Enzymes for carbohydrate and peptide syntheses

New practical procedures have been developed for the enzyme-catalyzed synthesis of carbohydrates and peptides. Aldolases have been shown to be effective catalysts for the synthesis of uncommon sugars, particularly azasugars. Enzymatic methods for the large-scale synthesis of oligosaccharides have been developed with the use of glycosyltransferases coupled with the regeneration of sugar nucleotides. Engineered subtilisin variants that are stable and active in anhydrous dimethylformamide and in aqueous solution have been developed for peptide segment coupling. As many enzymatic methods become available for the stereocontrolled synthesis of c h i d synthons, attention has been extended to the enzymatic synthesis of molecules with increasing complexity. One class of such complex molecules are carbohydrates, especially glycoconjugates that exist on cell surfaces (1). These molecules are involved in many types of biochemical recognition phenomena. They have been difficult to isolate, characterize and synthesize, and are the least explored of the major classes of biomolecules. Carbohydrate-related structures therefore offer new opportunities for the study of molecular recognition and for the development of therapeutic agents. The recently identified tetrasaccharide sialyl Lewis x as the ligand of endothelial leukocyte adhesion molecule (2) stimulates new interests in the development of practical methods for the synthesis of carbohydrates and mimetics or analogs as ligands, antagonists or inhibitors of specific glycoenzymes or glycoreceptors. It is of our interest to develop enzyme-based technology for the synthesis of carbohydrates and related substances, to make these materials readily available to study their structure-function relationships and to evaluate their therapeutic potential. SYNTHESIS OF U N C O M M O N AND AZA SUGARS BASED O N ALDOLASES Catalytic asymmetric aldol condensation is of current interest in synthetic organic chemistry. Enzyme-catalyzed aldol condensation holds great potential in this regard (3). More than 20 aldolases are known (Figure 1); many of them have been explored for synthesis. A particularly important application of aldolases is the synthesis of deoxyazasugars a class of molecules useful as glycosidase inhibitors. We have examined five aldolases, i.e. fructose-l,6-diphosphate aldolase from rabbit muscle or E. coli (4), rhamnulose (Rham) and fuculose (fuc) 1-phosphate aldolases (5,6), 2-deoxyribose-5-phosphate aldolase @EM) (9, and sialic acid aldolase (7), for their utility in asymmetric aldol condensations. All these aldolases possess two common features: first, they are highly specific for the donor substrate but flexible for the acceptor component; second, the stereoselectivity is controlled by the enzyme not by the substrate, with some exceptions observed in the sialic acid aldolase reactions. A general strategy was therefore developed for the synthesis of deoxyazasugars based on aldolase-catalyzed condensation of azidoaldehydes followed by Pd-mediated reductive amination. Both thermodynamic and kinetic approaches have been explored for the aldolase reactions. Figure 2 illustrates a general strategy for the synthesis of azasugars based on this combined chemical-enzymatic approach. With this straightforward stereocontrolled process, a number of azasugars have been synthesized and evaluated as glycosidase inhibitors. This research has led to a new direction of inhibitor design (4-6). The structure-inhibition studies indicate that the five-membered-ring aza sugars are good mimics of the transition state of glycosidic bond cleavage due to their positively charged half-chair conformation (6). We believe these aza sugars will be useful building blocks for the synthesis of many sequence-specific glycosidase and glycosyltransferase inhibitors, and the synthetic strategy illustrated in these processes should be applicable to the synthesis of other types of azasugars based on different aldolases. 1197 1198 C.-H. WONG eta/.