Page 158 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

134 6 Chemo-Enzymatic Cascade Reactions for the Synthesis of Glycoconjugates

Therefore, de novo synthesis strategies for glycoconjugates are in demand and
pose a considerable challenge because of the targets’ chemical complexity. Reactions
with fine control of regio- and stereoselectivity have to be translated into fast and
inexpensive processes generating glycoconjugates in high yields and high quality.
These requirements are difficult to achieve by chemical synthesis because of
multiple protection and deprotection steps leading to moderate yields and long
processes. Biocatalysis, employing glycosyltransferases (GTs), glycosidases, and
glycosynthases, offers unique advantages in stereo- and regioselectivity for fast
reactions at ambient temperature in aqueous solutions. An expanding array of
pathway enzymes from pro- and eukaryotic origin are available for the design
of in vitro synthesis strategies [22, 23]. They include enzymes for transfer and
modification of carbohydrates as well as for co-factor and substrate regeneration
[24]. In addition, site-directed mutagenesis or directed evolution is applied to adapt
enzymes for the conversion of chemically modified sugar substrates [25–27]. The
combination of various enzymes either in a sequential or ideally in a multi-enzyme
one-pot synthesis has been proved to be superior strategy for the synthesis of
glycoconjugates. In particular, a combination of enzymatic and chemical processes
is a suitable choice for the synthesis of highly pure and complex glycoconjugates.
With merging well-known chemical synthesis strategies and an ever-evolving
plethora of enzymatic biocatalysts we have the toolbox at hand to fulfill the demands
for the synthesis of well-defined glycoconjugates. In this respect, we highlight in
this chapter recent advances in chemo-enzymatic synthesis of glycoconjugates.
Summarizing first the principles of the used biocatalysts we focus on cascade
reactions including biocatalytic and chemocatalytic steps in different combinations.
Different concepts of cascade reactions are herein illustrated by recent exemplary
studies; the scope of the chapter is not meant to provide a comprehensive treatment.

6.1.2
Biocatalysts for the Synthesis of Glycoconjugates

6.1.2.1 Glycosyltransferases
GTs (EC 2.4.1.) are the working horses of natural glycosylation and the most
widely used enzymes for glycan syntheses in vitro. Owing to their stereo- and
regioselectivity as well as their substrate promiscuity, they are excellent choices for
the formation of complex glycan structures [25, 28–31]. GTs transfer glycans from
activated donor substrates to a broad set of acceptor substrates creating specific
glycosidic bonds. The acceptors reach from natural molecules like other glycans,
peptides, and so on to artificial synthetic compounds. Several reviews depict the
variety of applications for GTs [22, 32–35].
A peptide sequence based classification of this diverse group of over 65 000
enzymes divided into over 89 families is found within the CAZy (Carbohydrate-
Active enZYmes) database [25, 36]. The group with the highest synthetic potential
and impact for the synthesis of glycoconjugates are the Leloir-GTs and are therefore
in the focus of this chapter.