Solid Support Oligosaccharide Synthesis

Figure 12.1 Oligosaccharide synthesis.

principles of combinatorial chemistry have been extended beyond the biological oligomers to the assembly of small organic molecules and analogs.7,8 There are, however, only few reports of a combinatorial chemistry approach for the preparation of oligosaccharide libraries.9–15

There are, in principle, two possible approaches to create a library of molecules. One strategy is based on the synthesis of structurally defined molecules independent of the method of synthesis, while the other strategy relies on the creation of a mixture of compounds. The former strategy is more straightforward and seems to be less problematic to assay since only one molecule is involved in each assay. The advantage of the latter strategy, on the other hand, is the greater number of compounds that can be obtained in the same number of reactions. In the case of peptides for example,

12.1 INTRODUCTION 241

multiple units (X,Y,Z) where each unit consists of multiple compounds

(x1, x2, . . . , xn; y1, y2, . . . , yn; z1, z2, . . . , zn) can be used to create a library of mixtures (X−Y−Z) (Fig. 12.2). Although the number of compounds in a mixture could

be close to the theoretical maximum, more focused libraries may be advantageous to avoid difficulties in assaying the mixtures.16

In order to obtain a “good mixture” with equal distributions of all compounds, it is essential that the reactivities of individual molecules or functional groups in a reaction are equal. A possible problem is the fact that an observed activity might be the result of a synergistic effect of multiple compounds in the assay, or the activity could be affected by the presence of inhibitors. Mindful of these limitations a mixture library of oligosaccharides can be synthesized. Since carbohydrates have multiple hydroxyl groups in a molecule, one may be able to obtain a completely random mixture of products consisting of all possible positional isomers if all hydroxyl groups are equally reactive during the glycosylation reaction.

The natural mammalian sugar monomers, Glc, Gal, Man, Xyl, GlcNAc, GalNAc, Fuc, GlcA and NANA (Fig. 12.3) all carry at least three hydroxyl groups that can undergo glycosylation. Extensive branching and other functionalization such as sulfation or phosphorylation can therefore occur. New stereocenters are formed at the anomeric position during glycosylation reactions. These difficulties, inherent properties of this class of molecules, cannot be circumvented, and render the preparation of useful oligosaccharide libraries a formidable challenge. Oligosaccharides are quite different from the other linear biopolymers, nucleic acids, and peptides. The combinatorial number of trisaccharides possibly formed using nine carbohydrates is 119,736, whereas 20 amino acids or 4 nucleotides can form 8000 and 64 trimers, respectively. It is easily understood that the generation of oligosaccharide

Figure 12.2 Creating a molecule library using multiunit mixtures from the three basic Casrtesian coordinates (X, Y, Z).

242 CARBOHYDRATE LIBRARIES IN SOLUTION

OH

OH

COOH

AcHN O OH

HO

OH

N-acetylneuraminic acid

Figure 12.3 Structures of the nine natural mammalian sugar monomers.

libraries is therefore difficult. This number impressively underscores the enormous amount of information an oligosaccharide can contain in a limited space, thus explaining why this class of molecules is used to deliver information in cell-cell interactions.

12.2 STRATEGIES FOR OLIGOSACCHARIDE LIBRARY GENERATION

Before creating an oligosaccharide library, an important question has to be asked, which is how many carbohydrate residues are involved in the carbohydrate

recognition processes. Many systematic studies on the binding of oligosaccharides with proteins have been performed17,18 and the general trend to emerge is that the size

of the ligand bound by a protein is usually only a trior tetrasaccharide, or even smaller.

12.2.1 Libraries of Structurally Defined Compounds

The synthesis of libraries of structurally defined compounds can potentially be achieved either by split–mix synthesis or by parallel synthesis of individual compounds. The synthesis requires a reliable methodology of oligosaccharide synthesis, where stereochemistry and regioselectivity have to be achieved unlike other library approaches. Development of synthetic methodologies that can provide access to any oligosaccharide structure is underway.

Nevertheless, the strategy is most straightforward and the starting point leading to the parallel synthesis of defined compounds is to have all synthetic intermediates with all the hydroxyl groups suitably protected. Figure 12.4 illustrates such a strategy,

244 CARBOHYDRATE LIBRARIES IN SOLUTION

Figure 12.5 Branching of starlike pentasaccharides.

where all the glycosyl donors and acceptors consisting of galactose, glucose, and mannose are individually synthesized prior to the coupling reactions. Regardless of the method used to control stereospecificity of each glycosylation reaction, (4 × 3 × 2)n individual compounds are formed after n cycles of coupling and deprotection.

An alternative idea for the preparation of oligosaccharide libraries is based on orthogonal protection19,20 where orthogonally protected synthetic units are required to generate all possible regioisomers. This orthogonal strategy would require the handling of an unmanageable number of protecting groups because even at the disaccharide stage, up to seven different orthogonal protecting groups would be required. Furthermore, many combinations of these protecting groups would have to be installed on the same monosaccharide to enable the construction of a complete set of trisaccharides. An approach emerged using orthogonal protection to synthesize highly branched starlike pentasaccharides, where the central sugar unit carrying four orthogonal protecting groups served as glycosyl acceptor after deprotection of one of the protecting groups (Fig. 12.5).14

12.2.2 Creating a Mixture Library by Random Glycosylation

Knowing the current difficulties in creating a structurally defined oligosaccharide library, one may find a way to obtain a mixture library. One of the ideas to create such a library is to use an acceptor molecule that does not carry any protecting group. In this strategy only the glycosyl donor but not the glycosyl acceptor is protected (Fig. 12.6). In principle, several glycosylation reactions can be performed with or without protecting group manipulations in order to control the type of library (linear, branched, or both). Before attempting such an approach, one has to answer the question as to whether the reactivities of all hydroxyl groups are equal. Traditional organic chemistry suggests that reactivities of hydroxyl groups differ, a feature that is essential to achieve selective reactions in total synthesis of natural products.21–26

Figure 12.6 Protection of a glycosyl donor but not the glycosyl acceptor.

12.3 ATTEMPTS TO CREATE A RANDOM MIXTURE

The difference in reactivity of hydroxyl groups is often influenced by the presence of nearby bulky protecting groups, and the results may not reflect the inherent reactivities of each hydroxyl group in oligosaccharides toward glycosylation. Intraand inter-molecular hydrogen bonding and/or aggregation of molecules may also be involved in such reactions essentially carried out in an apolar solvent. The results of performing glycosylation reactions on totally unprotected acceptors, which requires the use of polar solvents for solubility, could hardly have been anticipated.

The initial results are summarized in Figure 12.7.10 β-GlcNAc glycoside 1 equipped with a hydrophobic aglycon was used as the acceptor to facilitate product isolation by C-18 reverse-phase chromatography (Fig. 12.7A).27 Treatment of 1 with tetra-O-benzyl-galactopyranosyl trichloroacetimidate 221 afforded in DMF using BF3 etherate as the catalyst, at ~25% conversion, the deprotected disaccharide product mixture in the indicated ratios. Separation of acceptor that contains no benzyl group, disaccharides having four benzyl groups and trisaccharides having eight benzyl groups could be conveniently accomplished by reverse-phase chromatography using a step gradient. Hydrogenolysis of the resulting disaccharide fraction then gave the product mixture, which was adsorbed onto a C-18 column27 and then washed with water to remove byproducts formed by galactosyl donor degradation. The disaccharides were eluted with methanol and characterized by NMR spectroscopy after further partial purification. The anticipated high selectivity towards the C6 hydroxyl group was not observed, and all the regioisomers were present. Glycosylation using O-benzyl ethers as protecting groups on a donor is known to yield α-linked compounds as the major products. Remarkably, all three α-linked products were present in similar amounts (20%, 20%, and 30%). These early results showed

12.5 COMPOUND DISTRIBUTION DEPENDS ON REACTION CONDITIONS 247

Figure 12.8 Protein binding to two random glycosylation libraries.

trisaccharides, respectively.29 Incubation of the library with partially purified human milk enzymes in the presence of GDP-[3H]-Fuc resulted in the incorporation of labeled fucose into the library as assayed by a Sep-Pak assay (Fig. 12.8A).27 The enzymes recognized their substrates in the library mixture and transferred fucosyl residues to them. On the basis of NMR evidence that the H-type 2 sequence 8 was indeed present in the fucosylated LacNAc-library (library 2, Fig. 12.8B), incubation of the library with human serum from a blood group A donor, in the presence of tracer labeled UDP-[3H]-GalNAc was attempted. This serum contained the blood group A GalNAc-transfer enzyme for which the presence of the αFuc(1→2)βGal sequence present in 8 is an absolute requirement in order to transfer GalNAc.30 Disaccharide 3 does not function as an acceptor for this enzyme. Incubation of the fucosylated library as potential acceptor resulted again in the readily detected incorporation of GalNAc under standard conditions. It was therefore evident that the trisaccharide library could be used in biological screening experiments, which indicated that the H-type 2 trisaccharide 8 was present and could be converted to the blood group A–active tetrasaccharide 9.

12.5 COMPOUND DISTRIBUTION DEPENDS ON REACTION CONDITIONS

On the basis of the early results described above, random glycosylation as an approach to oligosaccharide library preparation has been further pursued. The product distributions in these early reactions were difficult to determine, and in subsequent work, the aglycon of the acceptor was changed to the p-methoxyphenoxy octyl

TABLE 12.1 Use of 2,3,4,6-Tetra-O-benzylgalactopyranosyl Trichloroacetimidate and Dibenzylphosphite in Galactosylation Reactions