Page 175 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

6.4 Convergent Syntheses 151

A versatile synthesis of a sialoside library was established using three enzymes,
[119] starting with a range of Man(NAc)-derivatives, subsequent actions of sialic acid
aldolase, CMP-sialic acid synthetase, and proper SiaTs generated novel sialosides.
Interestingly, this system was also used with diverse Lac(NAc)-derivatives expand-
ing the scope of the sialoside library [120]. Sialylation of Tn-antigen derivatives was
accomplished by use of α2,6SiaT [121].
Peptide glycosylation was also carried out in a one-pot fashion. A large scale
process was used for the synthesis of MUC6-Tn glycoconjugates [122]. In this syn-
thesis, a nondefined mixture of ppGalNAcTs from a preparation of MCF7 cells were
used for the glycosylation of peptides. Another approach combined ppGalNAcT-1,-
2, and -13 with in situ regeneration of UDP-GalNAc for the glycosylation of various
peptides for use as vaccines with defined glycosylation patterns [123].
One-pot reactions were also carried out with immobilized enzymes. The syn-
X
thesis of Le -antigen derivatives was performed with immobilized β4GalT and
α3FucT [124]. Both enzymes were immobilized via the action of Sortase A from
Staphylococcus aureus.
Striking examples for the use of glycosidases in one-pot approaches have been
quite rare up to this time. Coupling of the β-galactosidase transglycosylation
reaction with a α2,3SiaT was introduced to drive the transglycosylation equilibrium
toward the synthesis of sialylated Tn-antigen [125]. Additionally, a full in situ
regeneration cycle for CMP-Neu5Ac was included.

6.4
Convergent Syntheses

Convergent syntheses combine products of independent synthesis cycles in a final
reaction. Each of the synthesis cycles may include cascade reactions by themselves.
These might be sequential or one-pot cascades employing either chemical or
enzymatic methods.
Convergent reactions are beneficial in fulfilling different requirements toward
reaction conditions to obtain complex glycoconjugates. Chemical reactions require
organic solvents and high temperatures that are often not compatible with biocat-
alytic steps. Nevertheless, these reactions can be combined in a convergent process.
Indeed, the majority of the applications of this strategy lead to the synthesis of
complex or even branched glycoconjugate structures.
A recent example employed a chemical conjugation of oxidized poly-LacNAc
oligomers with a heptasaccharide to produce a complex, branched polysaccha-
ride capable of binding a variety of glycan binding proteins with higher avidity
(Figure 6.9) [126]. Here different oligosaccharide building blocks could be joined
in a final coupling reaction to produce an even more complex polymer.
Also donor and acceptor substrates for β-N-acetylhexosaminidase from T. flavus
were modified via enzymatic oxidation of GlcNAc by galactose oxidase and alkaline
epimerization of chitobiose. Transglycosylation reactions yielded complex HexNAc-
oligosaccharides with GlcNAc and ManNAc residues [52].