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Enzyme recovery (B) Immobilized enzymes:

I Nucleotide sugar I transferase 2

Transferase 1

Transferase 2

Transferase 3

Fig. 24.8. Approaches to automated enzymatic saccharide synthesis. In (A), the enzymes are left in solution and the growing saccharide is immobilized on the solid phase. This approach simplifies purification, but requires an enzyme recovery step to avoid losing the expensive catalyst. In (B), the growing saccharide is attached to a water-

soluble polymer, which is passed across columns of immobilized enzymes. Product recovery at the end can be accomplished by precipitating the polymer or by affinity techniques if the polymer is tagged with an affinity ligand (such as biotin). Alternatively, both enzymes and substrates can be used in free form.

thermoresponsive polyacrylamide [27] may find use in enzymatic oligosaccharide synthesis. One can thus envision a scheme in which glycosyltransferases (and, if necessary, the enzymes required for the regeneration of their substrates) are immobilized onto a resin and packed into different columns. The substrate, free or bound to a water-soluble resin with an affinity tag, is passed through the columns in sequence depending on the glycosyltransferases desired. Intermediate isolation, if necessary, can be achieved via capture of the substrate using, for example, affinity capture. Attachment of enzymes and substrates to a support is, however, not trivial. In the end, given the high yields observed with glycosyltransferase-catalyzed glycosidations and the simplicity of product isolation, the choice of the reactor configuration is probably not critical, and both solution and solid-phase methods can be used for automated synthesis.

NHAc li aro

C8H17

bH NHAc II

Galactosyltransferase UDP-Galactose

'C8H17

bH NHAc II

Sialyltransferase CMP-Sialic Acid

Pseudo-GM3

Fig. 24.9. Enzymatic synthesis of the ganglioside pseudo-GM3 on a polymeric support. Ceramide glycanase-catalyzed transglycosidation can be used to remove the

Sialyltransferase CMP-Sialic Acid

Pseudo-GM3

Fig. 24.9. Enzymatic synthesis of the ganglioside pseudo-GM3 on a polymeric support. Ceramide glycanase-catalyzed transglycosidation can be used to remove the

Ceramide Glycanase Ci6 Ceramide o

Ceramide Glycanase Ci6 Ceramide o

ganglioside from the support. Polymer-bound versions of GM3 have also been shown to be useful as picomolar inhibitors of influenza hemagglutinin [48].

ganglioside from the support. Polymer-bound versions of GM3 have also been shown to be useful as picomolar inhibitors of influenza hemagglutinin [48].

24.4

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