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146 6 Chemo-Enzymatic Cascade Reactions for the Synthesis of Glycoconjugates

The last examples of sequential cascade reactions focus on microfluidic devices.
The first one is a pressure-driven flow cell used to synthesize a glycosaminoglycan
linkage region tetrasaccharide [96]. Three GTs, namely β1,4GalT-I, β1,3GalT-II, and
β1,3GlcAT, were expressed in insect cells and after immobilization inserted into
a reaction chamber on a microfluidic microchip. The reaction mixture containing
the acceptor substrate Xyl-O-pNP and appropriate sugar nucleotides in suitable
buffer was pumped into each reaction chamber sequentially with a syringe pump.
This led to the first reported sequential glycosylation on a microchip.
With sequential cascade reactions with the vision of a ‘‘Golgi on a chip’’
already realized, the miniaturization of the reaction vessel is of on-going interest.
Although only the enzymatic sulfation with 3-O-sulfotransferase isoform 1 (3-
′
′
OST-1) in the presence of 5 -phosphoadenosine 3 -phosphosulfate (PAPS) of an
immobilized heparin-sulfate trisaccharide is reported [97], the importance of this
work is outstanding. The combination of ultralow-volume digital microfluidics and
glycobiology opens an automated platform for future applications like substrate
screening or miniaturization of ligand screening in union with ligand synthesis.
It may be that nowadays cascade reactions in a sequential way are at their dawn
in glycan synthesis, but the combination of automated on-chip synthesis with
glycobiotechnology will be one of the future tasks to translate glycan synthesis into
a multiplexed high-throughput application.

6.3
One-Pot Syntheses

Enzymatic one-pot syntheses for the production of glycoconjugates are character-
ized by the use of multiple enzymes in one reaction vessel, therefore comprising
multiple catalytic steps in situ. In comparison to sequential syntheses, the one-pot
approach is the most favorable to optimize reaction time and space-time yield.
However, as multiple enzymes are combined directly, the conditions must be
carefully evaluated to suit all necessities of the involved biocatalysts. As in the
previous part of this chapter, we take a look at some examples for the syntheses of
nucleotide sugars and glycan structures.

6.3.1
Nucleotide Sugars

Starting from sucrose, one-pot reactions are suitable to work as regeneration cycles
for nucleotide sugars as demonstrated for UDP-Glc, UDP-Gal, and dTDP-deoxy-
sugars [98–103] (Scheme 6.4). Sucrose synthase (SuSy), known for the reverse
reaction of a GT, is used in combination with different enzyme module systems.
Nucleoside diphosphate (NDP) from GT modules are substrates of SuSy catalyzed
synthesis of activated glucose (UDP-Glc, dTDP-Glc) which enter enzyme modules
to generate the donor substrates of GTs.