CHEMICAL BIOLOGY

Key Strategies for Glycan Synthesis

Rodrigo B. Andrade, Department of Chemistry, Temple University, Philadelphia, Pennsylvania

doi: 10.1002/9780470048672.wecb194

The biologic significance of glycans (i.e., carbohydrates and saccharides) and the need to obtain structurally defined material for study have resulted in a proliferation of strategies for the synthesis of this extraordinary class of biomolecules. Each strategy carries with it both advantages and disadvantages. The two major approaches, chemical and enzymatic synthesis, will be discussed with an emphasis on the former.

The synthesis of glycans is more challenging than the synthesis of peptides and nucleic acids, largely due to issues of structural diversity and stereochemical complexity. Furthermore, glycan biosynthesis is not template-driven. Discoveries in glycobiology and the arrival of the glycomics era (see the article “Major Techniques in Glycomics”) have served to place an even greater emphasis on the synthesis of glycans and on the development of novel strategies for glycan synthesis, as the end products are vital for the elucidation of function. The roles that glycans play in the onset, progression, and development of various disease states have provided opportunities for scientists in academia and industry to harvest this therapeutic potential in developing carbohydrate-based drugs (glycopharmaceuticals) and/or vaccines. Twenty-five years have elapsed since Paulsen’s seminal review on glycan synthesis was published (1), which proclaimed: “There are no universal conditions for oligosaccharide synthesis.” Since then, a great deal of progress has been made in the field, and this review will survey the key strategies associated with glycan synthesis.

Chemical Synthesis of Glycans

Glycans encountered in nature in the form of glycoconjugates (glycoproteins and glycolipids) mediate a remarkable variety of biologic events, including inflammation, fertilization, cell growth and development, tumor growth and metastasis, host-pathogen interactions, and the storage and transfer of information (see the article “Glycans in Information Storage and Transfer”) (2, 3). To study the structure and function of these biomolecules, they must be obtained in pure form. As isolation from natural sources often yields impure material by virtue of microheterogeneity (4), recourse is made to either chemical or enzymatic synthesis (or a combination of the two referred to as chemoenzymatic synthesis).

Linear synthesis

The glycosylation reaction is fundamental to glycan synthesis (see the article “Key Reactions in Glycan Synthesis”). The iterative addition of monosaccharide units in a synthetic sequence constitutes a linear glycosylation strategy. With respect to overall synthetic efficiency, this strategy is not optimal, particularly for large targets (5). The classic Koenigs-Knorr reaction, which was discovered in 1901, signaled the advent of chemical oligosaccharide synthesis (6). The preparation and use of glycosyl bromides and chlorides as glycosyl donors in combination with appropriately protected glycosyl acceptors and heavy metal activators/promoters (typically silver or mercury salts) remains to date a formidable methodology for glycan construction despite toxicity and high cost. Because of the general instability of these intermediates, coupled with the harsh conditions necessary for their preparation, glycosyl bromides have been limited largely to a linear strategy, as opposed to a more efficient convergent (block) approach.

Convergent synthesis

The development of novel glycosylation methodologies in the 1970s and 1980s featuring more stable glycosyl donors such as orthoesters (7), fluorides (8), trichloroacetimidates (9), thioglycosides (10), n-pentenyl glycosides (NPGs) (11), glycals (12), and sulfoxides (13), among others, enabled access to larger oligosaccharides by virtue of a convergent approach. Mild preparation methods of these donors coupled with chemical stability (for purification and storage) greatly increased the overall efficiency of glycan synthesis. Routine conversion of oligosaccharide intermediates (blocks) into glycosyl donors and subsequent coupling with various acceptors has enabled chemists to access more complex structures in an efficient manner; for example, Schmidt et al. (14) have efficiently applied the block synthesis strategy in the preparation of various glycans.

Selective, two-stage and latent-active strategies

The necessity to recruit and use protecting groups on both the glycosyl donor and acceptor is synthetically cumbersome (see the article “Glycan Synthesis, Protection and Deprotection”). Additional synthetic manipulation of oligosaccharide intermediates before coupling lowers overall yield. In an ideal synthesis, the number of protecting group operations should be held to a minimum. Toward this goal, various strategies have been introduced that take advantage of selective activating conditions (Fig. 1). The selective activation strategy is depicted in Fig. 1. A generalized example of this method is the selective activation of a glycosyl donor (halide, trichloroacetimidate) in the presence of an acceptor bearing a potential leaving group (thioglycoside or NPG). After the coupling event, the disaccharide is reacted with another acceptor in the presence of a suitable activator. This strategy cuts down the number of protecting group operations in the synthesis. Thioglycosides and n-pentenyl glycosides are unique donors in that they are stable to traditional protecting group manipulation and can be activated selectively in the gly- cosylation event under appropriate conditions. A recent example of this strategy was Barchi’s preparation of a tumor-associated T antigen building block (15).

In 1984, the Nicolaou group introduced a two-stage glycosylation strategy (16), which is also represented in Fig. 1. In this approach, thioglycoside intermediates are converted into glycosyl fluorides with N-bromosuccinimide (NBS) and (diethylamido)sulfur trifluoride (DAST) (Stage 1) and are coupled subsequently (Stage 2) with a thioglycosyl acceptor. This process can be repeated, offering quick access to large structures. The Nicolaou group (17) has leveraged this methodology to prepare a host of complex glycans. Another powerful two-stage glycosylation approach is Kahne’s glycosyl sulfoxide methodology wherein a phenyl thioglycosides is used as a stable anomeric protecting group, which is oxidized to the anomeric sulfoxide with m-chloroperbenzoic acid (MCPBA). Subsequent triflation activates the anomeric center for glycosylation. The Kahne group (18) has demonstrated the utility of this method in the synthesis of several blood group antigens.

Roy (19) introduced a latent-active glycosylation strategy in 1992 to describe the use of a stable (latent) anomeric group that can be converted into a reactive (active) anomeric group. In this context, a 4-nitrophenyl thioglycoside bearing a free hydroxyl (acceptor) can be coupled selectively with a 4-N-acetylphenyl thioglycoside donor. The nitro group can then be reduced and acetylated, and this procedure can be repeated. By modulating the electronics of the thioarene, selective glycosylation can be accomplished. Another fine example of this strategy is the use of O-allyl glycosides (latent) by Boons (20), which are converted into O-vinyl glycosides (active) and activated subsequently with trimethylsilyl triflate (TMSOTf) in the presence of a suitable acceptor (Fig. 1).

Figure 1. Various strategies in chemical glycan synthesis.

Orthogonal strategy

An orthogonal glycosylation strategy was outlined in 1994 by Ogawa (21) that featured the use of two glycosyl donors (phenyl thioglycosides and fluorides) meeting the following two criteria: (1) Either anomeric group should be activated selectively in the presence of the other; and (2) both anomeric groups should remain compatible with subsequent protecting group manipulation (Fig. 1). This strategy was utilized in the highly efficient synthesis of an extended blood group B determinant (22).

In 1997, Boons (23) reported a highly convergent and efficient synthesis of Group B Type III Streptococcus hexasac-charide using three unique donor sets (ethyl thioglycosides, cyanoethylidenes, and NPGs) that required no protecting group steps beyond those used in building-block synthesis.

Chemoselective glycosylation

Fraser-Reid (24) introduced a chemoselective strategy (armed-disarmed glycosylation) in 1988 after making the observation that NBS-promoted hydrolysis of NPGs bearing electron-donating protecting groups (alkyl ethers such as benzyl) on the C-2 hydroxyl proceeded much faster relative to those bearing electron-withdrawing protecting groups (esters such as acetyl). This strategy led to the experiment in which two monosaccharides bearing identical leaving groups (4-pentenyloxy) are coupled wherein a “disarmed” monosaccharide bearing a free hydroxyl bears an ester on the C-2 position and an “armed” donor possesses an alkyl ether substituent on the C-2 position. In the event, no self-coupled product was formed (Fig. 2). This strategy differs from the orthogonal strategy in that the same anomeric group is used. Chemoselectivity is achieved by modulating the electronics of the incipient oxocarbenium ion. By proximity, the C-2 protecting group exerts a powerful inductive effect, and a lower electron density about the cationic anomeric center results in a slower relative rate. In this way, the disarmed acceptor does not self-couple, and the armed donor is formed rapidly, which ensures a chemoselective reaction. Another extension of this armed-disarmed method is the use of different promoters in this strategy, which thus obviates the need to convert the C-2 ester of the coupled product into an alkyl (benzyl) ether. More specifically, the less-reactive promoter iodonium dicollidine perchlorate (IDCP) can be used in the first coupling, whereas the more powerful N-iodosuccinimide (NIS)/triflic acid (TfOH) or NIS/triethylsilyl triflate (TESOTf) combination is sufficient to glycosylate disarmed donors (25).

In addition to changing the electronics of the C-2 protecting group, Fraser-Reid et al. have introduced the use of cyclic acetal protecting groups (1,3-dioxanes and 1,3-dioxolanes) to “disarm” glycosides. This process works by placing torsional strain on the pyranose scaffold, as the oxocarbenium ion is best stabilized by a dihedral angle of 0° (C₅O₅-C₁C₂). Conformational restraints placed by cyclic protecting groups on the saccharide preclude achieving the desired angle (26). Ley et al. have expanded on this torsional control element with the introduction of dispiroketals (27) and cyclohexane-1,2-diacetals (28). By using different promoters in the sequencing of these glycosylations (mild promoters for early coupling, strong promoters for later coupling), the number of synthetic operations performed on advanced intermediates is reduced (Fig. 2). Friesen and Danishefsky (29) has applied the chemoselective strategy to glycals for the preparation of 2-deoxyglycosides.

Chemoselective glycosylations can also be realized by changing the steric environment about the anomeric center (aglycon). Boons and colleagues (30) has shown that ethyl thioglycoside donors can be activated selectively in the presence of sterically encumbered dicyclohexylmethyl thioglycoside acceptors with IDCP. This methodology has been exploited in the synthesis of various glycan targets. The resulting coupled product can be activated again with the more powerful NIS/TfOH system in the presence of a suitable acceptor (Fig. 2).

Figure 2. Chemoselective strategy.

One-pot glycosylation

The assembly of complex glycans via either a linear or a block approach is often subject to tedious purification (chromatography) steps that lower the overall chemical yield. It would be desirable to use an approach that strives to lower the number of purifications, particularly as the synthesis advances. Toward this end, many groups have developed and refined one-pot glycosylation strategies in which the bulk of the purification is concentrated in the linear assembly of appropriate building blocks and in final protecting group removal steps. Proper sequencing of donor and activator addition, followed by a final purification event, furnishes the glycan in protected form.

One-pot glycosylations (OPGs) fall into three different categories. Each category is based on the particular strategy being employed, which includes reactivity-based (chemoselective), selective (including orthogonal donor sets), and iterative one-pot glycosylations (Fig. 3). Historically, Kahne and coworkers in 1989 disclosed the first OPG strategy for the synthesis of a cyclamycin trisaccharide, which was based on 1) the relative reaction rates (chemoselectivity) of electronically distinct phenyl-sulfoxides and 2) the use of a TMS ether as a latent acceptor in the second glycosylation. The desired trisaccharide was isolated in 25% overall yield (31). In 1994, Ley and coworkers demonstrated the use of 1,2-diacetals and glycoside tuning (torsionally armed-disarmed glycosides) in a one-pot two-step synthesis of a trisaccharide derived from the common polysaccharide antigen of a group B Streptococci. Coupling of an armed ethyl thiorhamnoside with first a torsionally disarmed ethyl thiorhamnoside acceptor (by virtue of a cyclohexane-1,2-diacetal) in the presence of NIS/TfOH followed by a reducing end acceptor in the presence of more NIS/TfOH resulted in a trisaccharide product with an impressive overall yield of 65% (32).

In the same year, Takahashi and coworkers demonstrated that various glycosyl donors could be sequenced in a one-pot strategy; namely, glycosyl bromides, fluorides, or trichloroace- timidates were first coupled with a thioglycosyl acceptor (33). The addition of a second, reducing end acceptor and a thiophilic promoter (NIS) resulted in trisaccharide formation. Yields as high as 84% for the overall process were realized, showcasing the efficiency of this strategy as well as the promoter compatibility, which can be a problem (Fig. 3). The method has been extended to branched structures as well (34). Takahashi has advanced the orthogonal OPG strategy in an impressive synthesis of the heptasaccharide phytoalexin elicitor (HPE) in 24% overall yield (35).

Ley and coworkers were the first to quantify the reactivity of various ethyl thioglycosides (36). In 1999, Wong and coworkers disclosed a programmable one-pot glycosylation strategy in which a database of relative reactivity values (RRVs) was established for numerous monosaccharides and disaccharides bearing the p-methylthiophenyl (STol) anomeric group (37). With this information in hand, they could optimize a one-pot synthetic sequence in which a series of donors bearing free hydroxyls (with the exception of the first unit, which is fully protected) would be sequentially reacted in the order of decreasing RRV terminating with a “reducing end” cap (Fig. 3). Disaccharides (or larger oligosaccharides) can be sequenced to offer access to branched oligosaccharides. A software program, OptiMer, was developed to select the appropriate building blocks from the database to perform an optimal one-pot oligosaccharide synthesis. Coupling of STol glycosides can be affected with a variety of thiophilic reagents. This software program has enabled the synthesis of a variety of important glycans, including poly-A-acetyllactosamines (38), Fucose GM₁ (small-cell lung cancer epitope) (39), the Lewis Y hexasaccharide (colon cancer epitope) (40), and Globo H (breast cancer epitope) (41) in addition to combinatorial carbohydrate libraries (42). As of 2005, 600 thioglycoside building blocks have been entered into the database with RRVs ranging from 1 to >10⁶ (43). Wong and colleagues have introduced a novel activator, N-(phenylthio)caprolactam/Tf₂O, for programmable one-pot synthesis (44).

Yu and co-workers synthesized several saponins in a one-pot fashion using monosaccharide and disaccharide trichloroacetimidates and thioethyl glycosides (45). In 2000, Mukaiyama and colleagues established that glycosyl fluorides or phenyl-cabonates could be used in concert with thioglycosides for the synthesis of several trisaccharides (46). This methodology was used in the synthesis of HPE using fluorides and thioethyl glycosides (47).

The iterative OPG strategy combines favorable elements of both chemoselective and orthogonal approaches in that it uses similar activating conditions for each coupling step yet does not rely heavily on differentiating building blocks with protecting groups to modulate and optimize reactivity (armed-disarmed concept), respectively. Danishefsky et al.’s glycal assembly method (48), Gin’s chemoselective dehydrative glycosylation (49), and Yamago et al.’s bromine-activated selenoglycosides (50) all reflect the utility of the iterative glycosylation strategy in glycan synthesis.

Crich and co-workers have demonstrated elegantly that the stereoselective synthesis of β-mannosides with glycosyl sulfoxides proceeds via anomeric α-triflate intermediates (51). Crich and Sun (52) observed that premixing the mannosyl sulfoxide with the promoter system (triflic anhydride and a hindered pyridine base) before the addition of the acceptor, as opposed to direct triflation of both donor and acceptor, led to higher β:α ratios. The authors theorized that the increased selectivity originated from an S_N2 displacement reaction of the acceptor and that the α-triflate derived from the sulfoxide. Crich et al. (53) also showed that when thioglycosides and anomeric bromides were subject to triflation, the same a-triflate intermediate was observed via low-temperature NMR studies. In summary, Crich and colleagues had demonstrated the viability of the donor preactivation concept. These findings account for the feasibility of the iterative OPG strategy, which is generalized in Fig. 3.

Figure 3. Reactivity-based, selective, and iterative one-pot glycosylation strategies.

In iterative OPG, the glycosylating species (donor) is treated with the promoter system in the absence of the acceptor. A priori, the activated species must be sufficiently stable so as not to decompose before glycosylation and yet reactive enough to undergo reaction with the acceptor. In the event, a glycosyl donor is first activated with the promoter, followed by the addition of an acceptor bearing a stable anomeric group (alkylthio moiety). The glycosylation occurs irrespective of the protecting group ensemble of the acceptor. This process removes the burden of electronically tuning each glycoside via protecting group manipulation; moreover, protecting groups can be selected so as to streamline deprotection steps (endgame). The process can be repeated (iterated) and terminated with a final “cap” acceptor (Fig. 3).

In 2003, the van Boom/van der Marel group cleverly used 1-hydroxy and thioglycosides in a sequential OPG strategy in which the powerful Ph₂SO/Tf₂O/TTBP (2,4,6-tri-tert-butylpyrimidine) (54) promoter system was leveraged to synthesize an a-Gal epitope trisaccharide in 80% yield (stepwise synthesis was accomplished in 69% overall yield) (55). Initial activation of the 1-hydroxy donor with the above promoter system followed by reaction with a thioglycoside acceptor yielded a disaccharide intermediate. As a by-product of the reaction is Ph₂O, more Tf₂O was introduced to activate the thioglycoside in the presence of an acceptor to furnish the target trisaccharides (56, 57).

In 2004, Huang et al. outlined a general, iterative OPG strategy based on “pre-activation” of the glycosyl donor for the synthesis of several trisaccharides and tetrasaccharides using STol glycosides and ap-TolSCl/AgOTf/MS-AW300 (in situ preparation of p-TolSOTf) promoter system. This approach obviates the need to tune electronically or torsionally each donor, which is critical in reactivity-based OPG (58). This strategy was applied in a four-component, one-pot synthesis of a-Gal Pentasaccharide, which is a glycan epitope responsible for the shortcomings of pig-to-human xenotransplantation. Yields for the one-pot sequence ranged from 39% to 41% (59). Recently, Huang et al. (60) introduced a novel arylsulfinylamine, benzenesulfinyl morpholine (BSM), for iterative OPG processes. The novel reagent was used in the synthesis of several trisaccharides.

Solid-phase synthesis

The advent of automated oligopeptide and oligonucleotide synthesizers revolutionized the fields of protein and nucleic acid chemistry, respectively. These technologies have greatly enabled the study and understanding of structure and function by making routine access to these structures straightforward. At the heart of these methodologies lies a solid-phase strategy (61) for which Merrifield was awarded the 1984 Nobel Prize in Chemistry. Solid-phase methodologies possess several advantages: purification is made easy (filtration as opposed to chromatography), excess reagents can be employed to drive reactions to completion via mass action as purification is rendered trivial, and the process lends itself well to automation. Critical to a successful solid-phase oligosaccharide synthesis are the proper selection of the following items: 1) polymer support such as insoluble polystyrene or soluble polyethylene glycol (PEG), 2) orthogonal protecting groups sets, 3) linker (which can be regarded as a resin-bound protecting group), and finally 4) building blocks. In addition, it is desirable to have “on-resin” analytical techniques available so as to monitor the progress of the synthesis. Toward that goal, various techniques, including high-resolution magic angle-spinning NMR (62), gated-decoupling ¹³C NMR spectroscopy using ¹³C-enriched protecting groups (63), fluorinated protecting groups and ¹⁹F NMR spectroscopy (64), FT-IR (65), MALDI-TOF MS (66), and colorimetric assays have been developed (67).

Solid-phase strategies for glycan synthesis can be broken down into three types: donor bound, acceptor bound, and bidirectional (Fig. 4). In the donor bound mode, an excess acceptor is used to drive the reaction to completion (or to maximize yields). As the donor in a typical glycosylation reaction is the “reactive intermediate,” unproductive side reactions correspond to a direct loss in overall yield as the material is bound to the solid support. The acceptor bound approach, which is the most popular of the three, uses an excess of donor in solution. This parallels Merrifield’s strategy for peptide synthesis in which the reactive species is in solution (and in excess); hence, any undesired side reactions along with unreacted donor are simply washed away. The bidirectional strategy represents a hybrid of the two approaches and is well suited for the synthesis of branched glycans (68).

With the success of solid-phase methods for peptide synthesis, early studies of solid-phase oligosaccharide synthesis were launched in the 1970s with the pioneering work of Frechet and Schuerch (69) who prepared several disacchardies and trisaccharides using Merrifield’s resin and glycosyl bromides. Van Boom and colleagues (70) reported on the solid-phase synthesis of a heptagalactofuranoside in 1987 using Merrifield’s resin and glycosyl chlorides in an impressive 23% overall yield. Both approaches used an acceptor bound strategy. The field underwent a renaissance in the 1990s as powerful new glycosylation methods and protecting group strategies for oligosaccharide synthesis emerged (71).

Danishefsky and coworkers (72) have used the glycal assembly method in a donor bound approach for the solid-phase synthesis of various glycans, including among others the Lewis B hexasaccharide, a blood-group determinant that has been identified as a mediator in the binding of pathogen Heliobacter pylori to human gastric epithelium and is implicated in the onset of peptic ulcers (73). Attachment of a glycal monosaccharide to a polystyrene resin via a silyl linker was followed by epoxidation with dimethyldioxirane (DMDO) and reaction with a glycal acceptor in the presence of ZnCl₂. Protection (esterification) of the newly formed C2 hydroxyl (or coupling with another glycosyl donor, which is a unique advantage of the glycal assembly method) and subsequent iteration of this process leads to a resin-bound, fully protected glycan. Cleavage or removal of the material from the resin and final protecting group removal steps furnishes the desired oligosaccharide. To address the issue of slower reaction rates on the solid-support as compared with solution-phase, Krepinski et al. (74) used the soluble polyethylene glycol (PEG) polymer in an acceptor-bound approach with glycosyl bromides. The PEG strategy was used by van Boom et al. (75) to prepare the heptasaccharide phytoalexin elicitor (HPE).

As the solid-phase paradigm began to attract interest, a host of research groups began transferring their respective glycosylation technologies onto the solid-support, with each using the acceptor-bound strategy. Kahne et al. (76) prepared a trisaccharide on Merrifield’s resin with glycosyl sulfoxides and a thiophenyl linker, and they demonstrated that sulfoxides could be used to install stereoselectively α-fucosidic linkages. The power of this technology was demonstrated in the combinatorial synthesis of a library of 1300 disaccharides and trisaccharides (77). Rademann and Schmidt have shown that trichloroacetimidates, which is one of the most powerful and popular donors to date, have been effective in the solid-phase paradigm (78). Schmidt et al. have introduced a variety of linker systems (79) has and have used different supports over the past decade (80). Recently, they disclosed the synthesis of a library of N-glycans (81). Nicolaou et al. (82) used phenolic polystyrene as a solid support to synthesize several oligosaccharides. Thioglycosides were employed as donors as well as a photolabile linker for the synthesis of HPE and a protected dodecasaccharide related to the phytoalexin elicitor family with a block strategy. Fraser-Reid et al. (83) translated the NPG method to the solid support (polystyrene) functionalized with a photolabile linker. A trisaccharide was prepared in which the chloroacetyl (ClAc) was used as the temporary protecting group between couplings. Seeberger et al. (84) have leveraged glycals for the rapid preparation and utility of glycosyl phosphates, which is a powerful class of glycosyl donors. A series of glycans has been prepared with this method (85). In 1999, Seeberger et al. (86) reported on the development and application of a 4(Z)-1,8-octenediol linker, which was used in the preparation of several linear oligosaccharides. Cleavage from Merrifield’s resin with Grubbs et al.’s first-generation ruthenium catalyst (87) under an atmosphere of ethylene performed a cross metathesis reaction to yield an NPG, which can be used for additional glycosylation in solution or can be modified to access neoglycoconjugates (88).

The development of an automated oligosaccharide synthesizer would be highly beneficial to glycobiologists and would serve to drive glycomics, as the synthesis of glycans has been largely restricted to specialized groups. It would allow for routine assembly of desired targets and would accelerate the pace of discovery in glycoscience. Toward this end, Seeberger and coworkers (89) used their solid-phase methodology and disclosed the first automated oligosaccharide synthesizer in 2001. A modified ABI peptide synthesizer was used to prepare a protected heptsaccharide (HPE) and a protected dodecasaccharide using monosaccharide and disaccharide phosphate donors. The synthesis of the latter was accomplished in 16 hours after purification by high-performance liquid chromatography. The synthesizer has also been used to prepare glycans related to the branched Leishmania cap tetrasaccharide (90), a synthetic anti-toxin malaria vaccine (91), a core N-linked pentasaccharide that is common to all N-linked glycoproteins (92), the Lewis X pentasaccharide, the Lewis Y hexasaccharide, and the Le^y-Le^x nonasaccharide (93). Glycosyl phosphates and trichloroacetimidates have emerged as privileged donors in this regime.

Figure 4. Donor-bound, acceptor-bound, and bidirectional solid-phase strategies.

Enzymatic Synthesis

Nature has evolved carbohydrate-processing enzymes for the efficient assembly of glycans. Glycosyltransferases and glycosidases perform highly regioselective and stereoselective glyco- sylations, which thus obviates the need for tedious protecting group manipulation and controls the stereospecific installation of glycosidic linkages (two key issues in chemical glycan synthesis) (94). Moreover, the reactions are carried out under mild and green (nontoxic, environmentally friendly) conditions. As such, a growing trend has occurred in the synthetic carbohydrate community to leverage these enzymes in order to streamline glycan synthesis (95). These reactions are particularly useful for difficult chemical glycosylations (e.g., installation of sialic acid residues).

Glycosyltransferases

The biosynthesis of oligosaccharides is mediated by glycosyltransferases, which transfer either monosaccharide nucleotide monophosphates (e.g., CMP-Neu5Ac) or monosaccharide nucleotide diphosphates (e.g., Glc-UDP) to acceptors with either retention or inversion at the donor’s anomeric center (Fig. 5). These enzymes have evolved to be highly regiospecific and stereospecific. The high cost and availability of both enzyme and substrate are major drawbacks of the enzymatic approach. In addition, nucleotide diphosphate generated during the enzymatic glycosylation inhibits the enzyme. The issue of substrate cost has been addressed with elegant nucleotide donor recycling strategies introduced by Wong et al. (96), which have been translated to other glycosyltransferase systems (97). Feedback inhibition has been tackled by including a phosphatase (98). Wong et al.’s (99) synthesis of sialyl Lewis X, which is a glycan involved in the inflammation cascade, is a testament to the power of this strategy. The enzymatic glycosylation of sialic acid has found widespread use in glycan synthesis, as the chemical installation of this monosaccharide is difficult. Many glycans have been prepared enzymatically, including one-pot and solid-phase enzymatic approaches (100).

Glycosidases

During glycoprotein synthesis, glycosidases are involved in processing the glycans via hydrolysis of glycosidic linkages. These enzymes have been used for in vitro glycan synthesis under the appropriate conditions and make use of readily available, inexpensive donors such as nitrophenyl glycosides (Fig. 5). In addition, glycosidases are more stable than glycosyltransferases and more compatible with organic solvents. Although highly stereospecific, glycosidase are not regiospecific and hence result in a lower yield of desired oligosaccharide. Nevertheless, these highly useful bioreagents have been employed in the synthesis of various glycans (101). Finally, a considerable amount of effort has gone into the engineering of novel glycosyltransferases and glycosidases that feature desirable characteristics (e.g., thermostability) as well as in preparing novel structures (e.g., thioglycosides). These strategies will certainly strengthen the ability to prepare more efficiently complex structures enzymatically (102).

Figure 5. Glycosyltransferases and glycosidases in enzymatic glycan synthesis.

Other strategies

To address issues of large-scale oligosaccharide synthesis, Koizumi et al. (103) at Kyowa Hakko (Kogyo Co. Ltd.) disclosed the utility of multiple metabolically engineered microorganisms containing all of the necessary genes for nucleotide generation and glycan synthesis; moreover, inexpensive orotic acid was used as a UTP precursor. The strategy resulted in a highly efficient synthesis of the globotriose epitope. Wang and coworkers (104) have also addressed the sugar nucleotide issue with the development of “superbeads,” in which enzymes involved in the glyconconjugate biosynthetic pathway are expressed in recombinant Escherichia coli strains, isolated, and immobilized on an agarose resin. Additional glycosyltranferases could be used in solution as well. In 2002, Wang et al. (105) engineered an E. coli strain, which they termed “superbug,” containing all of the biosynthetic genes necessary for the synthesis of a-Gal epitopes. This process obviates issues associated with isolating the individual enzymes (as in the superbead approach). The “superbug” technology was used in the large-scale synthesis of globotriose trisaccharide Gb₃ (106).

Finally, the hybrid chemical and enzymatic approach (chemoenzymatic strategy) has also been popular for the synthesis of many glycans (107). The chemical approach with its drawbacks still allows for greater flexibility in the synthetic scheme (e.g., carbohydrate-based drugs and unnatural glycans). Moreover, relatively straightforward components of the glycan can be assembled readily with chemical synthesis, which leaves the difficult glycosylations (sterically and/or stereochemically demanding) to be carried out enzymatically. A highly illustrative example of this strategy is Unverzagt’s chemoenzymatic synthesis of dodecasaccharide N-glycans of the “bisecting” type. Chemical synthesis is used to prepare an octasaccharide that is treated with UDP-Gal in the presence of a galactosyltransferase to deliver regiospecifically and stereospecifically two Gal units. Treatment of the decasaccharide acceptor with CMP-Neu5Ac (CMP-sialic acid) and β-galactoside-α-2,3-sialyltransferase delivered the target dodecasaccharide (108). Recently, Chen and co-workers (109) reported on an efficient one-pot, three-enzyme chemoenzymatic synthesis of various sialic acid-containing trisaccharides.

Future Outlook

The biologic significance of glycans and their potential as therapeutic agents has energized the field of glycochemistry. The prognosis for the development and implementation of novel strategies for the synthesis of glycans is very good as no general solution has been uncovered. Discoveries made in the chemical and enzymatic/biologic arenas will continue to “raise the bar” as the need to procure glycans for study persists.

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Further Reading

Boons G-J. Strategies in oligosaccharide synthesis. Tetrahedron 1996;52: 1095-1121.

Codee JDC, Litjens REJN, van den Bos LJ, Overkleeft HS, van der Marel GA. Thioglycosides in sequential glycosylation strategies. Chem. Soc. Rev. 2005; 34:769-782.

Davis BG. Recent developments in oligosaccharide synthesis. J. Chem. Soc., Perkin Trans. 2000; 1:2137-2160.

Demchenko AV. Strategic approach to the chemical synthesis of oligosaccharides. Lett. Org. Chem. 2005; 2:580-589.

Ernst B, Hart GW, Sinay P, eds. Carbohydrates in Chemistry and Biology, vols 1-4. 2000. Wiley, New York.

Sears P. Wong C-H. Toward automated synthesis of oligosaccharides and glycoproteins. Science. 2001; 291:2344-2350.

Seeberger PH, Werz DB. Automated synthesis of oligosaccharides as a basis for drug discovery. Nature Rev. Drug Disc. 2005; 4:751-763.

Wong C-H, ed. Carbohydrate-Based Drugs Discovery, vols 1-2. 2003. Wiley-VCH, Weinheim.

See Also

Glycans in Information Storage and Transfer

Glycan Synthesis, Key Reactions of

Glycan Synthesis, Protection and Deprotection Steps of

Glycomics, Major Techniques in