Page 168 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

144 6 Chemo-Enzymatic Cascade Reactions for the Synthesis of Glycoconjugates

Biotinylated analogs of gangliosides were synthesized using a cascade of α2,3SialT
and β1,4GalNAcT, starting from biotinylated lactose [87]. UDP-GalNAc was pro-
′
duced in situ by UDP-GlcNAc 4 -epimerase. Remarkably, both enzymes were used
in a mixture of methanol and water for increased solubility of the biotinylated
product.
Enzymatic synthesis of branched core 2 O-linked glycans was carried out with
GT enriched microsomes from CHO-K1 cells [88]. Sequential glycosylation led to
the conversion of core 1(Galβ1,3GalNAcβ-O-pNP) to a core 2 branched O-glycan
hexasaccharide. The involved enzymes were identified as a β1,6GlcNAcT, β1,3
and β1,4GalT, as well as α2,3SialT. Therefore, the final saccharide product could
be identified as NeuAcα2-3Galβ1-3[NeuAcα2-3Galβ1-3/4GlcNAcβ1-6]GalNAc. Each
sequential enzymatic reaction was induced by addition of the appropriate nucleotide
sugar to the mixture of membrane preparation, acceptor substrate, and suitable
buffer system.
A heparin oligosaccharide backbone, which was later prone to sulfation, was
synthesized in a cascade mode by a sequential combination of α3GlcNAcT (KfiA)
from Escherichia coli and β4GlcUAT (heparosan synthase-2, pmHS2) from Pas-
teurella multocida [89]. KfiA accepted the chemically modified sugar-nucleotide
UDP-GlcNTFA for transfer onto the chemically synthesized acceptor disaccharide
GlcUAβ1,3AnMan, where AnMan is a 2-deoxy derivative of Man. GlcNH was
2
obtained by deprotection before the addition of pmHS2 and UDP-GlcA. A crucial
point for the cascade was that no residual UDP-GlcNTFA was present in the mix-
ture, as pmHS2 is also capable of transferring this sugar-nucleotide. This would
lead to an uncontrollable polymerization and no definable oligosaccharide length.
In continuation, a remarkable complex sequence was used for the synthesis
of ultralow (1.5–3 kDa) molecular weight heparins and heparin oligosaccharides
(Figure 6.5) [90–92]. In a first cascade, a tetrasaccharide was synthesized with KifA
and pmHS2 as described above. The purified intermediate enters the second three-
step sequence of KifA, pmHS2, and again KifA, to yield a heptasaccharide. The
final cascade yields a heparin heptasaccharide by chemical deprotection producing
GlcNH and subsequent enzymatic O- and N-sulfation as well as C -epimerization.
2 5
This cascade synthesis is a brilliant example of a smart biocatalyst combination,
reaction, and process design to produce complex glycan structures with minimal
effort for intermediate product purification. As heparins are of great interest for
biomedical applications, this synthesis provides a great opportunity to produce
tailored heparin molecules.
Decoupling of enzymatic reaction steps can be easily achieved by immobilization
of the enzymes. Heparosan oligosaccharides and hyaluronan oligosaccharides were
synthesized with immobilized mutant enzymes derived from Pasteurella multocida
hyaluronan synthase (PmHAS) [93, 94]. PmHAS is a bifunctional biocatalyst
with two GT domains, which work in concert to produce polymeric heparosan
and hyaluronan. However, protein engineering was used to transfer this double-
action enzyme into two single-action biocatalysts. The enzyme domains were
2+
immobilized either by Ni -NTA (nitrilotriacetic acid) columns or by undirected