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378 17 Enzymatic Generation of Sialoconjugate Diversity

homology. Bacterial SiaTs are grouped into five GT families (GT4, GT38, GT42,
GT52, and GT80). On the other hand, all identified eukaryotic SiaTs belong to the
GT29 family, together with some viral SiaTs. Mammalian SiaTs are membrane
proteins made up from a soluble catalytic domain, which shows a relatively well-
conserved protein sequence homology among eukaryotic and viral SiaTs, and a
single transmembrane domain that localizes the enzyme in the Golgi lumen.
Mammalian enzymes often suffer from stability problems and normally cannot be
well produced from prokaryotic expression systems, which limits their application
for in vitro synthesis. On the other hand, bacterial SiaTs have less conserved protein
sequences among themselves, and none of them displays sequence homology to
mammalian SiaTs. Because bacterial SiaTs can be expressed more readily in soluble
form by an E. coli expression system, such enzymes have been developed recently
as efficient tools for sialoconjugate synthesis. Among these, a multifunctional
2,3/2,6SiaT from Pasteurella multocida [56, 57] as well as the 2,3- and 2,6SiaTs from
a number of marine Photobacterium sp. [58, 59] have immensely expanded the
synthetic potential originating in the bacterial SiaT family.
Several 3D protein structures of bacterial SiaTs have been determined in the
presence of CMP-Neu5Ac as a ligand [60]. Based on these crystal structures, from
which the active site organization and the catalytic mechanism have been elucidated,
SiaT enzymes can be predicted to tolerate rather flexibly structural variations in the
sialic acid part. This can be rationalized because only the nucleotide portion and the
sialic acid substructure around the anomeric center become buried into the active
site upon substrate binding and orientation toward the sialyl acceptor substrate,
whereas much of the remainder of the sialic acid portion remains oriented toward
the protein surface or even in contact with bulk solvent. This hypothesis has been
verified by a number of preparative studies [33, 47, 61].

17.3
Cascade Synthesis of neo-Sialoconjugates
17.3.1
Choice of Sialyl Acceptor

The development of new methods for oligosaccharide synthesis by using GTs
consumes expensive reagents and biocatalysts. Therefore, sensitive monitoring of
exploratory reactions is essential at the analytical scale to acquire reliable results at
the lowest possible expenses. For this purpose, the lactoside 36 [33] was developed
as a routine sialyl acceptor because it matches the SiaT acceptor specificity for
β-configured galactosides in combination with a rapid convergent synthesis from
simple building blocks, the presence of an allylic linker unit as a potential
cleavage site, and a fluorescent acridone tag as an ultrasensitive detection and
purification aid [62]. Also, the N-acetyl-d-lactosamine (LacNAc) moiety ubiquitously
found at the mature termini of oligosaccharides in glycoproteins and glycolipids
is an important natural substrate of SiaTs [63]. Therefore, the synthesis of a