Glycosyltransferases 2015 PDF

507
1.6.2 Glycosyltransferases
J. Voglmeir and S. L. Flitsch
General Introduction
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The emerging field of glycobiology has revealed the biological role of hundreds of
glycosyltransferases involved in the biosynthesis of the glycome. This knowledge about
the enzymatic function of glycosyltransferases also allows us to meet the increasing de-
mands for carbohydrates and their analogues for the elucidation of the exact function in
these biological processes. The stereo- and regioselective properties and the high selectiv-
ity of glycosyltransferases toward donor and acceptor substrates make these enzymes
highly attractive for synthetic applications. Various examples of glycosyltransferases ex-
pressed recombinantly demonstrate the versatility of both in vivo and in vitro syntheses
of oligosaccharides from milligram to kilogram scale. However, due to the enormous va-
riety of carbohydrate structures in living organisms, to date only a small proportion of
carbohydrate epitopes have been synthesized in a routine manner. This chapter summa-
rizes recent approaches to the application of glycosyltransferases in both preparative
sugar synthesis and biotransformation.
Whereas nucleic acid and protein synthesis has been widely achieved on an automat-
ed level for several decades, a general way to automate the synthesis of carbohydrates is
not yet available. Nevertheless, several elegant strategies have been applied to overcome
the bottlenecks in organic synthesis involving carbohydrates, such as the introduction of
novel protecting groups,[1,2] solid-phase synthesis,[3,4] the application of novel catalysts,
and reactivity-based one-pot assembly methods.[5,6] Despite all of these advances, the gen-
eral synthetic scheme still consists of the tedious preparation of a protected glycosyl
donor containing a leaving group and a glycosyl acceptor with a single unprotected hy-
droxy group, their coupling reaction, and consecutive deprotection steps. Each stage of
the synthetic scheme includes several steps and the level of complexity increases drasti-
cally with the number of desired glycosidic linkages. In general, the formation of a glyco-
sidic linkage may lead to a mixture of products with different ratios of Æ- or -configura-
tion, with the ratio depending on several factors, such as neighboring-group effects of the
donor/acceptor molecule and the reaction conditions. In contrast, stereoselective trans-
formations of an anomeric center can be achieved with high yields in a single-step reac-
tion using enzymatic or chemoenzymatic glycosylation approaches (Scheme 1).[7] To
achieve enzymatic conversion, the donor intermediate has to be “activated” by being
joined via a high energy hemiacetal phosphate ester, which allows the irreversible, and
therefore complete, transfer of the donor sugar to an acceptor nucleophile, which is gen-
erally an alcohol. The following sections give an overview of the biosynthesis and current
biosynthetic strategies toward glycans and glycan derivatives. Other enzymatic glycosyla-
tion strategies, such as transglycosylation and reverse glycolysis, are described in Section
1.6.1.
for references see p 539

508 Biocatalysis 1.6 Glycosides
Scheme 1 Comparison of Chemical and Enzymatic Glycoconjugate Synthesis[7]
OH
OH HO
OH HO
glycosyltransferase O OH
O + O HO
HO
HO OH HO HO
O O
OH HO OH
O HO
UDP OH
unprotected acceptor sugar activated donor sugar desired glycoside
deprotection
OPG
PGO
OPG OPG
PGO O OPG
O HO O PGO
LG + OPG PGO
PGO PGO O
O
OPG OPG PGO OPG
OPG
protected reaction product
OPG
PGO
O
PGO β OPG
PGO
α HO O
PGO OPG
OPG
PG = protecting group; UDP = uridine diphosphate
1.6.2.1 Sugar Nucleotides as Glycosyl Donors
With the discovery of new glycosyltransferases that can be easily expressed in high yields,
particularly those originating from microbial sources, the demand for simple and effi-
cient methods for synthesizing sugar nucleotides used for oligosaccharides and glycocon-
jugates has increased. Fully synthetic approaches are challenged by strict reaction condi-
tions, the presence of polar functional groups and therefore the low solubility of sugar
nucleotides in organic solvents, and the hydrolytic cleavage of the hemiacetal phosphate
ester bond.[8] On the other hand, purely multienzymatic approaches strictly following the
biosynthetic pathway are susceptible to inhibition from products or coproducts, requir-
ing in situ recycling, or further conversion. Therefore, combining chemically synthesized
(and eventually functionalized) sugar precursors which are enzymatically “activated”
with the nucleotide moiety in a final reaction step is widely applied.[3,9,10]
1.6.2 Glycosyltransferases 509
1.6.2.1.1 Commonly Known Nucleotide-Activated Sugars
Commonly known nucleotide sugars are generally regarded as those nine activated forms
1–9 that have been the focus of scientific research over the last five decades (Scheme 2).
They are present in all higher animals and can be easily classified based on the type of
sugar-linked nucleoside base such as uridine [UDP-galactose (1), UDP-glucose (4), UDP-xy-
lose (3), UDP-glucuronic acid (6), UDP-N-acetylgalactosamine (8, UDP-GalNAc), and UDP-
N-acetylglucosamine (5, UDP-GlcNAc)], guanine [GDP-mannose (7) and GDP-fucose (9)],
and cytosine [CMP-N-acetylneuraminic acid (2, CMP-Neu5Ac)].[11] With the exception of
UDP-xylose (3), all of these nucleotide sugars are commercially available, with prices (in
July 2014) ranging several magnitudes from <$100/g for UDP-glucose to >$200/mg for
GDP-fucose.
All of these activated sugars can theoretically be derived from glucose as a common
precursor from glycolysis. However, the specific biochemical route for each nucleotide
sugar, involving isomerization, transamination, transacetylation, and transphosphoryla-
tion reactions, varies substantially in length and cofactor requirement. Practical routes
for the synthesis of UDP-glucose (4), UDP-N-acetylglucosamine (5), and GDP-mannose (7)
usually start from the corresponding 6-phosphosugar precursors, which, after their isom-
erization to 1-phosphosugars, are nucleotide activated by specific phosphorylases [UDP-
glucose phosphorylase (EC 2.7.7.9), UDP-N-acetylglucosamine pyrophosphorylase (EC
2.7.7.23), GDP-mannose phosphorylase (EC 2.7.7.13)]. A highly efficient alternative bio-
chemical route for the synthesis of UDP-glucose (4), the activated precursor to synthesize
sucrose, exists in plants.[12] This reaction is catalyzed, at near equilibrium between sucrose
synthesis and degradation of sucrose, by the enzyme sucrose synthase (SuSy, EC 2.4.1.13),
and is therefore a good candidate for biocatalytic approaches where UDP-glucose (4) is fur-
ther processed by another enzyme.[13,14] Beside UDP, sucrose synthase can also transfer
other nucleotides such as ADP (10), thus allowing the synthesis of ADP-glucose (11), the
major glycosyl donor in starch biosynthesis (Scheme 3).[15]

Scheme 2 Common Nucleotide Sugar Structures
OH nucleotide sugar recycling OH OH

HO HO OH
via salvage pathways
O O CO2H
AcHN
HO
HO OH HO
galactose Neu5Ac
secondary nucleotide sugars
derived from
primary nucleotide sugars
OH OH CMP
HO OH O
HO
O O CO2H
HO AcHN
HO HO
O 2 CMP-Neu5Ac
UDP
1 UDP-galactose
primary nucleotide sugars
derived from glucose
OH OH
HO O HO O
HO HO
HO AcHN
O O
HO O UDP glucose UDP
HO 4 UDP-glucose 5 UDP-GlcNAc
HO
O
UDP
OH
3 UDP-xylose
HO
HO O
HO
OH
HO
O
GDP O
HO2C 7 GDP-mannose
O HO
HO
HO AcHN
O
HO UDP
O
UDP 8 UDP-GalNAc
6 UDP-glucuronic acid
GDP
O O
OH
OH
HO
9 GDP-fucose
OH
OH HO
O O
OH HO
OH AcHN
HO OH
fucose GalNAc
UDP = uridine diphosphate; GDP = guanosine diphosphate; CMP = cytidine monophosphate

Scheme 3 Synthesis of ADP-Glucose Using Sucrose Synthase[15]
OH
O
P OH
O
NH2 O NH2
HO P
O N O N
N N
HO P O HO P O myokinase
N + N
OH N O N
O O
OH OH OH OH
OH
HO O OH
NH2 HO OH
2 O
HO
O O N O
N
2 HO P O P O OH OH
N sucrose synthase
OH OH N
O
OH
OH
O
OH OH − 2 HO
10 OH OH
OH
NH2
HO O
HO O O N
N
HO
O P O P O
N N
O O O
OH OH
11
All other common nucleotide sugars including UDP-galactose (1), UDP-N-acetylgalactos-

amine (8, UDP-GalNAc), UDP-xylose (3), GDP-fucose (9), and CMP-neuraminic acid (2),
hereafter referred to as secondary nucleotide sugars, are generated by various other enzy-
matic reactions from the primary nucleotide sugars UDP-glucose (4), UDP-GlcNAc (5), and
GDP-mannose (7). UDP-Galactose (1) is simply derived from its isomer UDP-glucose (4) via
reversible isomerization by the enzyme UDP-glucose 4-epimerase, alternatively named
GalE (EC 5.1.3.2). The high price of UDP-galactose (1) makes this enzyme a valuable tool
in galactosylation reactions, allowing the utilization of multigram-scale reactions by
using less expensive UDP-glucose (4).[16,17] Similarly, the formation of UDP-GalNAc (8)
from UDP-GlcNAc (5) is catalyzed by the enzyme UDP-N-acetylglucosamine 4-epimerase
(EC 5.1.3.16) in the presence of NAD+.[18] Several of the characterized isoforms of these
4-epimerases are promiscuous for the epimerization of both UDP-glucose (4) and UDP-
GlcNAc (5), which makes them versatile candidates for various biocatalytic approaches.[19]
Alternatively, the chemoenzymatic synthesis of UDP-GlcNAc (5) and UDP-GalNAc (8) can
start from glucosamine (12)/galactosamine (14), which are phosphorylated (to 13 and 15,
respectively) and, after enzymatic nucleotide transfer, are chemically acylated as de-
scribed in Scheme 4. In the preparation of UDP-GalNAc (8), UDP-glucose pyrophosphoryl-
ase is used to regenerate UDP-glucose (4) from glucose 1-phosphate (16).[20,21]

Scheme 4 Chemoenzymatic Synthesis of UDP-GlcNAc and UDP-GalNAc from Glucosamine

and Galactosamine[20,21]
OH
HO O
HO OH
NH2
12
ATP
AcO−
ATP regeneration acetate kinase hexokinase
AcOPO32−
ADP
OPO32−
HO O
HO OH
H2N OAc
13 O N O
phosphoglucomutase
OH
O OPO32−
HO
HO
HO O
H2N
OPO32− HO OH
NHAc
UDP-glucose
Candida utilis
pyrophosphorylase
whole-cell extract
OH OH
HO O HO O
HO HO
H2N AcHN
O OPO32−
UDP
OAc UTP
O N O OH
inorganic
HO O pyrophosphatase
HO (removal of
AcHN pyrophosphate)
O PPi 2 Pi
UDP
5
UTP = uridine triphosphate; UDP = uridine diphosphate; PPi = inorganic pyrophosphate; Pi = inorganic phosphate
OH
HO
O
HO OH
NH2
14
ATP
AcO−
ATP regeneration acetate kinase galactokinase
AcOPO32−
ADP
OH
HO
O
HO
H 2N
OH OPO32−
inorganic
pyrophosphatase
15
HO O
(removal of HO
pyrophosphate)
HO
2 Pi PPi O
UDP
4 UDP-glucose
UDP-glucose galactose 1-phosphate
pyrophosphorylase uridylyltransferase
OH
UTP O
HO
HO
HO
OPO32−
16
OH
HO
O
HO
H 2N
O
UDP
OAc
O N O
OH
HO
O
HO
AcHN
O
UDP
8 34% (from 15)
UTP = uridine triphosphate; UDP = uridine diphosphate; PPi = inorganic pyrophosphate; Pi = inorganic phosphate
The biosynthesis of UDP-xylose (3) from UDP-glucose (4) is realized by two enzymatic
steps. UDP-Glucose 6-dehydrogenase (EC 1.1.1.22) catalyzes the oxidation of UDP-glucose
(4) to UDP-glucuronic acid (6) in the presence of NAD+, followed by the action of a UDP-
glucuronate decarboxylase (EC 4.1.1.35). Despite this relatively simple biosynthetic route,
the requirement of NAD+ as a cofactor and product inhibitory effects of UDP-xylose (3) on
the UDP-glucose dehydrogenase have so far prevented multienzyme approaches for the
synthesis of UDP-xylose (3) or xylosides on a preparative scale. Nucleotide activation of
N-acetylneuraminic acid (Neu5Ac) is performed by the enzyme CMP-N-acetylneuraminic

acid synthetase (EC 2.7.7.43), which, in the presence of the cofactor CTP, forms the final
product CMP-N-acetylneuraminic acid (2).[22] The synthesis of several CMP-sialic acid ana-
logues is realized using N-acetylneuraminate lyase (NanA, EC 4.1.3.3), which catalyzes the
reversible cleavage of N-acetylneuraminate to N-acetyl-d-mannosamine and pyruvate in a
preparatively unfavorable equilibrium; by coupling both reactions, synthesis of N-acetyl-
neuraminate [and consequently CMP-N-acetylneuraminic acid (2)] can be driven to com-
pletion.[23] The formation of GDP-fucose (9) from GDP-mannose (7) is biochemically real-
ized in a two-enzyme reaction. In the first step, GDP-mannose dehydratase (EC 4.2.1.47)
forms GDP-4-keto-6-deoxymannose from GDP-mannose (7). This reaction is followed by
epimerization and reduction catalyzed by GDP-keto-6-deoxymannose 3,5-epimerase/4-re-
ductase (EC 1.1.1.271) to yield the final product GDP-fucose (9).[24] Alternatively, fucose re-
leased from glycoconjugates can also be regenerated to GDP-fucose (9) via fucose 1-phos-
phate in the salvage pathway.[25] A bifunctional fucokinase/pyrophosphorylase Fkp from
the bacterium Bacteroides fragilis has been successfully applied in the synthesis of GDP-fu-
cose (9) using whole-cell transformations.[26,27]
The biosynthetic pathways of the common nucleotide sugars described above are
well established and a multitude of chemoenzymatic applications have been realized
over the last years. However, the high costs of the activated cofactors and a limited num-
ber of practical regeneration schemes make further process optimizations inevitable.
ADP-Glucose (11); Typical Procedure:[15]

A 50-mL soln containing AMP (8 mM), ATP (8 mM), sucrose synthase (100 U), myokinase
(10 U), and bovine serum albumin (BSA; 100 mg) was added to a buffer soln (50 mL) con-
taining sucrose (500 mM), dithiothreitol (DTT; 3 mM), MgCl2 (125 M), and HEPES/NaOH
(200 mM; pH 7.5). After sterile filtration, the soln was stirred gently for 4 h at 30 8C. The
concentration of formed ADP-glucose (11) was determined by ion-pair reversed-phase
HPLC [Hypersil ODS, KOAc (100 mM; pH 4.6) with 0.013% (v/v) octylamine and 8% (v/v)
MeOH]. The reaction was stopped when a yield of approximately 55% had been reached
(in reference to the combined starting amount of 0.8 mmol AMP and ATP). Isolation of
ADP-glucose (11) was facilitated by dephosphorylation of AMP, ADP, and ATP using alka-
line phosphatase (20 U) by incubating overnight at 30 8C with gentle stirring.
1.6.2.1.2 Unusual Nucleotide-Activated Sugars
Besides the activated nucleotide sugars described above, several other nucleotide sugars
are also known to exist in species other than man. Although widely distributed in nature,
these compounds are only investigated on the sidelines of current scientific efforts and
are therefore herein referred to as unusual nucleotide sugars. Compared to the studies re-
ported for the nine common nucleotide sugars, little has been published about the chem-
ical and chemoenzymatic synthesis of such nucleotide sugars. Some examples of the bio-
synthesis and applications will be given here.
CMP-Glycolylneuraminic acid is the activated form of glycolylneuraminic acid,
which is derived from CMP-N-acetylneuraminic acid (2) by the CMP-N-acetylneuraminic
acid hydroxylase (EC 1.14.18.2) for the synthesis of many glycoconjugates. Chemically
synthesized N-acylmannosamine analogues 17 can be condensed with pyruvate to neur-
aminic acid derivatives 18 by sialic acid aldolase and activated by CMP-sialic acid synthe-
tase. Similarly to other neuraminic acid derivatives 19, CMP-glycolylneuraminic acid 19
(R1 = CH2OH) can be chemoenzymatically prepared from mannosamine, succinimide hy-
droxyacetate, and pyruvate (Scheme 5).[23]
Scheme 5 Chemoenzymatic Synthesis of CMP-Neuraminic Acid Derivatives[23]
O R1 O O
N
NH2 O O CO2−
HO HO HN R1
sialic acid aldolase
HO O HO O
HO OH HO OH
17
OH OH CMP
OH OH CTP OH O
HO HO
sialic acid synthetase
O CO2H O CO2H
HN HN
− PPi
HO HO
O O
R1 R1
18 19
R1 = Me, CH2OH, CH2N3, CH2NHCbz;
CTP = cytosine triphosphate; CMP = cytosine monophosphate; PPi = inorganic pyrophosphate
CMP-Glycolylneuraminic acid is an important member of the sialic acid family in all mam-
mals studied so far, but cannot be synthesized in humans.[28] It has been reported that the
consumption of red meats and dairy products allows the incorporation of glycolylneur-
aminic acid into the human body, where it can be activated by endogenous CMP-N-acetyl-
neuraminic acid synthetase, similar to endogenous N-acetylneuraminic acid. Several
CMP-sialic acid derivatives 21 can be generated chemoenzymatically by direct activation
of the chemically synthesized free sialic acid derivatives 20 with Neisseria meningitidis
CMP-N-acetylneuraminic acid synthetase and CTP as cofactor, which allows the rapid
and inexpensive preparation of CMP-sialic acid derivatives 21 up to gram scale (Scheme
6).[29] Structure-guided site-specific engineering has allowed the isolation of mutant var-
iants which show up to 70-fold improved activity for sialic acid analogues, without com-
promising enzyme stability.[30]
Scheme 6 Preparation of CMP-Sialic Acid Derivatives[29]
OH OH
R5 CTP R5 CMP
OH O
R4 Neisseria meningitidis R4
CMP-sialate synthetase
R3 1 O CO2H R3 1 O CO2H
R − PPi R
OH OH
R2 R2
20 21
CTP = cytosine triphosphate; CMP = cytosine monophosphate; PPi = inorganic pyrophosphate
UDP-Galacturonic acid is a precursor for the synthesis of numerous cell-surface polysac-

charides in bacteria and plants. It is produced by the enzyme UDP-glucuronic acid 4-epi-
merase (EC 5.1.3.2) through the epimerization of the 4-hydroxy group of UDP-glucuronic
acid (6).[31] GDP-d-Rhamnose is a nucleotide sugar donor involved in the exopolysac-
charide biosynthesis of bacterial cell surfaces, and is synthesized from GDP-mannose (7)
in two steps similarly to the synthesis of GDP-fucose (9).[32] UDP-Arabinose, a nucleotide
sugar abundant in all plants, is generated via epimerization of UDP-xylose (3) by UDP-ara-
binose 4-epimerase (EC 5.1.3.5).[33]
The synthesis of UDP-galactofuranose, which is commonly found in plants, fungi,
and bacteria, is catalyzed by UDP-galactofuranose mutase (EC 5.4.99.9), with the thermo-
dynamic equilibrium far at the UDP-galactopyranose form.[34] Chemically synthesized fu-
ranosyl 1-phosphate precursors can be used as enzyme substrates for the consecutive nu-

cleotide activation using UTP as nucleotide donor and a galactose 1-phosphate uridylyl-
transferase (EC 2.7.7.12) with broad substrate specificity, leading to the synthesis of this
nucleotide sugar in pure furanose form.[35]
Several biosynthetic routes of other uncommon nucleotide sugar pathways follow-
ing a similar principle have been successfully elucidated, including those for variously
modified sugars such as amino- and deoxysugars. These may help to extend the enzymatic
toolbox for the generation of uncommon glycosyl donors.
CMP-Sialic Acid Derivative 21 (R1 = R3 = OH; R2 = R4 = H; R5 = CH2OH); Typical Procedure:[29]

A mixture containing sialic acid derivative 20 (R1 = R3 = OH; R2 = R4 = H; R5 = CH2OH;
402 mg, 1.5 mmol), CTP (858 mg), inorganic pyrophosphatase (25 U), and CMP-N-acetyl-
neuraminic acid synthetase (530 U), was added to 50 mM Tris-HCl buffer (pH 8.5; 15 mL)
containing MgCl2 (50 mM) and dithiothreitol (DTT; 0.2 mM). The reaction was monitored
by TLC, and the pH of 8.5 was maintained by dropwise addition of 2 M aq NaOH. If no fur-
ther product formation was observed by TLC, precipitated enzymes and insoluble phos-
phate salts were filtered off by syringe filtration (0.22-m pore size). After addition of
EtOH to the clear supernatant (9:1 v/v), the colorless solid product was isolated by centri-
fugation and dried under reduced pressure; yield: 783 mg (80%, assumed as disodium salt).
1.6.2.2 Other Sugar Donors
1.6.2.2.1 Glycosyl Phosphates
The use of glycosyl phosphates and glycosyl pyrophosphates has regained interest as an
alternative to nucleotide sugars in enzymatic glycosylation reactions. Under certain ex-
perimental conditions, sugar phosphorylases, which naturally favor the phosphorolysis
of di- and oligosaccharides in the presence of inorganic phosphate, can also catalyze the
formation of O-glycosidic bonds. Several microbial disaccharide phosphorylases, such as
sucrose phosphorylase (EC 2.4.1.7), maltose phosphorylase (EC 2.4.1.64), and cellobiose
phosphorylase (EC 2.4.1.20) are widely used as enzyme catalysts for the production of glu-
cose 1-phosphate from the corresponding disaccharides and inorganic phosphate. Glu-
cose 1-phosphate (16) can be further utilized as a substrate for UDP-glucose synthesis
and consecutive carbohydrate synthesis using nucleotide sugar dependent glycosyltrans-
ferases. In an advanced study, two sugar phosphorylases were combined, amongst other
enzymes, to synthesize lacto-N-biose (24) on a kilogram scale in a one-pot, four-enzyme
reaction (Scheme 7).[17] In the consecutive actions of sucrose phosphorylase [phospho-
rolysis of sucrose to glucose 1-phosphate (16) and fructose], UDP-galactose:glucose 1-phos-
phate uridylyltransferase [transfer of the nucleotide from UDP-galactose (1) to glucose
1-phosphate and generation of galactose 1-phosphate (22)], UDP-glucose 4-epimerase [epi-
merization of UDP-glucose (4) to UDP-galactose (1)], and lacto-N-biose phosphorylase [EC
2.4.1.211; reversible phosphorolysis from lacto-N-biose and inorganic phosphate to galac-
tose 1-phosphate and N-acetylglucosamine (23], 86% conversion of sucrose and N-acetyl-
glucosamine (23, GlcNAc) into lacto-N-biose (24) and fructose was achieved. UDP-Glucose
(4) and inorganic phosphate are only present in catalytic concentrations. Product purifi-
cation is conducted by final treatment of the reaction mixture with yeast to remove the
generated fructose as well as residual sucrose and N-acetylglucosamine.
Scheme 7 Kilogram-Scale Synthesis of Lacto-N-biose in a One-Pot, Four-Enzyme

Reaction[17]
HO
HO O OH
HO OH
HO O
O
OH OH
catabolic
OH treatment
SP OH with yeast
O
HO H2O + CO2 + EtOH
OH OH
OH fructose
HO O
HO
HO
OPO32−
16
GalT
OH
HO O
HO
HO
O
UDP
4
GalE
OH
HO
O
HO
HO
O
UDP
1
UMP
GalT OH
HO O
HO OH
OH NHAc OH OH
HO 23 GlcNAc HO
O LNBP O HO O
HO HO O OH
HO HO NHAc
OPO32−
22 24 86% (based on GlcNAc)
Pi
SP = sucrose phosphorylase; GalT = UDP-galactose:glucose1-phosphate uridylyltransferase;

GalE = UDP-glucose 4-epimerase; LNBP = lacto-N-biose phosphorylase
Similar strategies have been applied for the synthesis of galacto-N-biose [-d-galactopyran-
osyl-(1ﬁ3)-2-acetamido-2-deoxy-d-galactose] from sucrose and N-acetylgalactosamine
(GalNAc).[36] Another application describes the use of potato phosphorylases for the syn-
thesis of maltose oligomers from sucrose. A coupled enzymatic reaction of sucrose phos-
phorylase with potato phosphorylase (EC 2.4.1.7) gives high yields for the formation of an

Æ-1,4-glucopolysaccharide. The reaction equilibrium can be shifted toward polymeriza-

tion due to the catalytic concentrations of inorganic phosphate in the reaction mixture,
and the degree of polymerization can be controlled by the amount of acceptor sub-
strate.[37] Moreover, potato phosphorylase has been used to generate well-defined
branched and hyperbranched polysaccharides using star- and comb-shaped acceptor oli-
gosaccharides.[38,39]
Taking into account that starting materials for the generation of sugar phosphates,
such as starch or sucrose, are very inexpensive and abundantly available, glycosylation
reactions using glycosyl phosphate donors in specific cases have the potential to be pow-
erful alternatives to nucleotide sugar donor driven reactions.
Lacto-N-biose (24); Typical Procedure:[17]
A soln (10 L) containing sucrose (660 mM), GlcNAc (23; 600 mM), UDP-glucose (4; 1 mM),
phosphate buffer (30 mM; pH 7.0), MgCl2 (10 mM), sucrose phosphorylase (SP; 17 kU),
UDP-galactose:glucose 1-phosphate uridylyltransferase (GalT; 36 kU), UDP-glucose 4-epi-
merase (GalE; 31 kU), and lacto-N-biose phosphorylase (LNBP; 1.5 kU) was incubated at
30 8C. The concentration of lacto-N-biose (24) reached 500 mM after 600 h; yield: 83%
[based on residual GlcNAc (23)]. Diethylaminoethyl-cellulose (DEAE-cellulose; 50 g; equil-
ibrated in phosphate buffer, pH 7.0) was added to absorb the enzymes, and removed by
filtration after the mixture had been stirred at rt for 90 min. Bakers yeast (200 g) was
added to the mixture and incubated at 30 8C for 12 h to eliminate fructose and the residual
sucrose substrate. After removal of the yeast by centrifugation and concentration of the
mixture to 4.8 L by rotary evaporation, lacto-N-biose (24) was crystallized at 4 8C (12 h);
yield: 1.5 kg [65.3% based on GlcNAc (23)].
1.6.2.2.2 Di- and Oligosaccharides
Due to the low cost of the starting materials, enzymatic glycan synthesis based on non-
phosphoryl-activated disaccharide donor substrates shows great potential for applica-
tions in the food industry, as additives for dyes, and in cosmetics. Various microbial
glycosyltransferases isolated from Leuconostoc, Lactobacillus, Streptococcus, and other gen-
era have been used to demonstrate the successful synthesis of Æ-glucans from sucrose in
whole-cell transformations[40,41] and in vitro.[42] Isoforms of these enzymes isolated from
different microorganisms have allowed the synthesis of various Æ-glucans such as
mutan (Æ-1,3-glucose units), alternan (alternating Æ-1,3- and Æ-1,6-glucose units), and
amylose (Æ-1,4-glucose units) or dextrans (Æ-1,6-linked glucose units) with or without
Æ-1,2- and Æ-1,3-glucose side chains.[43]
1.6.2.2.3 Glycosyl Lipids
Lipid-linked glycosyl donors play an important role as sugar donors in membrane-based

glycosylation reactions. These donors consist of a lipid anchor, with structural features
such as length and saturation depending on the type of organism. Eukaryotes and archaea
lipid anchors are generally dolichol derivatives, whereas bacteria bear undecaprenols.[44]
In eukaryotes, the lipid carrier is phosphorylated in a first step by the action of a CTP de-
pendent dolichol kinase (EC 2.7.1.108).[45] In the second step, the transfer of glucosamine
1-phosphate is catalyzed by the enzyme UDP-N-acetylglucosamine:N-acetylglucosamine
1-phosphate transferase (EC 2.7.8.15) from a UDP-N-acetylglucosamine donor substrate.[46]
An additional lipid-linked carbohydrate bearing a mannose moiety abundant in animals
and yeast can be generated by the action of guanosine diphosphomannose:dolichol phos-
phate mannosyltransferase (EC 2.4.1.83) transferring mannose 1-phosphate from GDP-
mannose (7).[47] Both of these lipid-linked diphosphosugars are acceptor substrates for
the stepwise assembly of lipid-linked glycan precursors from further nucleotide sugars by
a series of glycosyltransferases.[48]
Several examples demonstrate the synthesis of dolichol-based glycosyl lipids. Howev-
er, one of the major limitations of preparing these structures is the low abundance and
heterogeneity of dolichol from natural sources.[49] This drawback can be effectively cir-
cumvented by substituting dolichol with the synthetically accessible isoprenoid analogue
phytanol, which provides comparable acceptor substrates for various mannosyltransfer-
ases (Scheme 8).[50,51]
Scheme 8 Chemical Synthesis of a Phytanol-Based Glycosyl Lipid and Subsequent Enzy-

matic Mannosylation[50,51]
OH
OH OAc
1. Ac2O OAc
HO O 2. H2NNH2•AcOH
O AcO O
HO O O
HO AcO O
AcHN AcO
AcHN OH AcHN
AcHN OH
O O
1. BnO P P OBn
O
BnO OBn OAc
OAc
LDA
2. Pd, H2 AcO O
O O
AcO
AcO
AcHN
AcHN OPO 2−
3
1. 2−
O3PO
H
4
O
N N OH
OH
N N
HO O
2. NaOMe O
HO O O O− O O−
HO
AcHN P P H
AcHN O O O
4
OH
HO
HO O
HO
O
GDP OH
OH OH
7 OH
β-1,4-ManT HO O
O O
HO O
HO O O O− O O−
HO
AcHN P P H
AcHN O O O
4
β-1,4-ManT = β-1,4-mannosyltransferase
1.6.2.2.4 Other Sugar Donors
Glycosyl fluorides, compounds which are normally used as substrates for glycosynthases
(see Section 1.6.1), have been successfully applied as alternative substrates for the synthe-
sis of oligosaccharides catalyzed by nucleotide diphosphate sugar dependent glycosyl-
transferases.[52] In the presence of catalytic quantities of UDP, Æ-galactosyl fluoride is suc-
cessfully transferred to fluorescently labeled lactosides and galactosides by a lipopolysac-
charide galactosyltransferase C from Neisseria meningitidis in quantitative yields. A second
application using a fluorosugar as donor substrate demonstrated that a sucrose phospho-

rylase from Leuconostoc mesenteroides catalyzes the enzymatic glycosylation using Æ-gluco-
syl fluoride as donor substrate in the presence of fructose.[53] Simple aromatic compounds,
such as 2-chloro-4-nitrophenyl -d-glucopyranoside, can be used as sugar donors using
oleandomycin glycosyltransferase from Streptomyces antibioticus for the generation of var-
ious nucleotide sugars.[54]
1.6.2.3 Glycoconjugate Products
Glycoconjugates are a massive group of sugar-derivatized organic substances that can

play critical roles in diverse biological events, with the sugar being the key functional
component. Glycoconjugates generally include free oligosaccharides, polysaccharides,
glycoproteins/glycopeptides, and glycolipids. Although the functional role of glycoconju-
gates is very important, many of these compounds exist only in minute amounts and
therefore isolation from natural sources is only possible in a limited number of cases. Syn-
thesis of these products in vitro on a large scale is therefore of considerable interest.
Enzymatic or chemoenzymatic synthesis of glycoconjugates has become a realistic
alternative approach to pure chemical synthesis methods.[55] Glycosyltransferases have
thus been extensively studied and methods and protocols have been developed for the fac-
ile utilization of these enzymes in chemoenzymatic synthesis for different glycoconju-
gates. Although genome-wide more than 300 glycosyltransferases have been identified
in humans,[56] the glycosyltransferases currently used for the synthesis of glycoconjugates
(mainly) include galactosyltransferases, fucosyltransferases, sialyltransferases, poly-
peptide N-acetylgalactosaminyltransferases, and N-acetylglucosaminyltransferases. Isola-
tion of these glycosyltransferases from natural sources is generally not applicable to ob-
tain sufficient enzyme for the synthesis of glycoconjugates on a preparative or large scale,
especially if the glycosyltransferase is of mammalian origin. The discovery of bacterial
glycosyltransferases, which are stable, can be well expressed, and have similar functions
to mammalian glycosyltransferases, enables the simple preparation of glycosyltransfer-
ases in large quantities.[57,58] Glycosyltransferases can be used for the synthesis of glyco-
conjugates either individually, if the substrate of the final synthetic step is available, or
in combination with other related glycoenzymes such as epimerases, synthases, or glyco-
sidases in a multistep approach. The latter can be realized by separate, consecutive reac-
tions for each step, or in a one-pot reaction. The synthesis of target glycoconjugates in a
one-pot reaction can be performed more efficiently with high yields, time- and cost-effec-
tiveness, reproducibility, and simple operation.[59] Until now, numerous publications
have described the application of glycosyltransferases for the production of naturally ex-
isting or synthetic glycoconjugates which have remarkable biological functions.
1.6.2.3.1 Free Oligosaccharides
A number of different glycosylation reactions have been applied for the enzymatic syn-
thesis of free oligosaccharides in vitro. The following examples are divided based on the
type of sugar moiety transferred.
1.6.2.3.1.1 Galactosylation
The simplest and most commonly studied galactose-containing disaccharides are lactose
and N-acetyllactosamine (25, LacNAc). LacNAc (25) is believed to be a biofunctional factor
in human milk oligosaccharides, responsible for the intestinal colonization of beneficial
Bifidobacteria in infants. Various enzymatic synthesis routes have been developed for the
preparation of these compounds. For example, LacNAc (25) is synthesized using in situ
cofactor regeneration starting from glucose 6-phosphate (26), GlcNAc (23), and phospho-
enolpyruvate (Scheme 9).[60] Also, whole-cell reactions by bacterial coupling have been em-
ployed for the production of LacNAc (25) using Escherichia coli cells overexpressing genes
responsible for UDP-galactose synthesis and the -1,4-galactosyltransferase of Neisseria
gonorrhoeae, with Corynebacterium ammoniagenes contributing the production of UTP,
yielding high concentrations of LacNAc (25).[61]
Scheme 9 Enzymatic Synthesis of LacNAc with In Situ Cofactor Regeneration[60]
OH
HO O
HO
AcHN OH
23
OH
HO
OH
O HO OH
HO β-1,4-GalT1
O
O
HO HO O
O HO
UDP OH
1 AcHN OH
25 LacNAc
GalE
UDP
OPO32−
OH CO2−
O PK
HO
HO cofactor regeneration
HO
O
UDP UTP O
4
CO2−
UGPP
PPi OH OPO32−
O PGM O
IPP HO HO
HO HO
HO OH OH
OPO32−
2 Pi
16 26
UTP = uridine triphosphate; UDP = uridine diphosphate; PPi = inorganic pyrophosphate; Pi = inorganic phosphate;
GalE = glucose 4-epimerase; β-1,4-GalT1 = β-1,4-galactosyltransferase; PK = pyruvate kinase;
UGPP = UDP-glucose pyrophosphorylase; IPP = inorganic pyrophosphatase; PGM = phosphoglucomutase
Various studies have investigated the possibility of conducting whole-cell biotransforma-

tions with transgenic E. coli strains overexpressing glycosyltransferases. The coexpression
of chitin pentaose synthase nodC (-1,4-GlcNAc-oligosaccharide synthase) isolated from
Azorhizobium, and -1,4-galactosyltransferase LgtB from Neisseria meningitidis in E. coli
yields a hexasaccharide product {-Gal-1,4[-GlcNAc-(1,4)]4-GlcNAc}. The finding that chi-
tin pentaose is an acceptor for -Gal-1,4-GlcNAc-Æ-1,3-galactosyltransferase suggested that
the expression of additional glycosyltransferases in E. coli may allow the production of
more complex oligosaccharides.[62]
1.6.2.3.1.2 Fucosylation
Similarly to other glycosylated biochemicals, fucosylated carbohydrates play crucial roles

in a variety of biological processes including tissue development, cell adhesion, inflam-
mation, and tumor metastasis. 2¢-Fucosyllactose, a component of human milk oligosac-

charides, can be successfully synthesized in a two-step biotransformation.[63] In a first

step, E. coli cell extracts overexpressing GDP-d-mannose 4,6-dehydratase and GDP-4-keto-
6-deoxy-d-mannose-3,5-epimerase-4-reductase catalyze the synthesis of GDP-fucose (9;
78 mg, 78% yield) from GDP-mannose (7). GDP-fucose (9; 35 mg) is then applied in a second
step as a donor substrate for the synthesis of 2¢-fucosyllactose (18 mg, 65% yield) using a
purified Æ-1,2-fucosyltransferase from Helicobacter pylori. Alternatively, 2¢-fucosyllactose
can be successfully produced from lactose using whole-cell biotransformations by hetero-
logously coexpressing Æ-1,2-fucosyltransferase.[64] By coexpression of fkp, a gene involved
in the biosynthesis of GDP-fucose (9) and futC, a gene encoding Æ-1,2-fucosyltransferase,
2¢-fucosyllactose biosynthesis can be achieved on a multigram/liter scale.[65]
Beside 2¢-fucosyllactose, many other fucosylated oligosaccharides such as lacto-
N-neofucotetraose (27), lacto-N-neodifucohexaose (28), and lacto-N-neodifucooctaose
(29) have been synthesized using E. coli whole-cell expression systems (Scheme 10). The
overexpressed enzymes responsible for the synthesis of the products 27–29 include
Æ-1,3-fucosyltransferase from Helicobacter pylori, and -1,4-galactosyltransferase and
-1,3-N-acetylglucosaminyltransferase from Neisseria meningitidis.[66]
Scheme 10 Structures of Lacto-N-neofucotetraose, Lacto-N-neodifucohexaose, and Lacto-

N-neodifucooctaose[66]
OH
HO OH OH
HO OH
O
O O
HO O O
HO O O
OH O
NHAc OH
OH OH
O
OH
OH
HO
27 lacto-N-neofucotetraose
OH
HO OH OH
HO OH
O
O O
HO O O
O O O
OH O
NHAc OH
O OH OH
OH O
OH OH
HO OH
HO
28 lacto-N-neodifucohexaose
OH
HO OH OH
HO OH OH
O HO OH
O O
HO O O O
O O O O
OH HO O O
NHAc OH O
O NHAc OH
OH OH OH
O
OH OH
HO
OH
HO
29 lacto-N-neodifucooctaose
Disulfated tetrasaccharide derivatives of sialyl-Lex are efficiently synthesized using re-

combinantly expressed human Æ-1,3/4-fucosyltransferase (FUCT3) in Pichia pastoris
whole-cell biotransformations.[67] In another whole-cell fucosylated oligosaccharide syn-
thesis system, a two-step large-scale fermentation has been developed to produce the Lew-
isx trisaccharide [Gal--1,4(Fuc-Æ-1,3)-GlcNAc]. This was realized through the coupling of E.

coli cells harboring various GDP-fucose biosynthetic genes and Corynebacterium ammonia-
genes cells contributing to the formation of GTP from GMP. Initially, GDP-fucose (9) was
synthesized at 18.4 g/L through a two-step reaction, and the target product Lewisx was sub-
sequently produced in multigram/liter scale after the addition of E. coli cells overexpress-
ing the Æ-1,3-fucosyltransferase gene.[68]
1.6.2.3.1.3 Sialylation
Several sialyltransferases with different substrate specificities have been identified from
various genetic sources. These enzymes play a key role in modifying glycoconjugates with
sialic acid groups at a terminal, nonreducing residue. Numerous studies on this type of
enzyme and their applications in large-scale production have been documented.
Recombinant microbial sialyltransferases required for the biosynthesis of sialylated
oligosaccharides can be easily purified and consequently used for in vitro reactions to-
gether with activated sugar donor and acceptor substrates. The gene encoding Æ-2,3-sialyl-
transferase from Neisseria meningitidis was fused with that for CMP-Neu5Ac synthetase
(NanA) and the purified fusion protein has been successfully applied to the enzymatic syn-
thesis of 3¢-sialyllactose (32) on a multigram scale within a reaction time of 6 days. Sialic
acid 30 (Neu5Ac), lactose monohydrate, phosphoenolpyruvate, and catalytic amounts of
ATP and CMP are employed as starting materials for this reaction.[69] Instead of isolating
and purifying the expressed enzyme from an in vitro biotransformation, whole-cell trans-
formation approaches have also been adopted. Such approaches have the advantage that
living cells have the capability to metabolically (re)generate cofactors such as CTP and
phosphoenolpyruvate. Furthermore, 3¢-sialyllactose (32) can also be produced on a large
scale by metabolic coupling of the UTP producing Corynebacterium ammoniagenes strain
with genetically engineered Escherichia coli strains harboring CMP-Neu5Ac synthetase,
CTP synthetase, and the Æ-2,3-sialyltransferase genes (Scheme 11).[70] In the case of
whole-cell transformations, cofactors such as CTP can be generated from orotic acid
(31), whereas this cofactor has to be provided stoichiometrically in cell-free enzymatic
coupling reactions.

Scheme 11 Comparison of Bacterial and Cell-Free Enzymatic Coupling[70]
bacterial coupling
OH OH
OH
HO
O CO2H
AcHN
HO
30 Neu5Ac
CTP-synthetase
in E. coli
UMP UTP CTP
CMP-Neu5Ac synthetase
in E. coli
C. ammoniagenes
OH CMP
O
HO OH
O O CO2H
AcHN
HO
NH
2 CMP-Neu5Ac
HO2C N O
H CDP
31 OH
HO OH
O
C. ammoniagenes HO O O
OH HO
CMP OH OH
2,3-sialyltransferase in E. coli
OH
OH HO OH
HO2C
OH
HO O
O O O O
AcHN HO
HO OH
OH OH
32 3'-sialyllactose
cell-free enzymatic coupling
CMP
OH OH CTP OH O
OH CMP-Neu5Ac synthetase
OH
HO HO
O CO2H O CO2H
AcHN − PPi AcHN
HO HO
30 Neu5Ac 2 CMP Neu5Ac
OH
HO OH
O
HO O O
OH HO OH
OH OH OH HO OH
HO2C
α-2,3-sialyltransferase OH
HO O
O O O O
− CMP AcHN HO
HO OH
OH OH
32 3'-sialyllactose
One-pot multienzyme reactions are currently the method of choice for glycoconjugate
synthesis, and have been used to a very great extent for the sialylation of oligosaccharides.
Various oligosaccharides have been reported to be successfully modified, on a preparative
scale, by sialic acids through an Æ-2,3-linkage by using a one-pot, three-enzyme sialylation
system containing a multifunctional sialyltransferase. The yields for preparative-scale
synthesis (>20 mg) are higher than 60%, with many being more than 90%.[71,72] A similar
synthetic strategy has also been applied to the formation of Æ-2,6-linked sialosides using
a corresponding Æ-2,6-sialyltransferase[73] and the generation of trisaccharide libraries
containing sialic acids.[74] To date, the one-pot enzymatic reaction has only been used for
the synthesis of short oligosaccharides such as di-, tri-, and tetrasaccharides. However, for
longer oligosaccharide products, such as sialyl lacto-N-tetraose (Neu5Ac-Æ-2,3-Gal--1,4-
GlcNAc--1,3-Gal--1,4-Glc), the synthesis has been carried out in a stepwise process start-
ing from lactose. Firstly, the LNT-2 trisaccharide (GlcNAc--1,3-Gal--1,4-Glc) is synthe-
sized from lactose and UDP-GlcNAc (5) by the action of -1,3-N-acetylglucosaminyltrans-
ferase (LgtA) from Neisseria meningitidis. Secondly, lacto-N-neotetraose (Gal--1,4-GlcNAc-
-1,3-Gal--1,4-Glc) is synthesized from LNT-2 and UDP-galactose (1) using -1,4-galactosyl-
transferase (LgtB) from Neisseria meningitidis. In the third step, the final product sialyl
lacto-N-tetraose is synthesized from lacto-N-neotetraose and 3¢-sialyllactose (32) using re-
combinant Trypanosoma cruzi Æ-2,3-transsialidase.[75] Size-defined polysaccharides with
sialic acid containing repeating units have been chemoenzymatically synthesized by a
bacterial sialyltransferase with flexible substrate specificity. The recombinant Æ-2,6-
sialyltransferase derived from Photobacterium damselae catalyzes a block transfer of pseudo
di- or -tetrasaccharides from their CMP activated forms to generate higher oligosacchar-
ides related to surface polysaccharides of pathogenic organisms.[76]
Derivatives of 3¢-sialyl LacNAc 33 and the potentially anti-inflammatory therapeutic
tetrasaccharide sialyl-Lewisx (34, R1 = H) are examples of chemoenzymatically synthesized
compounds that have been pursued on an industrial scale (Scheme 12).[77]

Scheme 12 Chemoenzymatic Synthesis of a Sialyl-Lewisx Derivative[77]
OH
HO
O
HO
HO
O
UDP OH
OH HO OH
1
β-1,4-galactosyltransferase O
HO O O O
OR1 HO OR1
HO − UDP HO
OH
NHAc NHAc
OH CMP
OH O
HO
O CO2H
AcHN
HO OH
HO OH
2 OH HO2C
α-2,3-sialyltransferase OH
HO O
O O
O O
− CMP AcHN HO OR1
OH
HO NHAc
33
GDP
O O
OH
OH OH
HO OH
9 HO
OH HO2C
α-1,3-fucosyltransferase OH
HO O
O O
O O
− GDP AcHN O OR1
OH
HO NHAc
O
OH
OH
HO
34
Sialyl-Lewisx Derivative 34 (R1 = CH2CH=CH2); Typical Procedure:[77]

A soln of Æ-1,3-fucosyltransferase (2 mL, 0.02 U) was added to a soln containing 3¢-sialyl
LacNAc derivative 33 (R1 = CH2CH=CH2; 0.031 mmol), GDP-fucose (9; 0.036 mmol), ATP
(0.025 mmol), MnCl2 (0.1 mmol), and HEPES (3 mL; 120 mM, adjusted to pH 7.5). The mix-
ture was gently stirred under argon for 5 d at 25 8C and then concentrated. The residue
was chromatographed (silica gel, EtOAc/iPrOH/H2O 2:2:1), and fractions containing the
title compound were further purified using Biogel P2 (H2O). The eluent was passed
through an ion-exchange resin (Dowex 50W-X8 H+, H2O). The flow through was neutral-
ized with NaOH, and lyophilized; yield: 18 mg (68%).
1.6.2.3.2 Glycolipid Oligosaccharides
1.6.2.3.2.1 Globosides
The Forssman glycolipid (also called Forssman antigen) is a glycosylceramide containing a

neutral pentasaccharide group (the pentasaccharide fragment of 36, Scheme 13).[78] It is a
member of the globoseries glycolipid family. The globotriose (Gb3, Gal-Æ-1,4-Gal--1,4-
Glc) and globotetraose (Gb4, GalNAc--1,3-Gal-Æ-1,4-Gal--1,4-Glc), known as Pk and P
blood group antigens, respectively, are biosynthetic precursors of the Forssmann antigen
(GalNAc-Æ-1,3-GalNAc-1,3-Gal-1,4-Gal-1,4-Glc). Gb4 is synthesized from Gb3 through the
addition of an N-acetylgalactosaminyl residue with the reaction being enzymatically cata-
lyzed by a globoside -1,3-GalNAc-transferase (EC 2.4.1.79).[79] The Forssman oligosacchar-
ide is completed by transferring another N-acetylgalactosaminyl residue to Gb4, catalyzed

by an Æ-1,3-GalNAc-transferase.[80]
Scheme 13 Chemoenzymatic Synthesis of a Labeled Forssman Antigen[81]
OH
HO
O
HO
HO
O
UDP
OH 1
HO OH NO2
α-1,4-GalT
O
O O 62%
HO
HO O
OH
OH
35
OH
HO
O
OH HO
HO AcHN
O
O UDP
HO 8
HO β-1,3-GalNAcT
OH
O OH NO2
95%
O
O O
HO O
HO
OH
OH
globotriose (Gb3)
OH
HO
O
OH OH HO
HO HO
AcHN
O
O O UDP
O 8
HO
HO OH α-1,3-GalNAcT
NHAc OH
O NO2
90%
O
O O
HO O
HO
OH
OH
globotetraose (Gb4)
OH
HO
OH OH
O HO HO
HO
O O
AcHN
O O
NHAc HO OH
O OH NO2
O
O O
HO O
HO
OH
OH
36
To synthesize the nitrophenyl-labeled derivative of the Forssman pentasaccharide 36

(Scheme 13), all three enzymes needed to be purified after recombinant expression in E.
coli. The synthesis starts from 4-nitrophenyl lactoside 35 and is performed in a three-step
chemoenzymatic reaction, with individual yields of 61.7% (Gb3), 95% (Gb4), and 90% (la-
beled Forssmann antigen 36), respectively. Similarly, the synthesis of this pentasac-

charide has also been realized by starting from a 4-nitrophenyl derivative of N-acetyl-
lactosamine (25, LacNAc) as acceptor substrate in 100-mol scale in sequential enzymatic
conversion with a final 12% yield for the Forssman antigen.[81] Another example of
Gb3 synthesis involves the coupling of recombinant E. coli overexpressing the Æ-1,4-
galactosyltransferase gene from Neisseria gonorrhoeae together with a nucleotide sugar re-
generation system in vivo using lactose as the acceptor substrate.[16]
1.6.2.3.2.2 Gangliosides
Gangliosides are glycolipids containing diverse sialylated molecules that exist in most
cells, but particularly in neuronal tissues. They have been found to play many important
biological roles, such as tumor antigens and receptors for growth factors, toxins, and vi-
ruses, facilitating the attachment of human melanoma and neuroblastoma cells. A che-
moenzymatic approach for the synthesis of a series of ganglioside oligosaccharides (e.g.,
37–39, Scheme 14), including gangliosides GM3, GD3, and GT3, respectively, and other
disialyl glycans containing terminal Neu5Ac-Æ-2,8-Neu5Ac moieties with different natural
and nonnatural sialic acids, has been established using a genetically improved enzyme
with enhanced Æ-2,8-sialyltransferase activity. In a first step, Æ-2,3- or Æ-2,6-linked mono-
sialylated oligosaccharides are synthesized using a one-pot, three-enzyme approach.
These compounds are then used as acceptors for the Æ-2,8-sialyltransferase activity of a
recombinant truncated sialyltransferase from Campylobacter jejuni to produce disialyl oli-
gosaccharides.[82]
Scheme 14 Oligosaccharide Structures of the Gangliosides GM3, GD3, and GT3[82]
OH
OH
OH HO2C HO
OH
HO O
O O O
AcHN O
HO
HO OH
OH OH
37
OH OH
OH
OH
OH
CO2H HO2C HO
OH
HO O
O O O O O
AcHN AcHN O
HO
HO HO OH
OH OH
38
OH OH OH OH
OH
OH
CO2H
OH
CO2H HO2C HO
OH
O
HO O O O O O O O
AcHN AcHN AcHN O
HO
HO HO HO OH
OH OH
39
1.6.2.3.3 Glycopeptides and Glycoproteins
1.6.2.3.3.1 N-Glycopeptides and Glycoproteins
The in vitro synthesis of glycoproteins/glycopeptides containing asparagine-linked glycan

structures has long been focused on chemical methods. To synthesize these molecules en-
tirely through enzymatic reactions still remains challenging due to the complexity of
their structure. Most progress for the synthesis of these structures has been achieved by
chemoenzymatic approaches, with the glycopeptide precursors synthesized in an initial

chemical step and then followed by enzymatic modification or elongation.
Based on the successful development of highly efficient methods for enzymatically
adding different terminal sugar units, such as sialic acid and galactose, in the presence of
alkaline phosphatase (which hydrolyses nucleoside diphosphates that potentially inhibit
glycosyltransferase activities) to an existing oligosaccharide acceptor,[83,84] a series of che-
moenzymatic approaches have been established and applied that permit the total synthe-
sis of glycosylated complex-type N-glycans and N-glycopeptides. An early approach for the
synthesis of N-glycopeptides containing asparagine-linked sialyl-N-acetyllactosamine side
chains was realized through a combined chemical and enzymatic approach. In a first step,
tert-butoxycarbonyl-protected -N-acetylglucosamine-asparagine residues were chemical-
ly incorporated onto the target peptide. After a deprotection step, the N-acetylglucos-
amine on the preliminary glycopeptide was then elongated with galactose through a bo-
vine -1,4-galactosyltransferase (-1,4-GalT1, EC 2.4.1.22) catalyzed reaction in the pres-
ence of UDP-galactose (1) and calf alkaline phosphatase (CIP, EC 3.1.3.1). The final desired
glycopeptide 40 (Scheme 15) was completed through sialylation by a recombinant
Æ-2,3 sialyltransferase (EC 2.4.99.–) and N-acylneuraminate cytidylyltransferase (EC
2.7.7.43).[85] Similarly, the complex sialylated biantennary asparagine-linked building
block 41, a partial structure of many glycoproteins, was obtained by total synthesis featur-
ing a combination of modern chemical and enzymatic methods. Compared to previous
approaches, this method reduced the overall number of synthetic steps (Scheme 15).[86]
Scheme 15 Chemoenzymatic Strategies for the Synthesis of an

N-Glycopeptide and an N-Glycoasparagine[85,86]
enzymatic
synthesis chemical synthesis
AcHN
(Gly)2
Neu5Acα Gal GlcNAc β Asn
6 β 2
(Gly)15
Neu5Acα Gal β GlcNAc β Asn
6 2
(Gly)2
HO2C
40
Neu5Ac α Gal GlcNAc Man α

3 β 2 β 2
Man GlcNAc GlcNAc Asn
3 β 4 β 4 β
Neu5Ac Gal GlcNAc Manα
α 3 β 2 β 2
41
This strategy has also been successfully applied for the generation of a glycopentapeptide
representing amino acid residues 21–25 from bovine ribonuclease B.[87] The glycopeptide,
obtained by solution-phase peptide synthesis, with unprotected saccharide units was
then used as the substrate for the enzymatic, stepwise sugar elongation to yield a complex
biantennary, sialylated N-glycosylated peptide. This was the first reported synthesis of a
glycoprotein fragment carrying a full-length asparagine-linked N-type glycan. The meth-
od was further advanced using 9-fluorenylmethoxycarbonyl as the protecting group, and
then used for glycopeptide synthesis both in solution and on solid phase. After chemical

deprotection, the glycopeptide was then elongated through the same enzymatic approach
using -1,4-galactosyltransferase and Æ-2,6-sialyltransferase in the presence of alkaline
phosphatase.[88] By combining a series of glycosyltransferases and chemical synthesis
using orthogonal protecting groups, the synthesis of a library of asymmetrical multian-
tennery N-glycans was achieved, which were useful as probes in array format.[89]
In a different strategy, endoglycosidases have been used to remove the heterologous
oligosaccharide part from ribonuclease B to leave only a single N-linked N-acetylglucos-
amine residue on the glycoprotein. Glycosyltransferases were then used to add carbohy-
drates in the desired sequence to build up novel glycoforms. Alternatively, the ribonu-
clease was digested by subtilisin BPN¢ into smaller peptide fragments, which were then
enzymatically religated with chemically synthesized glycopeptides by protease catalysis
to form the full-length glycoprotein.[90] In addition to the synthesis of N-linked glycopro-
teins, glycosyltransferases can also be used to modify naturally existing N-glycoproteins/
peptides. Due to the functional role of sugar structures attached to immunoglobulin G, it
is advantageous to have uniform glycoforms on immunoglobulin G. A soluble recombi-
nant -1,4-galactosyltransferase has been used to remodel heterogeneous glycans at-
tached to immunoglobulin G to obtain homologous biantennary glycoforms with termi-
nal galactose on a kilogram scale.[91]
1.6.2.3.3.2 O-Glycopeptides and Glycoproteins
Several attempts have been made to generate mucin-type O-linked glycoproteins and gly-
copeptides using glycosyltransferases. Heterologously expressed UDP-GalNAc:poly-
peptide GalNAc-transferases (ppGalNacT, EC 2.4.1.41) or extracted ppGalNacT from can-
cer cells have both been used to successfully transfer N-acetylgalactosamine onto
MUC6 peptides to generate MUC6-Tn antigens on a milligram scale.[92] Members of this
group of glycosyltransferases also have donor substrate promiscuity toward UDP-GlcNAc
(5), which has been demonstrated by the enzymatic synthesis of Æ-linked N-acetylglucos-
amine-containing glycopeptides on a milligram scale.[93] Further glycan elongation of
O-glycopeptides has been demonstrated with tumor-associated MUC1a¢ peptides. Core-
2 sialyl-Lewisx MUC1a¢ was synthesized using the combination of -galactosidase and
core-2--1,6-N-acetylgalactosaminyltransferase (EC 2.4.1.102), and subsequently a series
of other glycosyltransferases for further enzymatic decoration (Scheme 16).[94]
Scheme 16 Structural Representation of MUC1a¢

Peptide with Attached Core-2 Sialyl-Lewisx Glycan[94]
Neu5Ac Gal
α 6 β
4
Fuc GlcNAc
α 3 β
6
Gal GlcNAc
β 3 α
H 2N AlaHisGlyValThrSerAluProAspThrArg OH
A series of sialyl-Tn-MUC1 and sialyl-T-MUC1 glycopeptides (e.g., 43 and 44, respectively)

containing naturally occurring and nonnatural sialic acids have also been synthesized
chemoenzymatically from Tn-MUC1 glycopeptide 42 as an acceptor substrate. In situ gen-
eration of the sialyltransferase donor cytidine 5¢-monophosphate-sialic acid (CMP-Sia)
using a CMP-sialic acid synthetase in the presence of CTP in combination with Æ-2,3-sialyl-
transferase allows the quantitative synthesis of sialyl-Tn-MUC1 peptide 43 (Scheme 17).[95]
Scheme 17 General Chemoenzymatic Strategy for the Synthesis of Mucin-Type

O-Glycopeptides[95]
O O GalNAc
α
S
Ar1 NHAlaProGlySerThrAlaProProAlaNH2
42
α-2,6-sialylation β-1,3-galactosylation
Neu5Ac GalNAc
α 6 α Gal GalNAc
O O O O β 3 α
S S
Ar1 NHAlaProGlySerThrAlaProProAlaNH2 Ar1 NHAlaProGlySerThrAlaProProAlaNH2
43
α-2,6-sialylation
Neu5Ac Gal GalNAc

α 6 β 3 α
O O
S
Ar1 NHAlaProGlySerThrAlaProProAlaNH2
44
Me2N
Ar1 =
N N
Most research on O-glycopeptide synthesis is based on mucin-type structures, although

examples of other important O-glycan structures, such as O-mannosyl peptides, are
known. One example is the chemoenzymatic synthesis of glycopeptide 45 containing an
O-linked mannosyltetrasaccharide (Scheme 18). In the first step a mannosyl peptide was
chemically generated on the solid phase using 9-fluorenylmethoxycarbonyl-protected
mannosyl serine and mannosyl threonine building blocks, and the residual three sugar
units were attached by consecutive enzymatic glycosylations. This glycopeptide library
allows the study of the role of further enzymes involved in this glycosylation pathway
and can be also used as a standard for further genomic studies.[96]

Scheme 18 Chemoenzymatic Synthesis of a Glycopeptide Library with Mono-, Di-, Tri-, and
Tetraglycosylated Peptides[96]
Man
α solid-phase
O O Man
peptide synthesis α
O
OH
AcHNAlaThrProThrProValThrAlaIleGlyOH
NHFmoc
OH
HO O
HO
AcHN
O GlcNAc
UDP β
5 3
POMGnT1
Man
α
O
OH
HO
Gal
O β
HO 4
HO GlcNAc
O β
UDP
3
1 Man
α
β-1,4-GalT1
O
Neu5Ac
α
3
Gal
β
4
GlcNAc
trans-sialidase β
3
fetuin
Man
α
O
45
POMGnT1 = protein O-mannose N-acetylglucosaminyltransferase 1 (EC 2.4.1.-)

β-1,4-GalT1 = bovine β(1,4)-galactosyltransferase (EC 2.4.1.90)
An alternative approach to generate mucin-type O-glycoproteins is to use transgenic

plants as bioreactors. Transgenic tobacco overexpressing a human GalNAc-T2 gene to-
gether with genes encoding a UDP-GlcNAc-4-epimerase (EC 5.1.3.2) or a UDP-GalNAc trans-
porter has been cultivated. The results obtained indicate that LTBMUC1 (recombinantly
expressed E. coli enterotoxin B subunit: Homo sapiens mucin 1 tandem repeat derived pep-
tide fusion protein) is O-glycosylated by polypeptide N-acetylgalactosaminyltransferase 2
(GalNAc-T2).[97] Similar results are obtained by transient expression of a Pseudomonas aer-
uginosa GlcNAc-4-epimerase and a human polypeptide GalNAc transferase (GalNAc-T2/T4)
in tobacco leaves together with a human O-glycoprotein substrate gene (MUC) which is
consequentially O-glycosylated with N-acetylgalactosamine. These studies demonstrate
that engineered plants can be used for synthesizing human O-glycoproteins.[98]
1.6.2.3.4 Glycosaminoglycans
1.6.2.3.4.1 Poly-N-acetyllactosamines
Poly-N-acetyllactosamines are common terminal sugar moieties of many N- and O-linked

glycan structures present in glycoproteins and glycolipids, and the ability to synthesize
these compounds is therefore of considerable importance. With the concentrated effort
of several research groups, the gram-scale synthesis of 24 different poly-N-acetyllactos-
amine derivatives covering mammalian blood group and tumor-associated antigens
have been achieved using six different glycosyltransferases and four different nucleotide
donor substrates.[99] Starting with the chemically synthesized monosaccharide precursor
azidoethyl 2-acetamido-2-deoxy--d-glucopyranoside 46, the synthesis of poly-N-acetyl-
lactosamines was accomplished by adding the donor substrates UDP-galactose (1) and
UDP-N-acetylglucosamine (5) to the bacterial enzymes -1,4-galactosyltransferase (-1,4-
GalT1) and -1,3-N-acetylglucosaminyltransferase (-1,3-GlcNAcT), which generated in al-
ternating action the repetitive elongation of N-acetyllactosamine units (Scheme 19).
Scheme 19 Enzymatic Synthesis of Poly-N-acetyllactosamine Structures by the Alternating

Action of Two Glycosyltransferases[99]
OH OH
HO
O O
HO , HO
HO
HO AcHN
O O
UDP UDP
OH
1 5
O β-1,4-GalT1, GalE, β-1,3-GlcNAcT
HO
HO O
N3
NHAc
46
OH OH
HO OH
O O
O O O
H HO O O
HO
NHAc OH N3
NHAc
n
The poly-N-acetyllactosamines were then subjected to fucosylation using recombinant

Æ-1,2-fucosyltransferase or Æ-1,3-fucosyltransferase to generate fucosylated poly-N-acetyl-
lactosamines, and sialylation through Æ-2,3-sialyltransferase or Æ-2,6-sialyltransferase to
form sialylated poly-N-acetyllactosamines. The difucosylated Lewisy and Æ-2,3-sialyl-Lew-
isx structures were also synthesized on all terminal N-acetyllactosamine moieties. Poly-
N-acetyllactosamines have also been prepared with alternative -GlcNAc glycosides carry-
ing aglycones, by the concerted action of recombinant human -1,4-galactosyltransferase
1 enzyme, which exhibits a specifically high activity toward the acceptor substrate, cou-
pled with recombinant -1,3-N-acetylglucosaminyltransferase from Helicobacter pylori.[100]
1.6.2.3.4.2 Heparosan
Heparosan, consisting of linear repeating glucuronyl--1,4-N-acetylglucosaminyl-Æ-

1,4 units, is a precursor of a highly sulfated class of polysaccharides called heparan sul-
fates. Heparosan polysaccharides 47 have been prepared using the enzymes -1,4-glucu-
ronic acid transferase (-1,4-GlcUAT, EC 2.4.1.17) and Æ-1,4-N-acetylglucosaminyltransfer-
ase (Æ-1,4-GlcNAcT, EC 2.4.1.224, Scheme 20).[101,102]

Scheme 20 Enzymatic Synthesis of Fluorescein Di--D -glucuronide Labeled Heparosan

Analogues[102]
OH
O HO2C
HO O O
O , HO
R2O HO
O OR3 UDP O
1
R HN
UDP
α-1,4-GlcNAcT, β-1,4-GlcUAT
HO2C
HO O
HO O O O
OH HO2C
HO O
HO
OH
fluorescein di(β-D-glucuronide)
OH
HO2C O
H O
O O
2
R O O HO2C
HO O
OR3 O
NHR1 HO O O O
n OH
OH
HO2C
H O
O O HO2C
R2O O
HO O
OR3 O
HO
NHR1
OH
n
47 (n >100)
R1 = Ac, COEt, COPr; R2 = R3 = H, Ac
A recent review summarizes the progress in the production and use of specific N-sulfo-
transferases, O-sulfotransferases, and heparan sulfate C5-epimerases to convert heparo-
san into heparan sulfates and heparin.[103] The heparosan-synthesizing glycosyltransfer-
ases and the heparosan-modifying enzymes have been employed in the elegant produc-
tion of libraries of small heparin fragments, ranging from heptasaccharides to dodecasac-
charides, by starting from a disaccharide prepared by nitrous acid degradation of heparo-
san.[104]
1.6.2.3.5 Homoglycan Extensions
Chitooligosaccharides are natural oligosaccharide products with -1,4-linked N-acetylglu-

cosamine units that show diverse health-related functions.[105] The enzymatic synthesis of
these structures is catalyzed by a group of enzymes called chitooligosaccharide synthases
(EC 3.2.1.14). E. coli cells heterologously overexpressing the nodC gene from Azorhizobium
caulinodans, which encodes a chitooligosaccharide synthase, can be used to successfully
synthesize penta-N-acetylchitopentaose and tetra-N-acetylchitopentaose on a gram
scale.[106] This approach has been successfully extended to facilitate the synthesis of O-
acetylated and sulfated chitooligosaccharide derivatives of these two compounds, by co-
expressing nodC or nodBC with nodH and/or nodL, enzymes that encode chitooligosac-
charide sulfotransferase and chitooligosaccharide O-acetyltransferase, respectively.[107]
Sucrose is the most widely distributed disaccharide in nature. Due to the high cost
and low availability of nucleotide-activated sugars, the ability of sucrose to act as a
donor substrate for a range of sucrose-dependent glycosylation reactions to produce nat-
ural polysaccharides and oligosaccharides is of special value. As sucrose consists of glu-
cose and fructose, glycosyltransferases of the non-Leloir type, namely glucosyltransfer-
ases (or glucansucrases) and fructosyltransferases, can be used to form glucose-based oli-
gosaccharides (glucans) or fructose-based oligosaccharides (fructans), respectively.[108]
Fructosyltransferases transfer the fructosyl units of sucrose to acceptors with release of
glucose. The potential of using fructosyltransferases for the production of novel glycopy-
ranosyloligofructosides, such as -d-fructofuranosyl-Æ-d-galactopyranoside (48), has also
been investigated (Scheme 21).[109,110]
Scheme 21 Enzymatic Synthesis of Glycopyranosyloligofructosides Using Fructosyl- or

Glucosyltransferases[109]
O OH
OH
O
OH
O
OH OH
sucrose analogue
OH
OH
O O
− OH fructosyltransferase − HO glucosyltransferase
OH
OH OH
O
O OH
OH
O O
O O
O
O OH
O n
O O OH
O n OH
OH O
O
OH OH
OH
HO O OH
HO OH
HO O OH
O HO
OH
HO OH OH O OH
O fructosyltransferase HO OH
HO O
HO
OH O
OH OH
O OH OH
− HO
HO
OH OH 48 54%
Dextransucrase from Leuconostoc mensenteroides NRRL B-1226 shows a broad acceptor tol-
erance and allows the generation of a series of Æ-1,6-dextran-extended oligosaccharides
based on several acceptor substrates such as maltose, gentiobiose, glucose, or lactose
with high efficiency.[111] A transglycosylation reaction with a related dextransucrase gen-
erates oligosaccharides from sucrose and a cellobiose precursor. The obtained degree of
dextran polymerization to the acceptor ranged from 3 to 7, and the final yield reached
20% based on cellobiose.[112]

Cyclodextrins have a broad range of applications in food technology, pharmacology,

the chemical industry, and agriculture for the encapsulation of hydrophobic mol-
ecules.[113] Typically, these structures consist of cyclic Æ-1,4-linked oligosaccharides of 6,
7, or 8 glucose units (Æ-, -, ª-cyclodextrins, respectively), which are enzymatically gener-
ated from soluble starch by the action of microbial cyclodextrin glycosyltransferases
(CGTases, EC 2.4.1.19). These enzymes act as endotransglycosylases, cleaving off linear
Æ-glucans which are subsequently transferred to their own nonreducing end to form a cy-
clic product. Alternatively, CGTases can also transfer to nonreducing ends of other Æ-glu-
cans as short as maltotriose, yielding elongated Æ-glucans. To a small extent, CGTases also
catalyze the hydrolysis of the cleaved linear Æ-glucan.[114] Furthermore, engineered CTGas-
es can transfer the cleaved Æ-glucans to various alternative acceptor substrates.
CGTase mediated coupling reactions have been reported with various sugar accept-
ors such as sucrose,[115] isomaltose,[113] and anhydro-d-fructose.[116] Furthermore, sugar de-
rivatives such as phenyl -d-glucopyranoside,[117] sucrose laureate,[118] or rebaudioside[119]
are also acceptor substrates. Of special industrial interest is the transglycosylation to ste-
vioside, a low-calorie sweetener, which showed reduced bitterness and higher solubility
after CGTase treatment.[120] Several studies have also described the transfer of Æ-glucans to
non-sugar hydroxy groups. Glycosylation of the hydroxy group of the lactone ring of l-as-
corbic acid led to increased stability toward oxidation.[121] Several other studies on CGTase
glycosylated phytochemicals such as neohesperidin, naringin, and thiamine similarly re-
ported higher solubility, stability, or reduced bitterness.[122] In summary, the four enzy-
matic reactions catalyzed by CGTases open a wide field of applications for both academic
and industrial use. Over the last decade, an increasing number of CGTase isoforms have
been discovered and characterized, which will allow continuous improvement for specif-
ic transglycosylation reactions in the future.
-d-Fructofuranosyl-Æ-d-galactopyranoside (39); Typical Procedure:[109,110]

A soln containing sucrose (250 mM), galactose (500 mM), and Na2HPO4/KH2PO4 buffer (50
mM; pH 6.0) was incubated with fructosyltransferase from Bacillus subtilis NCIMB 11 871
(2.8 U/mL) at 37 8C for 100 min. The residual sucrose in the mixture was converted into
dextran and fructose using dextransucrase (1 U/mL; pH adjusted to 5.4, 30 8C, 2 h reaction
time), which allowed easy separation using cation-exchange chromatography (Purolite
PCR 6, Na+ form, 70 8C); yield: 54%.
1.6.2.3.6 Glycosylated Natural Products
The glycosyltransferase-catalyzed modification of natural products shows great potential

for applications in glycoconjugate synthesis. Oleandomycin glycosyltransferase from
Streptomyces antibioticus shows great flexibility using dTDP- and UDP-glucose donor sub-
strates toward a whole library of natural and synthetic acceptor molecules, such as genis-
tein, novobiocic acid, or oleandomycin (Scheme 22).[123,124]
Scheme 22 Acceptor Variability of Oleandomycin Glycosyltransferase of Streptomyces

antibioticus[123,124]
*
HO O
* O
HO
O OH O
OH OH
daidzein genistein
OH O
* HO
HO O
*
* O O OH
OH O O OH
OH O
kaempferol 4-methylumbelliferone 7-hydroxycoumarin-3-carboxylic acid
O
O
OH NMe2 OH
* OH
HO H
O O O N
O O * O
HO O O
OMe
O OH
oleandomycin novobiocic acid
*
OH = site of glycosylation
Several flavonoid glycosyltransferases have been identified over the last years, which do
not or not exclusively utilize UDP-glucose (4) as donor substrate. UGT89C1 from Arabidop-
sis thaliana also shows, in addition to UDP-glucose (4), promiscuity toward UDP-galactose
(1) and UDP-rhamnose,[125] whereas UGT78D3 and UGT79B1 from the same organism ex-
clusively transfer UDP-arabinose[126] and UDP-xylose (3) to flavonoids, respectively.[125]
Apart from the donor substrate promiscuity, several flavonoid glycosyltransferases have
been described to have broad range of regiospecificities toward various acceptor sub-
strates,[127,128] whereas other flavonoid glycosyltransferases have been described to have
glycosylate specific substrates at one specific hydroxy group.[129,130]
Expression of the C-glycosyltransferase urdGT2 in a lanGT2 mutant of the landomy-
cin A producer Streptomyces cyanogenus S136 resulted in the formation of a C—C bond be-
tween angucycline and d-olivose.[131] Expression of the aranciamycin biosynthetic gene
cluster in Streptomyces diastatochromogenes T6028 led to the accumulation of various gly-
cosylated aranciamycins.[132,133]
Natural products contain more than one hundred less-common sugars in addition to
the common sugars found in mammals.[134] These include deoxy-, methyl, amino deoxy-,
amino dideoxy-, and trideoxysugars which are transferred from their corresponding nu-
cleotides by microbial and plant glycosyltransferases.

1.6.2.4 Conclusions and Outlook
The application of glycosyltransferases has become a robust and versatile tool and is al-
ready an integral part of carbohydrate synthesis. The discovery of novel glycosyltransfer-
ases has been greatly facilitated by the genomic sequencing of dozens of eukaryotic and
hundreds of bacterial organisms during the past decade, and continuous improvement of
these biocatalysts in terms of substrate specificity and stability has been achieved. Fur-
thermore, enzymatic characterization, modification of functional properties via directed
evolution, and metabolic pathway engineering have continuously contributed to the fur-
ther improvement of glycosyltransferases. However, only a limited number of glycosyl-
transferases are commercially available.[55] With the exception of cyclodextrin glucosyl-
transferases, which find various industrial applications on the kiloton scale, for most
large- and preparative-scale glycosylation reactions, both the glycosyltransferases and
their donor substrates are too expensive. Therefore, a considerable amount of future
work is still needed to further improve the availability and economic feasibility of syn-
thetic applications of glycosyltransferases and their donor substrates.
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507

J. Voglmeir and S. L. Flitsch

for references see p 539

Scheme 1 Comparison of Chemical and Enzymatic Glycoconjugate Synthesis[7]

protected reaction product

PG = protecting group; UDP = uridine diphosphate

1.6.2.1 Sugar Nucleotides as Glycosyl Donors

1.6.2.1.1 Commonly Known Nucleotide-Activated Sugars

for references see p 539

Scheme 2 Common Nucleotide Sugar Structures

OH nucleotide sugar recycling OH OH

UDP = uridine diphosphate; GDP = guanosine diphosphate; CMP = cytidine monophosphate

Scheme 3 Synthesis of ADP-Glucose Using Sucrose Synthase[15]

All other common nucleotide sugars including UDP-galactose (1), UDP-N-acetylgalactos-

for references see p 539

Scheme 4 Chemoenzymatic Synthesis of UDP-GlcNAc and UDP-GalNAc from Glucosamine

ATP regeneration acetate kinase galactokinase

for references see p 539

ADP-Glucose (11); Typical Procedure:[15]

1.6.2.1.2 Unusual Nucleotide-Activated Sugars

Scheme 5 Chemoenzymatic Synthesis of CMP-Neuraminic Acid Derivatives[23]

Scheme 6 Preparation of CMP-Sialic Acid Derivatives[29]

UDP-Galacturonic acid is a precursor for the synthesis of numerous cell-surface polysac-

for references see p 539

CMP-Sialic Acid Derivative 21 (R1 = R3 = OH; R2 = R4 = H; R5 = CH2OH); Typical Procedure:[29]

1.6.2.2 Other Sugar Donors

1.6.2.2.1 Glycosyl Phosphates

Scheme 7 Kilogram-Scale Synthesis of Lacto-N-biose in a One-Pot, Four-Enzyme

SP = sucrose phosphorylase; GalT = UDP-galactose:glucose1-phosphate uridylyltransferase;

for references see p 539

Æ-1,4-glucopolysaccharide. The reaction equilibrium can be shifted toward polymeriza-

1.6.2.2.2 Di- and Oligosaccharides

1.6.2.2.3 Glycosyl Lipids

Lipid-linked glycosyl donors play an important role as sugar donors in membrane-based

Scheme 8 Chemical Synthesis of a Phytanol-Based Glycosyl Lipid and Subsequent Enzy-

1.6.2.2.4 Other Sugar Donors

for references see p 539

1.6.2.3 Glycoconjugate Products

Glycoconjugates are a massive group of sugar-derivatized organic substances that can

1.6.2.3.1 Free Oligosaccharides

Scheme 9 Enzymatic Synthesis of LacNAc with In Situ Cofactor Regeneration[60]

Various studies have investigated the possibility of conducting whole-cell biotransforma-

Similarly to other glycosylated biochemicals, fucosylated carbohydrates play crucial roles

for references see p 539

charides, can be successfully synthesized in a two-step biotransformation.[63] In a first

Scheme 10 Structures of Lacto-N-neofucotetraose, Lacto-N-neodifucohexaose, and Lacto-

Disulfated tetrasaccharide derivatives of sialyl-Lex are efficiently synthesized using re-

isx trisaccharide [Gal--1,4(Fuc-Æ-1,3)-GlcNAc]. This was realized through the coupling of E.

for references see p 539

Scheme 11 Comparison of Bacterial and Cell-Free Enzymatic Coupling[70]

cell-free enzymatic coupling

for references see p 539

Scheme 12 Chemoenzymatic Synthesis of a Sialyl-Lewisx Derivative[77]

Sialyl-Lewisx Derivative 34 (R1 = CH2CH=CH2); Typical Procedure:[77]

1.6.2.3.2 Glycolipid Oligosaccharides

The Forssman glycolipid (also called Forssman antigen) is a glycosylceramide containing a

ide is completed by transferring another N-acetylgalactosaminyl residue to Gb4, catalyzed

Scheme 13 Chemoenzymatic Synthesis of a Labeled Forssman Antigen[81]

To synthesize the nitrophenyl-labeled derivative of the Forssman pentasaccharide 36

for references see p 539

Scheme 14 Oligosaccharide Structures of the Gangliosides GM3, GD3, and GT3[82]

1.6.2.3.3 Glycopeptides and Glycoproteins

1.6.2.3.3.1 N-Glycopeptides and Glycoproteins