Primary contributions to this chapter were made by O. Hindsgaul (University of Alberta, Canada, and The Burnham Institute, La Jolla, California).

THIS CHAPTER ADDRESSES THE SYNTHESIS of oligosaccharides on a preparative scale (greater than milligram quantities) in the laboratory. Both organic chemical and enzymatic approaches are presented. Emphasis is placed on the scope and limitations of the existing methods, and an effort is made to provide an understanding of the amount of experimental work required to produce well-defined glycan samples for biological studies.

Background ^(1–3)

The interest of organic chemists in the synthesis of oligosaccharides arises from the challenge of stereospecifically assembling large highly functionalized complex molecules in an efficient manner. The interest of glycobiologists, on the other hand, is in obtaining samples of pure well-characterized oligosaccharides for use in biological studies. Such studies almost always probe the role of a glycan in some biological recognition process such as the binding of bacteria, toxins, or viruses to mammalian cell surface glycans or the specific recognition of a glycoprotein or glycolipid by a cell surface receptor. The glycans in question are often very large, but the protein-combining sites tend to be more limited in size and usually recognize epitopes presented by as few as 1–4 sugar units. For this reason, this chapter focuses on the synthesis of small oligosaccharides.

Mammalian glycans are formed from only ten monosaccharides, which are considered the “building blocks” (see Chapter 2). These are d-Glc, d-Gal, d-Man, d-Xyl, l-Fuc, d-GlcNAc, d-GalNAc, d-GlcA, l-IdoA, and Neu5Ac. Additional structural diversity can be present in glycans through acylation, sulfation, and phosphorylation. Each of these monosaccharides can in principle form glycosidic linkages with either the α or β configuration, although not all of the possible anomers have been discovered. In bacteria, a seemingly unending list of monosaccharides that includes deoxy and amino sugars is combined to produce highly complex and synthetically challenging molecules (see Chapter 21).

Oligosaccharide Synthesis Is More Difficult Than DNA or Peptide Synthesis ^(4–5)

The challenge of oligosaccharide synthesis is easily appreciated by comparing it with the much more developed areas of DNA and peptide synthesis, which are commonly performed on the solid phase using commercial instruments.

The general scheme used in the solid-phase synthesis of DNA oligomers is shown in Figure 39.1. A partially protected first nucleoside is immobilized by attachment to a solid support through its 3′-OH group. Its 5′-dimethoxytrityl group is then cleaved under mildly acidic conditions. An activated form of the next nucleoside, usually a 3′-phosphite derivative, is then added to make the 3′→5′ phosphite. The phosphite is then oxidized to the phosphate, usually with iodine in pyridine/water, and the phosphodiester linkage is completed. Removal of the dimethoxytrityl group then allows the cycle to be repeated with the next nucleoside derivative. The key features of the chemistry are that all of the required derivatized nucleosides are commercially available or are prepared in two to three steps. The time for cleavage of the dimethoxytrityl group is 3 minutes, that for phosphite coupling is 5 minutes, and that for oxidation is 2 minutes. Because all of the nucleosides couple at the same rate, there is no need to monitor each reaction. The total cycle time is under 20 minutes and the yield is near 99%, and thus many cycles can be performed. Clearly, the chemistry has been extensively developed and is extremely efficient, simplified by the fact that there are only two OH groups involved—a nonhindered primary 5′-OH group and a secondary 3′-OH group—and the linkage is always between the 3′-OH on one residue and the 5′-OH on the next residue. The final oligonucleotide is obtained after cleavage from the solid support (usually glass) by base treatment, which also removes the protecting groups on the nucleoside base and on the phosphodiester.

Figure 39.1

Synthetic protocol for the solid-phase synthesis of DNA oligomers (A) and peptide oligomers (B). (DMT) Dimethoxytrityl; (B) base (e.g., uracil); (Fmoc) fluorenemethyloxycarbonyl.

A general scheme for the solid-phase synthesis of peptides is shown in Figure 39.1. The most commonly used protocol involves amino acids that have their side-chain functional groups (amines, carboxylates, etc.) protected with acid-labile protecting groups (such as Boc groups) and the amino acid nitrogen protected with a base-labile Fmoc group. In the “normal direction,” the Fmoc-protected carboxy-terminal amino acid is coupled to a solid support through its carboxyl group using an acid-labile linker. The Fmoc-protecting group is cleaved with a base, e.g., morpholine, to expose a free reactive amine. The next amino acid is then added, either as a preactivated ester (such as a pentafluorophenyl ester) or as the free acid in the presence of a condensing agent such as a carbodiimide. Catalysts are often added, and the completion of the reaction can be monitored colorimetrically for the disappearance of the basicity of unreacted amino groups. When the reaction is complete, the Fmoc group of the newly added amino acid is cleaved and the next cycle is initiated. Unlike the case of DNA synthesis, different amino acids can react at different rates, and some sequences are particularly problematic, which is the reason colorimetric monitoring is desirable. With modern techniques, racemization is virtually eliminated, but reaction yields for the formation of the amide bonds are more commonly under 97%, and side reactions occur at the level of a few percent. In practice, a cycle can take from one to several hours, and after cleavage from the resin by acid treatment, which also cleaves the side-chain-protecting groups, the purity of the product peptide must be evaluated by analytical chromatography. Preparative HPLC is generally performed as a final purification step. Synthetic peptide synthesis is generally considered efficient for up to about 30 cycles.

All of the required natural protected nucleosides and Fmoc-protected amino acids for DNA and peptide synthesis are commercially available, as are the prepackaged reagents, solvents, and instruments that add reagents using pumps, which perform the washing and cleavage steps. The synthesis of DNA and peptides is therefore common in nonchemical laboratories.

Complexities and Challenges in the Chemical Synthesis of Oligosaccharides ^(1–3)

The above two cases contrast sharply with that of the chemical synthesis of oligosaccharides, which is far more complex. The differences are outlined in Figure 39.2, which shows the synthesis of a simple disaccharide made up of two glucose units. The mammalian monosaccharide building blocks all have either three or four OH groups. If a specific disaccharide sequence, such as a 1→4-linkage, is required, the remaining OH groups must be protected. Once this has been accomplished, the second sugar (the glycosyl donor) is added in an activated form, with a leaving group “L” at the anomeric center. The glycosyl donor then couples with the glycosyl acceptor having a single free-OH group. Formation of the glycosidic linkage creates a new asymmetric center which can have either the α or β configuration. Clearly, mixtures are not wanted; therefore, stereospecific methods are required to produce the correct anomer of the target disaccharide.

Figure 39.2

Synthetic protocol for a hypothetical Glc1-4Glc disaccharide. P is a generic hydroxyl-protecting group.

In normal cases, a glycosylation reaction takes 2–48 hours. Individual sugars can react at very different rates and give a very different ratio depending on the free-OH group that is being glycosylated. Yields are generally in the 60–80% range, with anomeric ratios rarely better than 20:1 and often 6:1 or worse. With such yields, chromatographic purification is essential at each step. Even more severe limitations exist if longer oligosaccharides are required. As seen in Figure 39.2, after the second sugar has been added to the first, seven potential sites are available for the addition of the next sugar to form a trisaccharide. In the case shown, a single generic protecting group “P” was used (for simplicity of presentation) such that no single OH group in the product disaccharide can be selectively removed for chain extension. If the next sugar must be added to the 2′-OH group, for example, then a glycosyl donor with its 2-OH group differentially protected must be used as the glycosyl donor. In other words, four differentially protected acceptors and four differentially protected donors must be prepared to be able to make each of the possible triglucosides. The situation clearly gets much more complex if a tetrasaccharide is envisioned and other monosaccharides are also involved. Very few of the required building blocks are commercially available.

The challenge of oligosaccharide synthesis then reduces to the requirement for elaborate OH group protection strategies and the need for stereospecific methods for glycosylation. As noted above, even in well-precedented systems, the yields and α/β ratios are poor by comparison with that of DNA and peptide synthesis, and they are also difficult to predict in new sequences. It is for these reasons that only 18 papers have appeared to date reporting the solid-phase synthesis of oligosaccharides (for review, see Reference 3).

A Representative Trisaccharide Synthesis ⁽⁶⁾

Figure 39.3 shows the synthetic scheme followed for the preparation of a glycosyltransferase acceptor that is used to measure the activity of the metastasis-associated enzyme N-acetylglucosaminyltransferase-V (see Chapters 17 and 35) in crude tissue extracts. This represents a highly efficient solution synthesis of a trisaccharide in a very well precedented system and is presented so that the reader can get a feel for an actual oligosaccharide synthesis procedure. The scheme requires 17 steps from commercially available starting materials; 8 intermediate compounds require purification by chromatography and typically 100 mg of the final compound is obtained. Initially, the development of the synthetic scheme required about 3 months of work for an experienced postdoctoral fellow. To repeat the sequence now takes about 1 month of full-time work.

Figure 39.3

Synthetic scheme used for the preparation of trisaccharide that is used in a glycosyltransferase assay.

Enzymatic Synthesis of Oligosaccharides ^(7–9)

The very high current level of interest in using enzymes to synthesize oligosaccharides on a preparative scale is in direct response to the severe limitations still present in the chemical approaches. Both glycosyltransferases (the enzymes that biosynthesize the oligosaccharides) and glycosidases (the enzymes that hydrolyze them) have been used. The attractiveness of enzymatic synthesis is that protecting groups are not required and that stereochemically defined glycosidic linkages, not α/β mixtures, are always formed.

Synthesis of Oligosaccharides Catalyzed by Glycosidases ^(10–12)

Glycosidases (see Chapter 18) were used well ahead of glycosyltransferases (see Chapter 17) for the preparative synthesis of oligosaccharides. This is because these enzymes are stable and easy to isolate, and they were therefore more generally available. Glycosidases normally catalyze the hydrolysis of glycosidic linkages and must therefore be “coaxed” into providing useful yields of oligosaccharides.

Most frequently, glycosidases are used in “transglycosylation” reactions where they transfer a monosaccharide from another readily available and inexpensive glycoside. For some glycosidases, the reactions are very regiospecific, but for others, mixtures are obtained. Figure 39.4 illustrates the use of lactose as the “glycosyl donor” for three different β-galactosidases that have different specificities. The enzymes cleave the terminal β-galactosidase residues from lactose, but, under appropriate experimental conditions involving high concentrations of added acceptors, yields of more than 20% of new Gal→GlcNAc disaccharides can be obtained. Hydrolysis (transfer to water) always competes and thus limits the yields, but all of the new disaccharides have exclusively the β configuration.

Figure 39.4

Examples of the use of β-galactosidases in transglycosylation reactions to produce disaccharides on a preparative scale. (a) Enzyme from bovine testes; (b) enzyme from Lactobacillus bifidus; (c) enzyme from Escherichia coli.

Another strategy used to increase yields in glycosidase-mediated oligosaccharide synthesis is to use p-nitrophenyl glycosides as thermodynamically high-energy donors. This is illustrated in Figure 39.5 where a β-hexosaminidase was used to synthesize the very linkage it normally hydrolyzes. Transfer of GlcNAc, from its p-nitrophenyl glycoside to free GlcNAc, resulted in a 55% yield of the 1-4-linked chitobiose derivative and a 22% yield of the 1-6-linked disaccharide. This reaction also illustrates the main limitation of glycosidase-catalyzed oligosaccharide synthesis: Mixtures of positional isomers are frequently, and unpredictably, obtained. In fact, this lack of regioselectivity has been used in the generation of oligosaccharide libraries using glycosidases.

Figure 39.5

Example of a single glycosidase producing two different disaccharides on transglycosylation using a p-nitrophenyl glycoside as the donor.

Synthesis of Oligosaccharides Using Glycosyltransferases ^{(7–9,13–14)}

Glycosyltransferases transfer monosaccharide residues from their activated forms, the sugar nucleotides, to growing oligosaccharide chains (Figure 39.6). Since oligosaccharides are biosynthesized by glycosyltransferases in vivo, it stands to reason that this class of enzymes can be used also for their in vitro preparative synthesis. This requires access to both the sugar nucleotides and the enzymes themselves, and this has until recently been a major impediment. The mammalian sugar nucleotides are UDP-Glc, UDP-Gal, UDP-Xyl, UDP-GlcNAc, UDP-GalNAc, UDP-GlcA, GDP-Man, GDP-Fuc, and CMP-Neu5Ac, and each exists in only one anomeric form. The sugars can be transferred with either retention or inversion of the anomeric configuration, inversion being the more common.

Figure 39.6

Use of two sequential glycosyltransferase reactions in the synthesis of the Lewis X (LeX) trisaccharide.

All of the mammalian sugar nucleotides are now commercially available, although they are usually expensive. For very large scales where economy is of concern, the sugar nucleotides can be produced in situ using very elegant enzyme recycling systems beginning with the free sugars themselves and high-energy drivers such as phosphoenol pyruvate (see Chapter 41). Until recently, only a bovine β1-4 GalT was commercially available in amounts sufficient to prepare milligram quantities of oligosaccharide (Figure 39.6). The tremendous progress made in the cloning of glycosyltransferases, however, has now yielded the sequences of scores of enzymes, and several recombinant galactosyltransferases, fucosyltransferases, and sialyltransferases have appeared on the market.

For glycosyltransferase-assisted oligosaccharide synthesis, the acceptor, donor, and enzyme are simply mixed in a buffer where the enzyme activity is at its optimum. Manganese ions are frequently required. The product oligosaccharide that is formed is controlled by the specificity of the enzyme so that protection is not required. The reaction also produces a nucleotide by-product, e.g., UDP in the reaction of a β1-4 GalT (Figure 39.6). The nucleotide by-product usually inhibits the enzyme, and alkaline phosphatase is sometimes added to destroy it.

Most glycosyltransferases do not act on simple monosaccharides but, as in vivo, require more elaborate structures for recognition and transfer, and the enzymes have a specified order of addition. For example, the Lewis X trisaccharide (Figure 39.6) must be prepared first by the addition of galactose to GlcNAc. Only then can the fucose residue be added to the GlcNAc residue.

Future Prospects ^(14–21)

The move toward solid-phase synthesis is long overdue. Despite the tremendous problems ahead, new methods for solid-phase synthesis are slowly appearing that are truly different and hold promise for simplifying oligosaccharide assembly. These methods should become sufficiently robust to permit the assembly of oligosaccharide and other carbohydrate libraries, an area of research that is in its infancy.

Numerous examples have appeared describing the use of glycosyltransferases in the synthesis of natural oligosaccharide sequences. The enzyme specificities are sometimes relaxed, and with sufficient enzyme available, analogs of the natural oligosaccharides can also be produced, particularly those containing deoxy sugars. The limitation of glycosyltransferases in synthesis will be their inability to prepare sequences not found in nature. The use of glycosyltransferases to “cap-off” oligosaccharides immobilized on the solid phase promises to further speed up the preparation of biologically active glycans.

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