Oligosaccharides play essential roles in many biological processes. It has been recognized that these compounds may provide important leads for drug development. Recent advances in the chemical and enzymatic synthesis of oligosaccharides now make it possible to reliably prepare a wide range of oligosaccharides which can be used for structure - function relationship studies. Synthetic procedures make it possible to prepare designed glycomimetics with improved pharmacokinetics and better binding affinities.

Glycoconjugates are the most functionally and structurally diverse molecules in nature. It is now well-established that protein- and lipid-bound saccharides play essential roles in many molecular processes impacting eukaryotic biology and disease(1,2). Examples of such processes include fertilization, embryo- genesis, neuronal development, hormone activities, the proliferation of cells and the organization of cells into specific tissues. Remarkable changes in the cell-surface carbohydrates occur with tumor progression, which

appears to be intimately associated with metastasis(3). Furthermore, carbohydrates are capable of inducing a protective antibody response and this immunological reaction is a major contributor to the survival of the organism during infection(4). Oligosaccharides have also been found to control the development and defense mechanisms of plants(5). At present there are several carbohydrate-based compounds marketed with important pharmaceutical applications(6) and many of these compounds can be classified as monosaccharides or simple disaccharides (Figure 1). For example, the monosaccharide Streptozocin is used to treat malignant insulinomas and Hodgkin's disease.

Another example of a simple sugar possessing a strong biological activity is the fructose sulfamate Topiramate, which is a new prototype of an anti-epileptic drug and is now in the late-phase of clinical trials. The compound 4-guanidino Neu-5Ac2en is an analogue of

neuraminic acid and is currently being developed as an anti-influenza A and B drug(7). The design of this compound was based on a crystal structure of influenza neuraminidase (sialidase) and 5-N-acetyl neuraminic acid. One of the first disaccharides to be applied as a therapeutic is luctulose, which is used against chronic constipation and hepatic coma. It should also be noted that many pharmaceutically important compounds are glycosylated. For example, the Avermectin antibiotics posses carbohydrate substituents that are crucial for their biological activity.

An increased appreciation and understanding of the roles of carbohydrates in biological processes coupled with the advances made in the analysis and chemical synthesis of oligosaccharides has stimulated the development of more complex carbohydrates as potential therapeutics. For example, recently it was discovered that selectins, which are membrane-bound adhesion receptors expressed on endothelial cells near an inflammation site, can recognize the tetrasaccharide antigen sialyl-Lewisx (SLex, Figure 2)(8). This saccharide has been synthesized chemically and enzymatically and is currently being developed as an acute anti-inflammation drug.

It should be recognized that several disadvantages are associated with the use of complex oligosaccharides (e.g. SLex) as therapeutic agents. In many cases, they display unfavorable pharmacokinetics and often have poor metabolic stability and poor oral adsorption. Apart from these qualities, many carbohydrates bind with low affinity to a protein (mmol or umol range), thus complicating their use as drugs.

These properties have stimulated the development of oligosaccharide analogues. Several oligosaccharides have been prepared in which one or more of the glycosidic oxygen atoms have been replaced by another atom (N, S, C). For example, a thio-SLex analogue has been

synthesized, however, this compound fails to exhibit any biological activity. It is believed that the replacement of an exocyclic anomeric oxygen atom with sulfur may alter dramatically the conformational properties of a saccharide, thus resulting in a loss of biological activity. It is, however, possible to make dramatic changes in the saccharide structure and still maintain or increase a required activity. For example, structure-function relationships and structural studies have shown that the carboxylic acid of the neuraminic acid and the C-2 and C-3 hydroxy groups of the fucoside are critical functionalities of SLex required for recognition (Figure 2). This information was used to design templates that present the required functional groups in their preferred orientation. The a-D-mannopyranosyloxybiphenyl substituted carboxylic acid, TBC265, is such a compound and displays these functionalities in the required orientation. This compound has a greater in vitro potency than the parent SLex tetrasaccharide and an in vivo efficiency in small animal models of inflammation (Figure 2)(9). Furthermore, compounds of this class have been shown to be orally bioavailable. It should be noted that the IC50 values of TBC265 for E, P and L selection inhibition remain at the mmol level. Other studies have demonstrated that the biological affinity of saccharide ligands can be tremendously enhanced by presenting them in clusters (multivalent saccharide ligands)(10).

The above mentioned developments will make it possible to design glycomimetics which may find application in areas in which selectin mediated mechanisms are thought to be important (e.g. reperfusion injury, psoriasis, septic shock, rheumatoid arthritis, asthma, cancer and inflammatory bowel disease).

Heparin sulfates constitute another class of saccharides with important biomedical applications. They are complex linear sulfated polysaccharides and the initial biosynthetic product is extensively modified by amino (N) and O-sulfation and uronate epimerisation. Heparin is widely used as an anticoagulant agent. It binds with high affinity to the plasma protein anti thrombin III (AT III), thereby accelerating its inhibitory activity towards factor Xa and thrombin, two serine proteases involved in blood coagulation. The AT III-binding region of heparin consists of a unique pentasaccharide domain. A synthetic analogue of this domain has been developed which accelerates the AT III-mediated inhibition of factor Xa, but not that of thrombin (Figure 3)(11). This pentasaccharide is produced exclusively synthetically (multi-kg scale) and is now in a late-phase of clinical trials. A growing body of literature also exists indicating important neurobiological roles for heparin sulfate proteoglycans. Examples include: neuroepithelial growth and differentiation, neurite outgrowth, nerve regeneration axonal guidance and branching, and the deposition of amyloidotic plaques in Alzheimer's disease and astrocyte proliferation(12). It is to be expected that synthetic analogues of heparin may find application in the treatment of several neurodiseases.

Carbohydrates may also be used in other pharmaceutical applications. Saccharides, which are recognized by cell-specific proteins, may be used as drug delivery systems(13). It has also been recognized that carbohydrate-based contraceptives may be developed by targeting lectins on the cell membrane of sperm cells(14). Neoglycopeptides may find application as novel vaccines and these compounds may have many advantageous properties over traditional vaccines(15). Derivatives of bacterial lipopolysaccharides (LPS) with endotoxin antagonist activity may provide drugs for the treatment of gram-negative Sepsis.

It may be evident that a wide range of well-defined oligosaccharides is required to study the structure-function relationships of the above-described processes. Organic and enzyme-mediated synthesis provides an important means to obtain these molecules. Combinatorial approaches have been developed to synthesize libraries of saccharides. Thus, in a relatively short period of time large numbers of compounds can be synthesized with applications in medicinal chemistry. It is anticipated that saccharides will play key roles in the development of drugs in this century.