University of Groningen
Enzymatic Glycosylation of Small Molecules
Desmet, Tom; Soetaert, Wim; Bojarova, Pavla; Kren, Vladimir; Dijkhuizen, Lubbert; Eastwick-
Field, Vanessa; Schiller, Alexander; Křen, Vladimir
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Chemistry : a European Journal
DOI:
10.1002/chem.201103069
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Desmet, T., Soetaert, W., Bojarova, P., Kren, V., Dijkhuizen, L., Eastwick-Field, V., Schiller, A., & Křen, V.
(2012). Enzymatic Glycosylation of Small Molecules: Challenging Substrates Require Tailored Catalysts.
Chemistry : a European Journal
,
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(35), 10786-10801. https://doi.org/10.1002/chem.201103069
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Enzymatic Glycosylation of Small Molecules: Challenging Substrates Require
Tailored Catalysts
Tom Desmet,
[b]
Wim Soetaert,
[b, c]
Pavla Bojarov,
[d]
Vladimir Krˇen,
[d]
Lubbert Dijkhuizen,
[e]
Vanessa Eastwick-Field,
[f]
and Alexander Schiller*
[a]
2012 Wiley-VCH Verlag GmbH& Co. KGaA, Weinheim Chem. Eur. J. 2012, 18, 10786 – 10801
10786
DOI: 10.1002/chem.201103069
Introduction
Besides being a source of energy and a structural compo-
nent of the cell wall, carbohydrates also mediate various rec-
ognition processes when attached to proteins or lipids.
[1]
Well-known carbohydrate motifs of such glycoconjugates
are the cancer epitope Sialyl Lewis X and the AB0 blood
group determinants. In addition, glycosylation is an impor-
tant source of structural diversity of natural products, such
as alkaloids, steroids, flavonoids, and antibiotics. Glycosides
typically display properties that differ from those of their
non-glycosylated aglycons.
[2]
A prime example is naring in, a
flavanone glycoside that is responsible for the bitter taste of
citrus fruits. Removal of the glycon part eliminates the
bitter taste, which is one of the main goals of enzymatic
treatment of grapefruit juice.
[3]
The opposite is true for gly-
cirrhizin, a terpenoid glycoside from sweetwood (Glycirhyza
glabra) that loses most of its sweetness upon hydrolys is.
[4]
Since the transfer of a glycosyl group can influence both
the physicochemical and biological properties of an organic
molecule, such processes may be used for a wide range of
applications. The most obvious advantage of introducing a
carbohydrate moiety is the increased solubility of hydropho-
bic compounds. This is nicely illustrated in flavonoids, the
pharmaceutical properties of which can often be efficiently
exploited only in the form of their hydrophilic glycosyl de-
rivatives.
[2]
Glycosylation may also be used to improve the
stability of labile molecules. A famous example is ascorbic
acid, a very sensitive vitamin, the long-term storage of
which can be drastically extended by glycosylation, resulting
in high-value applications in cosmetics and tissue culturing.
[5]
Another important application of glycosylation is the reduc-
tion of skin irritation caused by hydroquinone, employed in
cosmetics for its skin whitening effect.
[6]
Glycosides of fla-
vors and fragrances, in turn, can function as controlled re-
lease compounds. The a-glucoside of l-menthol, for exam-
ple, is only slowly hydrolyzed in the mouth, resulting in a
prolonged sensation of freshness.
[7]
Last but not least, it has
been possible to modulate the activity spectrum of glyco-
peptide antibiotics by varying their carbohydrate moiety, in
a process known as “glycorandomization”.
[8,9]
In view of these examples, the development of cheap and
efficient glycosylation technologies, useful both in the labo-
ratory and in industry, is highly desirable. In this review, the
challenges and recent innovations concerning the glycosyla-
tion of small, non-carbohydrate molecules are covered.
Chemical versus Enzymatic Glycosylation
Glycosylation reactions by conventional chemical synthesis
are used intensively in the field of glycochemistry. Despite
the variety of glycosylation protocols developed to date,
[10–25]
synthesis of glycosylated compounds largely relies on four
non-enzymatic reactions (Scheme 1). One of the first was fa-
mously developed by Koenigs and Knorr, in which glycosyl
halides, activated with silver salts, are used as glycosyl
donors.
[26–28]
Glycosyl trichloroacetimidates were later found
Abstract: Glycosylation can significantly improve the
physicochemical and biological properties of small mole-
cules like vitamins, antibiotics, flavors, and fragrances. The
chemical synthesis of glycosides is, however, far from trivi-
al and involves multistep routes that generate lots of
waste. In this review, biocatalytic alternatives are present-
ed that offer both stricter specificities and higher yields.
The advantages and disadvantages of different enzyme
classes are discussed and illustrated with a number of
recent examples. Progress in the field of enzyme engineer-
ing and screening are expected to result in new applica-
tions of biocatalytic glycosylation reactions in various in-
dustrial sectors.
Keywords: acceptor specificity · enzyme engineering ·
glycosylation · glycosyltransferase · high-throughput
screening
[a] Prof. A. Schiller
Friedrich-Schiller-University Jena
Institute for Inorganic and Analytical Chemistry
Humboldtstr. 8, 07743 Jena (Germany)
E-mail: alexander.schiller@uni-jena.de
[b] Prof. T. Desmet, Prof. W. Soetaert
University of Ghent
Centre for Industrial Biotechnology and Biocatalysis
Coupure links 653, 9000 Gent (Belgium)
[c] Prof. W. Soetaert
BioBase Europe Pilot Plant
Rodenhuizekaai 1, Havennummer 4200
9042 Gent (Belgium)
[d] Dr. P. Bojarov, Prof. V. Kr
ˇ
en
Institute of Microbiology
Academy of Sciences of the Czech Republic
Vden
ˇ
sk 1083, 142 20 Prague (Czech Republic)
[e] Prof. L. Dijkhuizen
Microbiology, University of Groningen
Groningen Biomolecular Science s and
Biotechnology Institute (GBB)
Nijenborgh 7 P.O. Box 11103
9700 CC Groningen (The Netherlands)
[f] Dr. V. Eastwick-Field
Carbosynth Limited
8 & 9 Old Station Business Park
Compton, Newbury, Berkshire RG20 6NE (UK)
Chem. Eur. J. 2012, 18, 10786 – 10801 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemeurj.org
10787
REVIEW
to be very powerful donor substrates with an excellent leav-
ing group.
[29]
Alternatively, more stable glycosides, such as
thioglycosides and n-pentenyl glycosides, can be used when
activated by ele ctrophilic reagents.
[30]
There are, however,
two major issues connected with the outcome of these reac-
tions, that is, the regioselectivity and the configuration of
the glycosidic linkage. The former can be solved by appro-
priate protection strategies, whereas the latter is strongly de-
pendent on the neighboring group participation of the C2
substituent. Additionally, solvents and catalysts have an im-
portant effect on the anomeric outcome of glycosylation re-
actions.
The chemical methods suffer from a number of draw-
backs: labor-intensive activation and protection procedures,
multistep synthetic routes with low overall yields, the use of
toxic catalysts and solvents and the amount of waste.
[31]
To
overcome these limitations, specific enzymes may be used
for the synthesis of glycosides. DeRoode et al. have calculat-
Tom Desmet received a Ph.D. in Biochem-
istry from Ghent University for work on
the structure–function relationships in
(hemi)cellulases. He is particularly interest-
ed in the engineering of carbohydrate-
active enzymes for use in biocatalytic proc-
esses, and has coordinated several projects
in that field. After a postdoctoral stay at
Wageningen University, he was appointed
Associate Professor at the Centre for In-
dustrial Biotechnology and Biocatalysis,
where he leads a team of about ten re-
searchers.
Wim Soetaert holds a Ph.D. in Applied Bi-
ological Sciences from Ghent University.
After working in the starch industry for
twelve years, he returned to the university
to become Associate Professor for Indus-
trial Biotechnology. His research interests
comprise the enzymatic and microbial con-
version of carbohydrates for the produc-
tion of added-value chemicals. Wim Soe-
taert also is the director of the Bio Base
Europe Pilot Plant as well as the founder
and chairman of Ghent Bio-Energy Valley.
Dr. Pavla Bojarov, born in 1978, graduat-
ed at Charles University in Prague, Faculty
of Sciences, in 2002, and obtained her
Ph.D. in biochemistry in 2006. She has
participated in several study stays abroad.
For her postdoctoral studies she joined Dr.
S. J. Williams group at University of Mel-
bourne, to work on time-dependent inacti-
vation of sulfatases. Since her undergradu-
ate years she has been working in the Lab-
oratory of Biotransformation with Prof. V.
Kr
ˇ
en. Her main research interests include
(chemo-)enzymatic synthesis of complex
glycostructures using glycoside hydrolases,
and analysis of these enzymes at the molecular level.
Prof. Vladimr Kr
ˇ
en, born in 1956 is, cur-
rently Head of Laboratory of Biotransfor-
mation, and a Head of Department of Bio-
technology of Natural Products at the In-
stitute of Microbiology, Academy of Scien-
ces of the Czech Republic. His research in-
terests cover the biotransformation of
natural products by enzymes and microor-
ganisms; glycobiology; supramolecular
chemistry; flavonoids and antioxidants;
secondary metabolites of fungi; and immo-
bilised microbial cells.
Lubbert Dijkhuizen is Professor of Micro-
biology, University of Groningen. He is
scientific director of the Carbohydrate
Competence Center, a public-private part-
nership with 19 companies and 6 knowl-
edge institutes (http://www.cccresearch.nl).
His research focuses on the characteriza-
tion and engineering of sterol/steroid con-
verting enzymes (Rhodococcus/Mycobac-
terium), and starch and sucrose acting en-
zymes (bacilli/lactobacilli). He is currently
involved in EU FP7 research projects NO-
VOSIDES and AMYLOMICS.
Vanessa Eastwick-Field is founder and
Managing Director of Carbosynth Limit-
ed, an SME specialising in the develop-
ment of carbohydrate-based entities for
high-value application. She received her
Ph.D. from the University of Warwick in
1990 in the electrochemistry of reduced
state conducting polymers and subsequent-
ly worked in the fine chemical industry.
Her career, spanning more than 20 years,
has involved in all aspects of managing in-
ternational SMEs including start-up, ac-
quisition, and divestment.
Alexander Schiller studied chemistry at the
University of Munich (LMU) and com-
pleted his Ph.D. in 2006 under the supervi-
sion of Prof. K. Severin at the cole Poly-
technique Fdrale de Lausanne (Switzer-
land). As a postdoc he worked together
with Prof. B. Singaram and Dr. R. Wes-
sling (University of California, Santa
Cruz) on fluorescent saccharide sensors.
At Empa (Switzerland) he was project
leader and head of laboratory for stimuli-
responsive materials. In 2009 he joined the
Friedrich-Schiller University of Jena as a
junior professor of the Carl-Zeiss founda-
tion. His present research interests include NO and CO releasing materials
and supramolecular analytical chemistry (http://www.jppm.uni-jena.de).
www.chemeurj.org 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Eur. J. 2012, 18, 10786 – 10801
10788
A. Schiller et al.
ed that enzymatic glycosylation reactions generate fivefold
less waste and have a 15-fold higher space–time yield, a tre-
mendous improvem ent in eco-efficiency.
[32]
Although oligo-
saccharides, such as isomaltulose, isomalto (IMO), galacto
(GOS) and fructo oligosaccharides (FOS), are synthesized
industrially with the use of enzymes,
[33–35]
this is not yet the
case for glycosides. A perspective on the enzymatic glycosy-
lation of small organic molecules will, therefore, be present-
ed in this review.
Several types of carbohydrate-active enzymes (CAZymes)
may be used in glycosylation reactions, each with specific
characteristics (Scheme 2).
[36,37]
Natures catalysts for glyco-
sylation reactions are known as “Leloir” glycosyl transferas-
es (GT). Although very efficient, these enzymes require ex-
pensive nucleotide-activated sugars (e.g., uridine diphos-
phate glucose) as glycosyl donors, which hampers their ap-
plication in the laboratory and industry. However, two spe-
cial types of glycosyl transferring enzymes are the
proverbial “exception to the rule” and are active with low-
cost donors. Glycoside phosphorylases (GP), on the one
hand, only require glycosyl phosphates (e.g., glucose-1-phos-
phate) as donors—compounds that can easily be obtained in
large quantities.
[38]
Transglycosidases (TG), on the other
hand, even employ non-activated carbohydrates (e.g., su-
crose) for the transfer of a glycosyl group.
[39]
Additionally,
glycoside hydrolases (GH) can also be used for synthetic
purposes when applied under either kinetic (transglycosyla-
tion) or thermodynamic (reverse hydrolysis) control.
[40]
In
the following sections, the glycosylation reactions catalyzed
by GH, GP, and TG will be described in more detail.
Glycoside Hydrolases
Glycosidases (O-glycoside hydrolases; EC 3.2.1.-) are in
vivo purely hydrolytic enzymes. Their subclass in the
IUBMB system (International Union of Biochemistry and
Molecular Biology) comprises over 150 entries. In the
CAZy database (Carbohydrate Active Enzymes, http://
www.cazy.org/) glycosidases are structurally divided into
over 130 families.
[42]
Glycosidases split saccharidic chains by transferring the
cleaved glycosyl moiety to water as an acceptor substrate
(Scheme 3). In laboratory conditions, however, the acceptor
molecule may be virtually any structure possessing a hydrox-
yl group, allowing the formation of a new glycosidic bond,
instead of the naturally occurring hydrolysis reaction. Two
strategies for such synthetic processes may be applied. First,
two reducing sugars react in a thermodynamically controlled
condensation process, usually called “reverse hydrolysis”.
This approach has been preferentially used for the glycosy-
lation of alcohols.
[43]
Besides primary and secondary alco-
hols, successful glycosylations of sterically hindered tertiary
alcohols were accomplished, such as of 2-methylbutan-2-ol,
2-methylpentan-2-ol or tert-butyl alcohol.
[44,45]
More complex
structures are efficiently glycosylated under kinetic control
in so-called transglycosylation reactions. In this case, glyco-
side donors require activation by a good leaving group; this
Scheme 2. Glycosylation reactions catalyzed by the various classes of car-
bohydrate-active enzymes (Copyright Wiley-VCH Verlag GmbH & Co.
KGaA. Adapted and reproduced with permission from reference [41]).
Scheme 1. Prominent glycosyl donors used in chemical synthesis (Ac=
Acetyl, NBS= N-bromosuccinimide).
[30]
Scheme 3. Synthetic and hydrolytic reactions catalyzed by glycosidases.
Chem. Eur. J. 2012, 18, 10786 – 10801 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemeurj.org
10789
REVIEW
Enzymatic Glycosylation of Small Molecules