Page 163 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

6.2 Sequential Syntheses 139

classes of enzymes are addressed: GTs, which use activated sugar donors, and
glycosidases. Cascade reactions may prove to be powerful in the case of GTs, as
nucleotide sugar (re)regeneration may be coupled with the actual transfer reaction
(Scheme 6.1). The preparative potential of glycosidases suffers mainly from product
hydrolysis, but cascade synthesis could be useful to drive the equilibrium toward
the product, thereby reducing or preventing hydrolysis (Scheme 6.2).

6.2
Sequential Syntheses
Sequential biocatalytic cascade reactions are characterized by the use of multiple
enzymatic steps involving various biocatalysts. One cascade reaction can consist
of an enzyme-module with several enzymes if substrate and inhibitor kinetics
are compatible with these combinations. Sequential use of such enzyme-modules
surpassing the work-up of intermediate products is the criterion for the idea of
cascade reactions we address here: on the one hand, the synthesis of nucleotide
sugars and their derivatives, on the other hand, the synthesis of glycan epitopes
with multiple GTs.

6.2.1
Nucleotide Sugars
dTDP-deoxyhexoses are an important class of activated donor sugars for GTs
in the synthesis of glycoconjugates with antitumor or antibiotic activity. Their
synthesis using a complex three-step sequential cascade strategy yielded dTDP-
2-deoxy-Glc, dTDP-2,6-dideoxy-4-ketoglucose, dTDP-l-olivose, as well as dTDP-d-
olivose [81]. The first reaction step yielded dTDP-2-deoxy-arabino-hexose and was
performed by an enzyme-module with phosphoglucomutase (PGM), dTDP-Glc
pyrophosphorylase (RmlA), and pyrophosphatase. Whereas the latter enzyme was
used to drive the equilibrium toward the desired product, PGM converted the
substrate 2-deoxy-glucose-6-phosphate to 2-deoxy-glucose-1-phosphate and RmlA
was responsible for the formation of the sugar-nucleotide. After removal of the
enzymes via ultrafiltration a second enzyme-module consisting of dTDP-Glc 4,6
dehydratase (RmlB) and alkaline phosphatase was used for the formation of
dTDP-2,6-dideoxy-4-ketoglucose. Alkaline Phosphatase was included to degrade
the RmlB inhibiting residual dTDP. Both steps were performed in a repetitive
batch process to increase the overall enzyme productivity. The intermediate was
successively converted to dTDP-l-olivose or dTDP-d-olivose by RmlC/RmlD (3,5-
epimerase/4-keto reductase) or by chemical reduction with NaBH , respectively
4
(Figure 6.1).
This complex synthesis was accomplished following the definition of sequential
cascade reactions combining multiple biocatalytic reactions without the necessity
of intermediate work-up. The central intermediate of dTDP-activated deoxysugar
synthesis was produced in an enzyme module system with sucrose synthase