on a gold surface with dextransucrase. (B) Wehner method for immobilizing carbohydrates on either polystyrene or gold surfaces.

tri(ethylene glycol). Glycosyltransferase β1,4-GalT successfully transferred Gal to the fourth position of the GlcNAc acceptor. The disaccharide was cleaved from the alkanethiolates by irradiation under UV. They discovered that density plays a critical role during glycosylation, with ligands immobilized at low densities being more accessible to enzymes than highdensity ligands. Š ardziḱ and co-workers277 also demonstrated glycosylation on a gold microarray platform by placing a mannopeptide on SAMs. Further, the monosaccharide Man was extended using a series of glycosyltransferases to obtain a tetrasaccharide mannosylpeptide, which is known to play a role in regulating the function of α-dystroglycan. (Figure 11B). Beginning in 2012, Xu and co-workers reported the use of bioviable poly(ethylene glycol) (PEG) on a gold surface as a support to perform glycosylations.278 They used PEG tethered to a gold surface as a spacer and also as a substrate for enzymatic transglycosylation. PEG brushes were treated with the βgalactosidase, which transferred the Gal to the hydroxyl group of PEG in the presence of lactose (Figure 12A). Later in 2015 they reported the dextransucrase (Dsase)-catalyzed synthesis of a polysaccharide by elongating the glucose/maltose acceptors immobilized on SAM surfaces.279 While there are several methods for immobilizing a glycan on a solid surface, most methods are tailored to a single surface. Wehner and co-workers described a method for preparing glycoarrays using a linker that can quickly be modified to suit either gold or polystyrene surfaces (Figure 12B).280 The substrate is immobilized on the gold-based array through a direct covalent linkage. To attach the substrate to a polystyrene array, a hydrophobic triphenylmethyl tag was installed to increase the affinity. The group sticks to the surface of the polystyrene through noncovalent interactions. This strategy had previously been shown to be robust against aqueous washes. 4.3.2. Solution-Phase Supports. Solution-phase synthesis, in which a substrate acceptor is bound to a water-soluble polymer or a special tag, can bypass the compatibility issue

between enzyme and solid supports, ensuring a higher conversion rate. Nevertheless, to achieve solution-phase automation of oligosaccharide synthesis, the development of a strategy to efficiently capture and release the substrate/product bonded water-soluble polymers or special tags is necessary. One well-developed option is the use of a special affinity tag such as a fluorous tag or an amino group, which can be captured directly from the solution by the appropriate resins.281 Another option is the incorporation of large water-soluble polymers, so that the product can be trapped by mechanical technologies such as ultrafiltration, ultracentrifuge, or thermal precipitation. 4.3.2.1. Fluorous-Tagged Glycosylation. Fluorous solidphase extraction (FSPE) is an attractive protocol for the automated synthesis of oligosaccharides. Compounds tagged with a CnF2n+1 moiety can be purified via a fluorous silica gel column that traps tagged compounds regardless of polarity during a water wash, releasing the compound only under a methanol wash. This procedure has been extensively studied for the past few decades with a wide variety of compounds,282,283 with a large number of researchers employing this technology in the chemical synthesis of oligosaccharides.284−293 It is important to consider that fluorous tags have electron-withdrawing properties that may be detrimental to the rapid growth of oligosaccharides and decrease overall yields. Additionally, fluorous tags are not overly soluble in reaction conditions and solvents that are common in routine glycosylations. To solve these problems, one of two approaches can be utilized: increase the solubility of the linker itself or introduce the soluble linker later in a “catch−release” procedure. These tags can be introduced into the glycosyl acceptor before or after glycosylation via a fluorous glycosyl donor. Pohl and co-workers investigated the use of a FSPE protocol that featured a tag that promoted solubility of the compound and linker by introducing an oxygen heteroatom into the linker separating the carbohydrate chain and the fluorous tag (Figure 13A).284 Linear and branched mannose oligosaccharides with P


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Figure 13. (A) Chemical synthesis of a linear mannosaccharide with via a FSPE procedure. (B) Fluorous tags in the synthesis of sialosides and galactosides.

Scheme 9. Synthesis of Heparan Sulfate Using a Fluorous Tag

up to five sugar residues, like compound 66, were both synthesized using this methodology. They noted that this approach had greater yields than similar reactions without an oxygen linker.288 With its general applicability and standardized reaction procedures, the authors endorsed this idea as a suitable method for automated synthesis of oligosaccharides.

Yang and co-workers performed a one-pot chemical glycosylation by employing a postglycosylation tagging strategy to isolate the final compound.285 Ketone-functionalized carbohydrates could be purified from the reaction mixture by reacting them with a fluorous-tagged hydrazide. The bound compound is isolated using FSPE, the tag is cleaved from the Q


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Figure 14. Ion-exchange purification used to synthesize HMOs.

Scheme 10. OPME Synthesis of a Pentasaccharide GSL

amino group can function as the special tag instead of conjugating a complex tag with several synthetic steps. However, one possible challenge of this technique is that the accumulation of salts after each ion-exchange step may affect subsequent enzymatic reactions, especially after multiple cycles. In their work, the disaccharide was subjected to enzymatic elongation with cation-exchange chromatography performed between each step to capture the product. The researchers noted that of the four glycosyltransferases used, each one had higher enzymatic activity when compared to similar reactions performed on solid supports. 4.3.2.3. Lipid-Linked Glycosylation. Attaching a lipid linker to a biomolecule is a similar technique to the previously discussed tagging techniques. Not only does the lipid tail facilitate quick and easy (typically about 30 min) separation by C18 cartridgea technique that is extensively used in the literaturebut also many oligosaccharides are naturally linked to lipids, meaning that a cleavage step is not necessary for many syntheses that utilize them. For this lipid technology, long-tail lipids are preferred for C18 separation. However, long-tail lipids can decrease the solubility of sugar−lipid substrates. On the other hand, extended sugar chains will decrease the efficiency of C18 separation because of increased hydrophilicity. It should be noted that this technique is good for large-scale syntheses of oligosaccharides with a limited number of sugars. Santra and coworkers 296 described a one-pot multienzyme (OPME) approach for the synthesis of glycosphingolipids (GSLs) like compound 79 (Scheme 10). The lipid tail was converted into a bilipid tail via acylation of free amine in 78 and purified by a Sephadex column. 4.3.2.4. Thermoresponsive Polymers. In 2001, Wong and co-workers reported the use of thermoresponsive polymers in enzymatic glycosylation reactions.297 These polymers exhibit inverse temperature-dependent solubility in water, being soluble at low temperatures yet precipitating out of solution once the

compound using TFA in acetone, and then the mixture is purified by running a FSPE column once again. Postglycosylation fluorous tagging has also been used by the Seeberger and Liu groups with success.289,290 Chen and co-workers studied the effect of fluorous tagging and linkage strategies on the performance of one-pot multienzyme glycosylations to synthesize a series of fluorous-tagged sialosides and galactosides (Figure 13B).294 They noted that short fluorous tags (e.g., −C3F7) allowed for solubility during the enzymatic reactions; however, larger oligosaccharides could not be retained on the column as efficiently, thus lowering yields and purity. Larger tags would keep the product on the column during the purification steps, yet the threat of the tag reducing overall solubility and enzymatic activity during reactions remains. By linking the larger tags, they tested (−C6F13 and −C8F17) with propylamide linkers and noted an increase in solubility; however, they still encountered issues with decreased enzymatic activity. Triethylene glycol (TEG) and hexaethylene glycol (HEG) linkers were ultimately used to significantly improve the yields of the glycosylation reactions. Linhardt and co-workers also demonstrated fluorous-supported enzymatic oligosaccharide synthesis (Scheme 9).295 Fluorinated tert-butyl dicarbonate (FBoc) tag 67 at the reducing end was used for the enzymatic oligosaccharide elongation without jeopardizing enzyme activities. Using the FBoc tag on the disaccharide, they could successfully synthesize the heparan sulfate tetrasaccharide and hexasaccharide 76 in a chemoenzymatic fashion in good yields using FSPE. 4.3.2.2. Ion Exchange. In a report from Fang and coworkers,281 an ion-exchange column was developed to assist in the synthesis of human milk oligosaccharides (HMOs) (Figure 14). An incredibly simple tag consisting entirely of a propylamine group was functionalized onto the anomeric end of lactose, the tag allowing for simple purification via ion-exchange chromatography. The advantage of this technique is that the R


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Figure 15. Thermoresponsive polymer used as a support to synthesize Lewis X and Sialyl Lewis X glycans.

Scheme 11. (A) Enzymatic Extension on a Water-Soluble Polymer and Transfer to Ceramide Linker; (B) Solid-Phase Sepharose Linked Glycosyltransferases Extend PAA-Linked Oligosaccharidesa

a

Reproduced with permission from ref 299. Copyright 2001 The Royal Society of Chemistry.

lower critical solution temperature (LCST) has been exceeded. This is due to the additional intramolecular hydrogen bonding within the polymer that is promoted at higher temperatures, which drives out internal water and encourages precipitation. Copolymers of N-isopropylacrylamide (NIPAm) have been the premier example of this phenomenon. A variety of enzymes, including glycosyltransferases, were immobilized onto NIPAm polymers and their activities measured. It was found that these enzymes retained the majority of their activities. The enzymes can then be recovered easily through gentle heating and centrifugation of the precipitated polymer. Additionally, these polymers proved useful for enzymatic glycosylation. A GlcNAc residue was functionalized onto the polymer via a hydrophilic linker 80 (Figure 15). A series of glycosyltransferases with the appropriate sugar nucleotides was introduced to synthesize both Lewis X and sialyl Lewis X polysaccharides in approximately 60% yields. Between each step, the oligosaccharide-polymer conjugates were gently heated to allow for efficient purification via centrifugation. The authors

suggest that with further refinement this technique can eliminate the need for size-exclusion chromatography for separation, simplifying purification overall. 4.3.2.5. Polyacrylamide-Supported Enzymatic Glycosylation. For many years, Nishimura and co-workers have been experimenting with clustering sugar acceptors on a water-soluble polymer. This allows for enzymes introduced to the reaction to take advantage of the polymeric glycoside-cluster effect, a phenomenon where multivalent glycosides experience higher affinity than more disperse glycosides, increasing yields and decreasing reaction times. Not quite a solid support and not exactly a tag, this polymer-based procedure does share some of the same hallmarks, namely, improved yield and ease of purification and recovery. In 1997, Nishimura and co-workers described the enzymatic synthesis of Ganglioside GM3 83 by clustering acceptors on a water-soluble polymer (Scheme 11A).298 Lactose was reacted with a ceramide mimetic “linker” that could be polymerized into a water-soluble cluster, taking advantage of the polymeric glycoside-cluster effect for the glycosylation reaction to obtain S


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Figure 16. Glycan array built by Etxebarria and co-workers using PEG-supported synthesis.

Scheme 12. MUC1 Glycopeptide Enzymatic Synthesis on a Peg-Chain Supporta

a

Reproduced with permission from ref 303. Copyright 2014 The Royal Society of Chemistry.

sialyl Lewis X derivative 94, was recovered. The total reaction took 4 days and resulted in a 16% overall yield. 4.3.2.6. Water-Soluble PEG Support. The poly(ethylene glycol) tag is one of the most widely used tags due to its solubility in aqueous and organic solvents and has been used in the solid-phase synthesis of peptides, nucleotides, and oligosaccharides. The insolubility of PEG in solvents like ethanol leads to a more facile purification, making it an attractive tool for automated synthesis. The presence of a PEG tag on a growing carbohydrate chain changes the solubility properties of the oligosaccharide, which lends itself well to purification and recovery of the final product. Additionally, the presence of a PEG tag appears not to interfere with the enzymes facilitating the glycosylation reactions, making them another attractive option for solid-phase synthesis. However, the efficiency of precipitating PEG with ethanol in the presence of some water is a possible challenge for this technology. Other additives such as sugar nucleotides and glycosyltransferases can also be precipitated out with a low portion of water at the same time. Moreover, high-speed centrifugation is needed for collecting precipitated PEG, which will increase the cost of the instrument design. Etxebarria and co-workers prepared 13 PEG101-tagged sugars using a combination of precipitation and ultrafiltration to purify and recover the final compounds (Figure 16).302 The large size of the substrate makes for easy purification by ultrafiltration. The glycan primers were first coupled to the bifunctional PEG tags and then elongated using the glycosyltransferases. Such glycan structures were directly printed onto the activated glass slides with good sensitivity and spot morphology. Lectins tended to favor the PEG-linked glycans over the C5-amino-linked homologues. This was especially true for the smaller glycans. Bello and co-workers303 used standard solid-phase peptide

82. In 2001, Nishimura and co-workers returned to the use of a polyacrylamide support for enzymatic extension of oligosaccharides,299 this time using immobilized glycosyltransferases on a sepharose bead to synthesize the sialoside 85 (Scheme 11B). Using a water-soluble polymer and having glycosyltransferases on a solid support allows for ease of purification and recovery of both the materials and the enzymes. It was noted that the relative activity of the immobilized glycosyltransferases was retained after each reaction and could be recycled for multiple reactions. It was also observed that the inhibitory effects of UDP on GalT were significantly reduced on the immobilized enzyme. The next year Nishimura and co-workers continued their work with water-soluble polymer-supported oligosaccharide synthesis,300 incorporating his technology with solid-phase glycopeptide synthesis to generate glycopeptides. In a 2010 report, Nishimura and co-workers demonstrated his Golgi Apparatus machine for the automated enzymatic synthesis of oligosaccharides, one of the first machines built for such a purpose (Scheme 14).301 This machine will be discussed in a later section. However, it is important now to note a few key components used by this machine, namely, (1) the synthesis of glycosyl acceptors with a heterobifunctional linker, (2) the ligation of the linkers with water-soluble polymers, (3) the enzymatic extension of the sugar moieties, and (4) the release and purification of the final products. The last two points were identified as key developments for the realization of automation. A poly(amidoamine) dendrimer (G7 PAMAM) was functionalized with a sugar residue on a linker that featured a tetrapeptide moiety 92 recognized as a cleavage site by BLase. One of the advantages of this work is the automation of the cleavage step. Glycosylation reactions were then carried out by the Golgi machine modified from regular HPLC, and the final product, a T


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Figure 17. Linkers and spacers in Supported Glycan Synthesis.

glycopeptide on a silica support. This initial peptide, with an additional six glycine segment, is built up by chemical synthesis on the silica. The final amino acid contains an N-linked GlcNAc which is extended enzymatically. The glycopeptide is cleaved off the support using α-chymotrypsin (Scheme 4A). The final glycosylation is then performed in solution. The synthesis of a range of glycopeptides using the same sort of linkage strategy was published by Nishimura and Matsushita and their coworkers.300,301,304 They prepared a monosaccharide-containing glycopeptide linked to an aminooxy-functionalized PAMAM dendrimer. The peptide contains an additional phenylalanine and glutamic acid at the N-terminus which is recognized and cleaved by a glutamic-acid-specific protease from B. licheniformis (Blase).305 4.4.2. Disulfide Linkers. A fairly gentle option is to use disulfide bonds as a coupling/cleavage strategy. This is the method used by Blixt and Norberg in 1997.272 A disaccharide on a thiol-functionalized linker was joined to thiopropyl−sepharose resin with a disulfide bond. After successful enzymatic extension the glycan with the linker was removed from the solid support using 2-mercaptoethanol. The researchers noted that resin and linker selection had an effect on the successful transfer of fucose, with potential outcomes varying drastically depending on the choice. The next year Blixt and Norberg reported the development of a new linker featuring a disulfide linkage connected to a sepharose solid support for use in solid-phase enzymatic syntheses (Figure 18).273 The design of the linker

synthesis to prepare a MUC1 peptide with an additional seven residue tobacco etch virus (TEV) protease recognition section and a PEG27 chain (Scheme 12). The peptide was then glycosylated and extended enzymatically to procure compound 88, with the PEG tag being used for purification and recovery steps. The PEG tag was removed through the use of a TEV protease that cleaved off the peptide fragment containing the PEG27 moiety to get glycopeptide 89. 4.4. Linker and Spacer

The second roadblock to automated enzymatic synthesis of oligosaccharides is the linker, which is essential for both solidand solution-phase strategies. As the linker is the direct connecting point between supports and substrates, it should be cleavable under mild condition without affecting the glycosidic bonds (Figure 17). The linker needs to remain robust to the normal operating conditions of the system until cleavage is desired. Additionally, the linker should not affect enzymatic reactions and the desired product structure can be obtained after cleavage without unwanted groups or chains on the products. The feasibility of constructing substrate−linker− support conjugate also needs to be taken into consideration. An enzyme which works very well on a glycan in solution may not work at all if the glycan is bound to a surface. A suitable spacer is sometimes needed, which can increase enzyme’s accessibility to substrates, especially for solid-phase support and water-soluble polymers. To this end, intelligent spacer design is often of great importance. A spacer puts distance between the glycan and the unnatural support, hopefully minimizing the impact of the support on the interaction between the enzyme and the glycan. 4.4.1. Protease-Cleaved Linkers. A number of researchers have used peptides as cleavable linkers. The peptide backbone is remarkably stable to reaction conditions. However, the inclusion of a protease recognition site can allow for easy enzymatic cleavage. In these cases, the target is a glycopeptide and the enzymatic extension occurs directly on an O- or N-linked glycan on the peptide. The peptide is engineered with a proteolytic cleavage site that is not a part of the desired peptide sequence. After the extension is complete the appropriate protease is used to cleave the glycopeptide from the support. The recognition of enzymes for glycopeptides is fairly high. The addition of a few additional residues at the end of a glycopeptide is unlikely to make the substrate unacceptable to an enzyme that would otherwise have acted on it. Also, if the target is a glycopeptide then solid-phase synthesis is probably being used anyway and adding a few more residues is not burdensome. The major disadvantage is that the product must be a glycopeptide. If the desired target is a free oligosaccharide a different strategy would be better. Also, in most cases it is necessary to first prepare a peptide with a small glycan by chemical means. Usually SPPS uses up to 10 equiv of amino acid to install each residue. While it is practical to use less at the cost of time and yield, ample glycosylated amino acid must still be prepared. These compounds are rarely trivial to make. An early example of this strategy is the work published by Wong and co-workers in 1994.274 They prepare a sialyl Lewis X

Figure 18. Linkage strategy used by Blixt and Norberg.

allowed for the high recovery of products, with greater than 95% product recovered when treated with DTT. Additionally, they experimented with the length of the linker and found that longer linkers produced the greatest yields, albeit with diminishing returns after a certain length; the authors declined to speculate about the cause of the increase but noted that similar results were observed by other laboratories. Individual saccharide transfers were measured in the 90%−98% range by NMR, and U


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the sialyl Lewis X tetrasaccharide was synthesized in a 57% yield over a few days. 4.4.3. Photocleavable Linkers. The use of a chemical cleavage system is limited by the need for orthogonality with any protecting groups in use. In simple systems this is usually not a problem. In more complex systems, however, the need for safe and efficient cleavage conditions sometimes encourages researchers to look to alternatives to chemical cleavage. Photocleavable linkers are growing in popularity, and there are examples of their use in chemical, but not enzymatic, glycan synthesis. In 2015 Bello and co-workers improved upon their prior work by the addition of a photocleavable auxiliary which facilitates both cleavage and the thiol needed for native chemical ligation (Figure 19).306 Wilsdorf (and Seeberger) use a

Figure 21. Modified Boc protecting group as a linker.

hydrophobicity of the linker two enzymatic extensions, one using β-1,4-galactosyltransferase and the other using α-2,3sialyltransferase, were performed successfully. 4.4.5. Safety-Catch Linkers as a Potential Option. Safety-catch linkers are linkers which must be activated before they can be cleaved. The two-step process improves stability when compared with a single-step process. The classic acylsulfonamide safety-catch linker was published by Kenner and co-workers in 1971.308 This linker has been used in the chemical synthesis of glycans309 but has not thus far been used in conjunction with enzymes (Figure 22). Similarly, Tanaka and co-workers used a phenyl ether linker as a safety-catch linker in the fluorous-assisted chemical synthesis of oligosaccharides.310 In their work, the phenyl ether linker is reduced to a vinyl ether which can be removed under mild acidic conditions. Again, this strategy does not appear to have been attempted in conjunction with enzymatic synthesis. 4.5. Automation Instruments for the Automated Synthesis

Figure 19. Bello’s photocleavable auxiliary. Reproduced with permission from ref 306. Copyright 2015 John Wiley and Sons.

Biopolymers, including nucleotides, peptides, and saccharides, are polymers produced by living organisms.311 These polymers play an important role in their bioactivities, and as such, it is useful to be able to prepare them as needed. However, it is slow, laborious work to manually synthesize them at any scale. The development of automated machines for the synthesis of biopolymers has saved researchers’ time, increased their output, and helped uncover biological mechanisms that might otherwise have remained elusive. The concept of biopolymer automation was pioneered by Merrifield and co-workers in the early 1960s.172 This basic principle of using a support to facilitate product recovery remains the basis of automation synthesis of biopolymer today. With advances in computer science, we see many areas experiencing considerable automation. Simplifying the task of performing a complex synthesis down to pushing a few buttons would further expand the scope of research undertakings which can be undertaken at present. With such a highly automated machine, even nonexperts could synthesize complex biopolymers. Over the past few decades peptide and gene synthesizers have been commercially available, and oligosaccharide synthesizers have been in use as well for over a decade. Their synthetic strategies all follow the general principle of SPPS, namely, the repeating reaction and wash cycles with the substrate bound to solid resin. These

photocleavable linkage along with another reducibly cleavable section to yield free reducing glycans after chemical solid-phase synthesis (Figure 20).307 4.4.4. Acid- or Hydrazine-Sensitive Linkers. Most often cleavage is performed by treatment with acids or bases. Cleavage by strong acid, usually TFA, is the standard for SPPS. Inevitably there is some carryover from this field if only in the synthesis of glycopeptides. This linkage strategy is risky if used with unprotected glycans. Palcic and co-workers270 used a short peptide spacer on standard acid-cleavable PEGA resin as did Withers.271 Glycosidic bonds are not usually very stable with respect to strong acids. To prevent any chance of damage to the glycan Withers performed a peracetylation prior to the cleavage. Cai and co-workers synthesized heparin sulfate oligosaccharides using fluorous tagging for purification between steps. The tag takes the form of a modified Boc-type protecting group (Figure 21).295 Similar FBoc tags are commercially available and may be installed and removed in much the same way as a standard Boc group. Halcomb and co-workers performed glycosylations with a carbon chain spacer and a hydrazine cleavable linker in 1994.275 Despite the simplicity and

Figure 20. Wilsdorf and Seeberger’s photocleavable/reducing strategy. V


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Figure 22. Acylsulfonamide safety-catch linker.

Superbeads approach. In this strategy all of the enzymes needed for the synthesis are bound onto solid beads (e.g., galK, galT, galU, and pykF), and these beads are used to perform glycosylation.338 4.5.3. Seeberger’s Synthesizer. In 2001, the first oligosaccharide synthesizer, a modified AB 433 peptide synthesizer, was set up by the Seeberger group.175 After that many researchers have built their own synthesizers, such as the HPLC-assisted automated synthesizer developed by Demchenko and co-workers312,326 and the syringe pump-based electrochemical synthesizer developed by Yoshida and co-workers.339 The most successful oligosaccharide synthesizer is the commercially available Glyconeer developed by Seeberger and co-workers. They use the acceptor-bound strategy for their synthetic process. As shown in Scheme 3, monosaccharide building blocks were first prepared, repeats of coupling and deprotection cycles were done on the solid phase, and the product is cleaved off and purified in the final step. To briefly describe the current machine, acceptor-bound resin is first loaded in the reaction vessel, the building block solutions and other reagents are added through valves, all of the reagents are removed through a filter after reaction is complete, and the resin is washed with solvent (Figure 23). The automated synthesis process is a simple repeat operation and can easily be monitored and controlled by computer. 4.5.4. Other Synthesizers. In 2012, Demchenko and coworkers developed an HPLC-based automated synthesizer using a similar strategy to that mentioned above (Scheme 3).312 The difference is that Demchenko and co-workers used an

synthesizers, including those machines developed by Seeberger and co-workers and Demchenko and co-workers,312,313 can accurately perform the coupling−wash−deprotection−wash cycles under computer control. In the near future, the first viable oligosaccharide synthesizers might be set up using modified peptide synthesizers. 4.5.1. Peptide Synthesizer. The solid-phase peptide synthesizer is a strong example of a highly automated production process that has been commercially available for a considerable length of time. The first such synthesizer was developed by Merrifield and co-workers in the mid-1960s. As a testament to its usefulness, they were able to prepare a 124 residue peptide with their machine.170,314 For decades researchers have had access to all sorts of different peptide synthesizers. Often these machines have different useful features, such as allowing for the synthesis of multiple peptides in parallel and the use of microwave synthesis.315−318 We can divide these machines into two broad classes: value systems and X−Y robotic platforms. The former is preferred for single syntheses and later for parallel syntheses, especially in the preparation of peptide libraries. With only a little training a technician can prepare crude peptides with little more than the push of button. 4.5.2. Early Progress toward Automated Oligosaccharide Synthesis. The case with oligosaccharide synthesizers is considerably more complicated. The first example in the literature was reported by Frechet and Schuerch and their coworkers in 1971.174 In 1994, several groups reported automated oligosaccharide synthesis based on enzymatic glycosylation and a solid-phase synthetic strategy.274,275 Over the past several decades, many different researchers have invested time and effort into setting up a convenient and effective SPPS-like automated system for oligosaccharides. A common strategy was to use beads with the enzymatic or chemical reactions occurring on the beads.319−332 The chemical solid-phase synthesis of oligosaccharides has two different strategies, either acceptor bound or donor bound on the solid phase.333 When the acceptor is bound on the solid phase, the donor solution serves as the mobile phase and is washed away after each glycosylation step. In the donor-bound method, the acceptor solution serves as the mobile phase and is removed after the glycosylation. The enzymatic solid-phase synthesis of oligosaccharides also has the same two basic strategies, with either the saccharide or the enzymes bound on the solid phase. Most of the researchers put the enzymes and sugar nucleotides in the mobile phase and bind the saccharide onto the solid phase.275,334−337 Others do solidphase enzymatic synthesis, which fixes the enzyme on the solid phase and puts the saccharides and sugar nucleotides in the mobile phase. One example of this approach is Wang’s

Figure 23. Schematic diagram of Seeberger’s oligosaccharide synthesizer. W


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5. AUTOMATED ENZYMATIC SYNTHESIS OF OLIGOSACCHARIDES

unmodified HPLC as the machine for the automated synthesis (Figure 24). Briefly, acceptor-bound resin is first loaded into the

5.1. Artificial Golgi Apparatus

In 2010, Nishimura and co-workers developed an artificial Golgi apparatus for the fully automated enzymatic synthesis of oligosaccharides.301 The machine they used was derived from an HPLC.340,341 Its major parts included buffer bottles, a fraction collector, a computer with dedicated software for controlling the operation of Golgi, a hollow fiber ultrafiltration (UF) module, a UV detector for the retained solutes from UF module, a UV detector for solvent and small solutes from UF module, a pump, and a programmable autosampler unit. To achieve automated synthesis, water-soluble poly(amidoamine) dendrimers were used as the supporting polymer. In addition to the relative ease of attaching surface ligand groups, poly(amidoamine) dendrimers also have a high monodispersity and spherical molecular shape. Recovery tests using a UF module showed that monodisperse G6 and G7 poly(amidoamine) dendrimers exhibited a similar profile to BSA protein. This allows the separation of oligosaccharides when attached to dendrimers via cleavable linker from smaller molecular impurities such as sugar nucleotides present during the synthetic process. The linker had a reactive ketone and a tetrapeptide moiety, Phe-Glu-Phe-Gly, which was cleaved by BLase from Bacillus licheniformis.342 However, although a bigger molecular dendrimer has a better recovery efficiency, it also has poorer solubility which decreases the enzymatic reaction efficiency. Indeed, among the four basic functions of the automated synthesis system (reaction, separation, detection, and repetition), separation is an especially key step as it is performed in every cycle and the overall yield of the synthesis is exponentially dependent on the yield of the individual cycles. By using this artificial Golgi apparatus system, Nishimura and co-workers successfully synthesized sialyl Lewis X oligosaccharide 98 (Scheme 14). The total reaction took 4 days and resulted in a 16% overall yield. The low yield is mainly due to product loss during the purification steps. Indeed, purification is the main obstacle that prohibits the automation of enzymatic synthesis. The assembly of glycans on solid support can address this issue, but the incompatibility between the solution-phase enzymes and the solid support is a major obstacle as glycosyltransferases have difficulty acting on substrates that are bound to such supports.343

Figure 24. Unmodified HPLC used for oligosaccharide synthesis. Reproduced with permission from ref 312. Copyright 2012 American Chemical Society.

column; the building block solutions, other reagents, and wash solution are treated as the mobile phase and added as necessary with the pump under computer control. The reaction is monitored and controlled in real time by a computer. In 2013, Yoshida and co-workers made use of a divided electrolysis cell for automation synthesis.339 They used a synthetic strategy which differed considerably from the usual SPPS model. As shown in Scheme 13, the reaction temperature Scheme 13. Automated Synthesis of Oligosaccharides by Using Iterative Electrochemical Assemblya

5.2. Combination of Automated Solid-Phase and Enzymatic Oligosaccharide Synthesis

Chemical glycosylation has many disadvantages as discussed above. Incompatibility between enzymes and solid supports has hindered the application of automated enzymatic solid-phase synthesis. To address these challenges, the combination of automated glycan assembly and enzymatic glycan synthesis might offer a solution. In 2015, Seeberger and co-workers reported a synthetic strategy that combines automated solidphase chemical synthesis and enzymatic sialylation to afford sialylated oligosaccharides (Scheme 15).321 Although significant progress has been made in developing efficient chemical sialylation reactions,344−348 the automated assembly of sialylated oligosaccharides is still a challenge since no participating group can be placed at the neighboring C-3. Moreover, the quaternary anomeric center contains an electron-withdrawing carbonyl that reduces reactivity. In previous automated syntheses, sialic acid−galactose disaccharide building blocks were used to produce α(2,3)- and α(2,6)-sialylated glycans. The

a


was extremely low (−78 °C), so it might be difficult to generalize to other systems. Their machine itself consisted of a syringe pump, a dc power supply, a temperature-controlling system, a magnetic stirrer, an electrochemical reaction system equipped with a divided electrolysis cell, and a system controller. X


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Scheme 14. Schematic Procedure of Automated Glycan Synthesis by Artificial Golgi Apparatusa

a


of general utility and represents a potential solution to address the challenges of automated chemical or enzymatic synthesis.

use of disaccharides as building blocks is not ideal compared to the use of monosaccharide building blocks. In automated chemoenzymatic synthesis, the oligosaccharide GM 1b tetrasaccharide backbone was constructed from monosaccharide thioglycoside building blocks using solid resin appended with a photocleavable linker. Successive glycosylation cycles were performed by a glycan synthesizer (Scheme 15). UV irradiation of the resin gives the protected tetrasaccharide, followed by deprotection manipulations to give completely deprotected oligosaccharides. Then enzymatic glycosylation was performed using α(2,3)-sialyltransferase from Pasturella multocida (PmST1) to give GM 1b. In a similar manner, several other oligosaccharides were also prepared. This work is expected to be

5.3. Novel Automated System for Oligosaccharide Synthesis

Researchers have successfully performed enzymatic reactions on solid-phase supports. It has been established over the past few decades of study that conventional solid bead-type supports dramatically decrease the activity of transferases. Hence, a common strategy has been to choose water-soluble polymers, such as PEG, as supports. However, these polymers cannot be separated from the reaction system by quick and simple filtration through a frit filter as in SPPS. Y


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Scheme 15. Automated Glycan Assembly Synthetic Strategy Illustrated for GM1b

Scheme 16. Automated Enzymatic Synthesis of GM 1

synthesizer to carry out the synthesis of compound 108 (10 mg) via fully automated enzymatic synthesis over 2 days. The details will be reported elsewhere later. Our work provides another path for automated oligosaccharide synthesis based on enzymatic glycosylation and a commercially available machine. It is foreseeable that commercially available oligosaccharide synthesizer machines based on the same principle will be available in the near future.

To overcome these difficulties, our lab has tried to design long linkers to minimize the deleterious effect of the presence of the support on the activity of the enzymes. After 2 years, despite trying a range of beads and linkers, we did not solve this problem to a satisfactory degree. Therefore, we shifted our focus to watersoluble polymers. After having tried many resins or polymers, we successfully achieved fully automated enzymatic synthesis of oligosaccharides by employing a polymer called PNIPAM.349 This polymer is thermoresponsive, and its solubility in water decreases with temperature. If it were used as a carrier then it would be possible to separate the product from the reaction system by simple filtration after increasing the temperature of the reaction vessel. An example of our synthetic cycle is illustrated in Scheme 16. To demonstrate the feasibility of PNIPAM for automated synthesis, we used the Liberty Blue microwave peptide

6. SUMMARY AND OUTLOOK Over the past few decades, significant progress has been made in developing synthetic methodologies for the construction of structure-defined oligosaccharides and glycoconjugates. These advances have greatly aided fundamental studies in glycobiology. Nevertheless, oligosaccharide synthesis is a lengthy process that generally requires a large team of skilled chemists and Z


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will have a revolutionary impact on the understanding of glycans in biological systems and on the development of carbohydratebased therapies.

biochemists. The invention of automated systems for oligonucleotide and oligopeptide synthesis allowed for nonspecialists to rapidly and easily access these biopolymers, providing a significant advance to the science. In an attempt to replicate these breakthroughs, researchers have endeavored for decades to develop a comparable general automated system to synthesize oligosaccharides. Pioneering work by Seeberger, Wong, Nishimura, and many other groups has advanced the state of the art in the field of automated glycan synthesis. However, the main challenge to overcome in achieving routine automated synthesis is the lack of general synthetic methods for glycan synthesis. Unlike oligonucleotides and oligopeptides, glycans can be branched and each new linkage creates a new chiral carbon. Therefore, glycan assembly needs careful control of both regiochemistry and stereochemistry to form correct glycosidic bonds. Chemical glycosylation and enzymatic glycosylation are the two main methodologies for oligosaccharide synthesis. As mentioned above, enzymatic glycosylation can proceed stereoand regiospecifically without using protecting group manipulation procedures. Moreover, reaction conditions are extremely mild compared to chemical glycosylation. Current methodologies toward the automated synthesis of oligosaccharides are focused on chemical glycosylation. Alternatively, automated enzymatic synthesis is conceptually possible yet not well developed. To achieve automated enzymatic synthesis of oligosaccharides, focus should be set on the development of appropriate enzymes, resins, linkers, and machines. There is an increasing number of enzymes including glycosyltransferases and glycosidases/glycosynthases being characterized for glycan synthesis. Notably, many human glycosyltransferases were recently cloned and expressed by the Moremen and Jarvis groups.206 Moreover, large-scale preparations of glycosylation donors such as sugar nucleotides have been achieved.350 Onepot multienzyme strategies employing nucleotide sugar generation systems have also been developed for large-scale synthesis of oligosaccharides.112 These efforts provide powerful support for producing diverse glycan structures via enzymatic methods. In addition, progress in material science has provided several reliable and practical polymer supports for enzymatic synthesis of oligosaccharides. As we can see, more and more mature technologies such as ion-exchange, His-tag-nickel binding, biotin/desthiobiotin-streptavidin binding, ultrafiltration, and ultracentrifugation will be established for the support of automated synthesis. Reliable and easy-to-use linkers continue to be developed with rapidly growing synthetic methodologies, mirroring the progress scientists have made with the development of nearly ideal linkers for solid-phase peptide synthesis.351,352 The design of an ideal instrument for automated enzymatic synthesis should be based on enzymatic reaction features and should have basic functions including a temperature control system, a pH control system, a separation system, a detection system, and an autoinjection system. With these issues addressed in the near future, the automated enzymatic synthesis of oligosaccharides will provide an attractive alternative for automated glycan synthesis. In the near future, it is expected that practical automated glycan synthesis will be realized and made readily accessible to provide various complex oligosaccharide structures for the whole scientific community, much like the automated synthetic systems of oligonucleotides and oligopeptides provide access to those biopolymers. The development of such automated systems that are easily accessible even to an unskilled researcher

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Madhusudhan Reddy Gadi: 0000-0003-3668-4646 Peng George Wang: 0000-0003-3335-6794 Author Contributions ‡

L.W., G.E., C.G., J.Z.: These authors contributed equally to this work. Notes

The authors declare no competing financial interest. Biographies Liuqing Wen received his undergraduate degree in Biotechnology from Henan Agriculture University in 2009. He has two master’s degrees in microbiology (2012) and chemistry (2015). He was awarded his Ph.D. degree in Chemistry at Georgia State University in 2017 for his research on the development of an enzymatic platform for the facile synthesis of rare sugars and the development of chemoenzymatic tools for detection of complex glycan epitopes on cell surface. He is currently working in Georgia State University for his postdoctoral training with Peng George Wang. Garrett Edmunds received his B.S. degree in Biochemistry from Brigham Young University in 2013. He is currently pursuing his Ph.D. degree in Chemistry under the direction of Peng George Wang at Georgia State University and is a Center for Diagnostics & Therapeutics Fellow. His research focuses on the chemical synthesis and enzymatic extension of common glycan core structures with a goal of incorporating this technology into automated oligosaccharide synthesis. Christopher A. Gibbons graduated from the Georgia Institute of Technology with his B.S. degree in Chemistry in 2013. In 2015, after a brief stint in chemical education, he joined the Peng George Wang lab as a Ph.D. student with an interest in learning organic methods and peptide synthesis. His work is mostly on the synthesis of glycosylated amino acids, glycopeptides, and polymeric drug carriers. Jiabin Zhang, a native of China, is a second year Ph.D. student in the Department of Chemistry at Georgia State University. Before coming to GSU, he received his B.S. degree in Organic Chemistry from Sichuan University in 2006 and M.S. degree in Pharmaceutical Engineering from Southwest Jiaotong University in 2015, both in China. His research mainly focuses on organic chemistry to synthesize biologically important and structurally interesting carbohydrates and glycopeptides, including automated enzymatic oligosaccharide synthesis. Madhusudhan Reddy Gadi was born in Guntur, India. He obtained B.S. degree in Chemistry from Acharya Nagarjuna University, India, in 2006. Later, he joined the School of Chemistry, University of Hyderabad, India, for postgraduate education in Chemistry and received his M.S degree in 2009. He continued in the same university for doctoral studies under the guidance of Perali Ramu Sridhar and received his Ph.D. degree in 2015. His main research focus was on the use of 3-C-branched glycals to construct bioactive structures, natural products, and developing new synthetic methodologies. In 2016, he joined Peng George Wang’s research group in the Department of AA


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Chemistry at Georgia State University for his postdoctoral studies. His current research focus is on the synthesis of O-GalNAc glycans. Hailiang Zhu was born in Hebei, China. He received his B.S. degree in Chemistry from Lanzhou University, China, in 2009. Later, he obtained his M.S. degree in Synthetic Organic Chemistry from Lanzhou University in 2012. After he graduated from Georgia State University with his Ph.D. degree in 2018, he was appointed as an assistant professor of Chemistry at Dordt College. His work of his Ph.D. project was mainly on decoding the function of N-linked glycosylation with chemical methods and automated enzymatic synthesis of oligosaccharides. Junqiang Fang obtained his B.S. degree in Pharmaceutical Engineering from Shandong University, China, in 2002. Later, he obtained his Ph.D. degree in Microbiology in Shandong University in 2010. He then joined National Glycoengineering Research Center, Shandong University, and became an associate professor in 2017. His research focuses on chemoenzymatic synthesis of glycosaminoglycans and their biological applications. Xianwei Liu received his B.S. degree in Biology from Shanxi University, China, in 2003. Later, he obtained his Ph.D. degree in Microbiology from Shandong University in 2009. During the last 2 years as a Ph.D. candidate he worked as a visiting scholar in the Departments of Biochemistry and Chemistry at The Ohio State University. He then took a position at the National Glycoengineering Research Center, Shandong University, and became an associate professor in 2016. His current research focus is on the development of chemical biology methods to understand glycosylation and synthesize important glycans. Yun Kong obtained her B.S. degree in Biotechnology from Shandong University, China, in 2007. Later, she received her M.S. degree in Microbiology from Shandong University in 2010. In 2014, she was awarded her Ph.D. degree in Molecular and Genetic Medicine at Copenhagen University in Denmark. She has been working as an assistant researcher in Shandong University since 2015. She is focusing on probing the substrate specificities and functions of glycosyltransferases involved in O-linked glycosylations including O-GalNAc and OMannose pathways. Peng George Wang obtained his B.S. degree in Chemistry from Nankai University, China, in 1984 and his Ph.D. degree in Organic Chemistry from the University of California, Berkeley in 1990. He then conducted postdoctoral research in the Scripps Research Institute and became an assistant professor in 1994 in the University of Miami. From 1997 to 2003 he was on the faculty at Wayne State University. In 2003 he took a position in the Departments of Biochemistry and Chemistry at The Ohio State University as an Ohio Eminent Scholar in Macromolecular Structure and Function. In 2011 he became Professor and Georgia Research Eminent Scholar in Chemical Glycobiology in the Department of Chemistry at Georgia State University.

ACKNOWLEDGMENTS We thank the National Institutes of Health (U01GM116263) for financial support of this work. REFERENCES (1) Yadav, P.; Yadav, H.; Shah, V. G.; Shah, G.; Dhaka, G. Biomedical Biopolymers, Their Origin and Evolution in Biomedical Sciences: A Systematic Review. J. Clin. Diagn. Res. 2015, 9, ZE21. (2) Varki, A.; Cummings, R. D.; Esko, J. D.; Stanley, P.; Hart, G. W.; Aebi, M.; Darvill, A. G.; Kinoshita, T.; Packer, N. H.; Prestegard, J. H.; Schnaar, R. L.; Seeberger, P. H. Essentials of Glycobiology, 3rd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 2015. AB


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Toward Automated Enzymatic Synthesis of Oligosaccharides

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