Chemical Strategies For Introducing Carbohydrate
Molecular Diversity Into The Drug Discovery Process

Michael J. Sofia, Ph.D.

Transcell Technologies Inc.
2000 Cornwall Rd.
Monmouth Junction, NJ 08852



http://www.netsci.org/Science/Combichem/feature12.html

Introduction

Combinatorial chemistry has precipitated a paradigm shift in the drug discovery process.[1] No longer can the medicinal chemist accept that the identification of an active or that the evaluation of the structure-activity relationship of a screening hit will occur one compound at a time. In a very short period of time, the identification of potent enzyme inhibitors and receptor ligands from peptide, oligonucleotide, and small molecule combinatorial libraries has become common, thus making combinatorial chemistry a widely accepted tool for drug discovery lead generation and optimization. This success resulted from the feverish development of engineered solid and solution phase chemistries for the rapid generation of large numbers of compounds, the invention of associated technologies such as split-mix and automated parallel synthesis, informatics, and library deconvolution strategies and the ability to rapidly screen these compounds using high-throughput strategies. However, even with the rapid development of this technology and despite its recent successes, combinatorial chemistry has been able to explore only a fraction of the vast realm of molecular diversity. An area of significant importance which would provide a new dimension to the field of molecular diversity and drug discovery is the area of carbohydrates. Saccharides and glycoconjugates play fundamental roles in biological functions.[2] As constituents of known drugs, saccharide units are critical to the biological response these drugs elicit.[3] In addition, the conformationally rigid and functionally rich carbohydrate system is unparalled in its value as a molecular scaffold.[4] Thus effective methods and strategies for generating carbohydrate-based combinatorial libraries would expand the utility of the combinatorial drug discovery paradigm.

A comprehensive carbohydrate-based combinatorial capability requires the ability to link sugars, attach sugars, and modify sugars (Figure 1). However, the execution of such a comprehensive strategy is complicated by the chemical complexity of carbohydrate-based systems and the issues related to their construction and modification. A simple monosaccharide contains multiple reactive sites and an anomeric center with difficult-to-control stereochemistry (Figure 2). Traditionally, site selective reaction is controlled by complicated protecting group schemes. In addition, the construction of multiple consecutive glycosidic linkages or glycosidic linkages to differing substrates has had to rely on the use of different glycosylating reagents because of the lack of a single generalizable glycosidic bond forming reaction that could be applied reliably to a wide variety of substrates. Therefore, the success of any carbohydrate-based combinatorial program will depend on how effective chemists are at solving the unique chemical problems posed by carbohydrates. However, these problems are not insurmountable and progress has now been made in addressing the fundamental synthesis problems associated with the generation of libraries of carbohydrates and glycoconjugates using both solid phase and solution approaches.

carbohydrate synthesis scheme

Figure 1



sugar attachment points

Figure 2

Linking Sugars

The application of solid-phase synthesis technology to the rapid and efficient construction of polymer-bound molecules has become one of the keys to the development of the combinatorial library revolution. Therefore, since linking of monosaccharide units through a glycosidic bond is fundamental to the synthesis of oligosaccharides and some glycoconjugates, the development of carbohydrate-based combinatorial technology requires the construction of glycosidic bonds on the solid phase. Several strategies have been reported which attempt to address the problem of solid-phase synthesis of oligosaccharides.[5] Early reports demonstrated the feasibility of solid phase glycosidic bond construction and paved the way for more recent breakthroughs.[6] Recent developments using glycosyl sulfoxide glycosyl donor technology on the solid phase demonstrated that the use of a single glycosyl donor technology has the ability to provide a general solution to the problem of the efficient and stereoselective construction of glycosidic linkages.[7] Construction of both and ß glycosidic linkages and glycosylation of both 1° and 2° alcohols on the solid phase sets this technology apart from the other reported methods. Glycal glycosyl donor technology was shown to provide another solution to the solid phase synthesis of oligosaccharides.[8] However, this method seems to be restricted to the efficient construction of ß-1,6-glycosidic linkages. More recently, other chemical methods for the solid phase construction of oligosaccharides through the construction of glycosidic bonds have been disclosed in abstract form.[9]

Solid phase construction of oligosaccharides employing enzyme technology provides an efficient and stereospecific strategy for the formation of glycosidic linkages especially glycosidic linkages to sialic acid.[10] Unfortunately, this method is severely limited by the availability of needed glycosyltransferases and sugar-phosphate-nucleotide cosubstrates. Additional limitations arise from the reduction in enzymatic reaction rates observed with polymer bound substrates. Consequently, the development of enzyme technology for the generation of carbohydrate libraries requires that several significant technical problems be solved before a generalized method can be implemented.

The synthesis of small molecules on soluble polyethylene glycol polymers has been shown to be useful in the synthesis of small molecule libraries. [11] This soluble polymer synthesis method has also been applied to the synthesis of small oligosaccharides. The trichloromethylimidate glycosyl donor technology and the thioether donor technology have been used with the soluble polymer strategy for the synthesis of small oligosaccharides.[12]

Although the natural linkage between two sugar monomers is the glycosidic bond, the construction of oligomers of modified sugar monomers on solid supports using other types of artificial links have appeared.[13] One group has reported homologating sugars on the solid phase through the construction of a 2,6 amide link between 2-amino-2-deoxy-6-carboxylic acid pyranosides thus taking advantage of well established solid phase amide bond formation.[13b]

Although solution synthesis strategies are not as prominent as solid phase strategies for generating combinatorial libraries, they have made an important contribution to the field.[14] In the area of carbohydrates, solution strategies are also being explored for the rapid generation of libraries. The "random glycosylation" solution strategy attempts to produce mixtures of products that contains all of the possible oligosaccharides.[15] This approach depends on equal reactivity of all of the hydroxyl groups on a completely unprotected glycosyl acceptor. However, the feasibility of deconvoluting such a randomly glycosylated mixture of oligosaccharides remains to be seen.

Although detailed reports in the literature have not been forthcoming on the generation of an oligosaccharide library utilizing solid phase glycosylation, current solid phase chemical technologies can effect the construction of such libraries.[16]

Attaching Sugars

Introducing diversity into the carbohydrate subunits of glycoconjugates requires the ability to attach sugars or their derivatives to a variety of aglycone chemical systems. These aglycones can range form peptides to lipids to small molecules as in the case of known drugs which have been obtained from natural sources. Depending on the nature of the aglycone moiety, attaching a saccharide unit can be accomplished either by direct glycosylation of the aglycone or by construction of a non-glycosidic linkage between the sugar and the aglycone subunit. The strategies to accomplish these sugar attachments will depend on the problem at hand.

Construction of glycopeptide conjugates on the solid phase is the most developed of conjugate strategies for application to combinatorial constructions. In fact, the construction of several glycopeptide libraries have been reported. Two approaches which have been successful implemented for library generation are the "building blocks" and "convergent" strategies.[5]

The "building blocks" approach uses preformed glycosylated amino acids and relies on the formation of the peptide bond between each amino acid.[17] This approach allows diversity to be introduced by varying the nature of the glycosylated amino acid in a fashion similar to the generation of a standard peptide combinatorial library. The construction of glycopeptide libraries employing the building blocks strategy have been reported.[18]

Successful demonstrations of the "convergent" approach for the construction of glycopeptides attach the sugar unit to the peptide through an amide bond construction. These demonstrations have either the peptide or the saccharide attached to the solid support.[5] The alternative approach, requiring site-selective glycosylation of a polymer bound peptide, has not been successfully demonstrated for the formation of peptide conjugates. The two glycopeptide libraries which have been disclosed maintain the peptide bound to the solid support. By using unprotected 1-amino-1-deoxy oligosaccharides, a small library of glycopeptides was produced by formation of an amide linkage between the amino group of the 1-amino-1-deoxy oligosaccharide and an activated side chain carboxylate group of the peptide.[19] A lipo-peptidoglycan library was constructed using a spatial array format in which carboxylic acid containing carbohydrate units were attached to the amino terminus of a small peptide library bound to a solid support.[20]

Modifying Sugars

Subsequent to linking or attaching sugars, further modification of the carbohydrate moieties would be very valuable for exploring and optimizing receptor binding site interactions. In addition, since monosaccharides and their higher oligomers are conformationally rigid, chiral, and highly functionalized molecules, they provide a unique molecular system for appending functionality in a well defined orientation. Therefore, they have substantial value as either designed molecular scaffolds from which combinatorial lead optimization can proceed or scaffolds for the construction of primary screening libraries. For either application, the ability to regiospecifically functionalize a carbohydrate system is required. To achieve this using either a solid phase or solution phase combinatorial synthesis approach, translation of well established carbohydrate protecting group strategies will be necessary. Subsequently, the use of the existing chemistry which has been developed for the generation of small molecule libraries can be used to build molecular diversity around a carbohydrate nucleus. Although libraries constructed using the carbohydrate scaffold theme have not yet been produced, the value of the molecular scaffold approach is expected to generate wide interest in the near future.

Conclusion

The power of the combinatorial drug discovery paradigm relies on the ability of the medicinal chemist to access wide expanses of molecular diversity. The ability to link sugars, attach sugars, and modify sugars in a combinatorial fashion will have a major impact not only on those targets where carbohydrates clearly play an important biological or functional role but also on expanding the accessible realm of molecular diversity critical to the success of combinatorial library drug discovery efforts.

References

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