Large scale enzymatic synthesis of oligosaccharides and a novel purification process

Article abstract of DOI:10.1016/j.bmcl.2010.11.011

Herein we report the practical chemo enzymatic synthesis of trisaccharide and derivatives of iGb3 and Gb3, and a novel purification process using immobilized yeast to remove the monosaccharide from the reaction mixture. High purity oligosaccharide compoun

Full text of DOI:10.1016/j.bmcl.2010.11.011

Bioorganic & Medicinal Chemistry Letters 21 (2011) 311–314
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Large scale enzymatic synthesis of oligosaccharides and a novel
purification process
Guangyan Zhou a, , Xianwei Liu d, Doris Su b,c, Lei Li d, Min Xiao d, Peng G. Wang b,c,
⇑
⇑
a Department of Basic Science, Touro University-California, Vallejo, CA 94592, USA
b Department of Chemistry, The Ohio State University, Columbus, OH 43210, USA
c Department of Biochemistry, The Ohio State University, Columbus, OH 43210, USA
d The State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, Shandong 250100, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein we report the practical chemo enzymatic synthesis of trisaccharide and derivatives of iGb3 and
Gb3, and a novel purification process using immobilized yeast to remove the monosaccharide from the
reaction mixture. High purity oligosaccharide compounds were achieved in large scale. This study repre-
sents a facile enzymatic synthesis of and novel purification process of oligosaccharide.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 26 September 2010
Revised 27 October 2010
Accepted 1 November 2010
Available online 9 November 2010
Keywords:
Oligosaccharide
Enzymatic synthesis
Purification
Glycosyltransferase
Isoglobotrihexosylceramide (iGb3) was regarded as primary
endogenous agonist ligand for invariant natural killer T(iNKT) cells,
because iGb3 stimulates both human VR24 NKT cell and mouse
VR14 NKT cells.1 iNKT cells regulate a variety of microbial, allergic,
autoimmune, and tumor conditions, through the rapid secretion of
cal synthesis of the lipid part has been well documented in recent
years,12,13 the chemical preparation of the oligosaccharide donors
were tedious, because of selective protections, deprotections, and
glycosylations in totally 17–19 steps. Generally, enzymatic glycosyl-
ation has several advantages over its chemical counterpart, being
one of the most practical methods for oligosaccharide synthesis be-
cause of its high stereo- and regioselectivity under mild aqueous
conditions.14–16 Therefore, taking advantage of the high efficiency
of enzymatic synthesis of oligosaccharides, we explored the
enzymatic synthesis of these trisaccharides in large scale.
interleukin-4 (IL-4), interferon-c (INF-c), and other cytokines and
chemokines.2 Such cells specifically recognize microbial and endog-
enous glycolipid antigens in a CD1d-dependent way.3,4 In contrast,
globotrihexosylceramide (Gb3) with a terminal Gal-
instead of Gal- -(1 ? 3)Gal in iGb3 has no stimulatory ability to-
ward iNKT cells.
It is alsonoticed that thehexasaccharideofGlobo-H (amemberof
the globo series of antigenic carbohydrates) has the motif of Gal-
a-(1 ? 4)Gal
a
According to Leloir pathway,17 application of glycosyltransfer-
ase to preparative synthesis, two factors must be considered:
(1) availability of the enzymes; (2) availability of nucleoside phos-
phate sugars, which serve as activated sugar donors, normally are
expensive from commercial source.
a
-
(1 ? 4)Gal-b(1 ? 4)Glc. This carbohydrate antigen is found to be
highly expressed on many types of human cancer cell lines, includ-
ing breast, colon, lung, ovarian, and prostate cancers.5–8 Therefore,
there has been high demand for iGb3, Gb3, and Globo-H for immu-
nology research and preclinical investigation. Figure 1 shows the
structures of iGb3, Gb3, and Globo-H.
Current research on this field is hampered by the rather limited
access to these compounds. Purification from natural sources not
only requires unique biochemical and analytical apparatus, but also
gives milligram quantities of the product.9 Chemical syntheses of
iGb3 and Gb3,10 Globo-H11 had been reported. Although the chemi-
The most promising approach of enzymatic synthesis of trisac-
charide of isoglobotrihexose and globotrihexose is directly using
UDP-galactose (UDP-Gal) and lactose catalyzed by galactotransfer-
ase. However, commercially available UDP-Gal is too expensive to
be used in large scale oligosaccharide synthesis. Multiple-enzyme
sugar nucleotide regeneration systems had been developed to avoid
using costly stoichiometric amounts of sugar nucleotides.18,19
Dr. Wang had reported the enzymatic synthesis of globotrihexose
and isoglobotrihexose using superbead or superbug techniques.20,21
Nowadays, UTP can be used in stoichiometric amounts to generate
UDP-Gal because of its commercially low price. Considering the
Gal-1-phosphate is much more expensive than Glc-1-phosphate, a
two enzymes UDP-Gal generation strategy was used to produce
⇑
Corresponding authors. Tel.: +1 707 638 5492; fax: +1 707 638 5255 (G.Z.).
E-mail addresses: guangyan.zhou@tu.edu (G. Zhou), wang.892@osu.edu (P.G.
Wang).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.011

312
G. Zhou et al. / Bioorg. Med. Chem. Lett. 21 (2011) 311–314
OH
HO
O
OH
O
OH
OH
HO
HN
(CH2)24CH3
(CH2)12CH
O
O
HO
O
O
O
3
HO
OH
OH
OH
O
OH
HO
HO
iGb3
O
HO
HO
OH
OH
O
HN
(CH2)24CH3
(CH2)12CH3
O
O
O
O
HO
OH
OH
OH
O
OH
OH
OH
HO
O
HO
O
HO
HO
Gb3
O
O
O
NHAc
HO
O
OH
OH
O
HN
(CH2)14CH
3
O
O
OH
O
O
O
(CH2)12CH3
HO
HO
OH
HO
OH
OH
OH
GlobO-H
Figure 1. Structures of iGb3,Gb3, and Globo-H.
UDP-Gal in large scale.22 Scheme 1 shows the strategy to prepare
UDP-Gal in large scale.
transfer the galactose residue from UDP-Gal onto a variety of accep-
tors. Two lactose derivatives (synthesized in our lab) with linkers at
the reducing end were used as acceptor. Synthesized compound Gal-
Briefly, glucosyl-1-phosphate reacted with UTP catalyzed by
GalU (E.C.2.7.7.9) to yield UDP-Glc, pyrophosphatase here was used
to decomposed the PPi into Pi to push the first step of this reaction
forward. Then UDP-Glc was converted to UDP-Gal by epimerase,
GalE (E.C.5.1.3.2).23 Two kinds of galactotransferases expressed in
Escherichia coli BL21(DE3) were employed in the following reactions.
The activities of the enzymes were assayed according to previously
publishedprotocols.24,25 Oneunit (U)ofenzymeactivitywasdefined
a-(1 ? 4)Gal-b(1 ? 4)Glc-1-OBn was used as precursor for Globo-H
synthesis, compound Gal- -(1 ? 4)Gal-b(1 ? 4)Glc-1-(OC2H4)3-N3
a
was used as array of glyconanoparcels study.
In order to utilize UDP-Glc sufficiently, two times acceptor was
added into the reaction mixture. For example, the enzymatic synthe-
sis reaction of compound 2 was set up with 100 mM lactose, 50 mM
UDP-Glc, 10 mM MnCl2, 10 U GalE, 10 U GalT in Tris–HCl buffer
(100 mM, pH 7.0, 100 mL) in 200 mL flask. The reaction mixture
was kept at 37 °C under argon atmosphere for 2 days. After the
UDP-Glc was used up as monitored by TLC (thin layer chromatogra-
phy) (isopropanol/H2O/NH4OH = 7/3/2 (v/v/v)), the mixture was
as the amount of enzyme that catalyzed the conversion of 1
substrate per minute at 37 °C.26 For the synthesis of isoglobotrihex-
ose, (1 ? 3) galactotransferase was used; and for globotrihexose
synthesis,
lm of
a
a
-(1 ? 4) galactotransferase (LgtC) was used. LgtC could
O
HO
P
O-
2
O
N
-
O
OH
HN
pyrophosphatase
O
O
HO
HO
O
P
O-
O
P
O
O
P
O
P
O-
O
O
GalU
HO
O
O
O
HO
P
O-
O
O-
P
OH
HO
O
-
O
O-
O-
HO
OH
O
N
O
N
OH
OH
O
OH
HN
O
HN
HO
HO
HO
O
P
O-
O
O
P
O
P
HO
O
HO
O
O
O
O
P
O-
O
O
O
GalE
O
O
O-
O-
HO
OH
HO
OH
UDP-Gal
UDP-Glc
Scheme 1. Strategy to prepare UDP-Gal.

G. Zhou et al. / Bioorg. Med. Chem. Lett. 21 (2011) 311–314
313
Table 1
The oligosaccharides synthesized
Entry Acceptor
Enzyme/reaction volume
Product
Qty.
25 g
Yield
(%)
1
2
Glc-1-P, UTP
50 U GalU/500 mL Â 5
UDP-GLC
70a
10 U GalE
10 U LgtC/100 mL
2.2 g
90b
1 U GalE
1 U LgtC/7.5 mL
3
0.2 g
81b
45 U GalE
45 U LgtC/450 mL
4
5
10.2 g 76b
30 U GalE
30 U
a(1 ? 3) galactotransferase/300 mL
5.0 g
66b
LgtC/100 mL
a
Yield was based on Glc-1-P.
Yield based on UDP-Glc.
b
worked up. The protein was removed by centrifugation after the
mixture was heated at 100 °C for 10 min, then the solution was
passed through Dowex Cl anion-exchange resin, only acceptor and
trisaccharide were remaining inside the filtration. The following tri-
saccharides in Table1 were prepared according to the same method.
Purification of oligosaccharides mixtures was conventionally
proceeded by chromatography using activated charcoal and ion-
exchange resins or Bio-Gel P-Gels column, actually these proce-
dures were not only complicated, but also could hardly separate
the remaining lactose from the filtration, especially in large scale.
It was worthy noted that an efficient way of producing high-purity
oligosaccharide was carried out by immobilized yeast cells
(Saccharomyces cereviside L1) under fermentation condition. This
yeast cells consumed only monosaccharide, such as glucose and
galactose.27 Inspired by this new technology, a novel purification
strategy was developed: using b-galactosidase to digest lactose
into glucose and galactose, then using this immobilized yeast to
consume the monosaccharide, so there will be only trisaccharide
remaining in the solution. The S. cereviside L1 was cultured in med-
ium containing 10 g malt extract in 1000 mL water (pH 6.4) at
30 °C. Cells were harvested at log phase growth by centrifugation
at 10,000 rpm for 20 min and were immobilized in calcium algi-
nate. Five grams crude cells were mixed with 10 mL sodium algi-
nate solution (4%) and then extruded into a 3.0% CaCl2 solution
to form beads at 4 °C. The beads were washed with distilled water
until no Ca2+ was detected in the wash. The syrup of the oligosac-
charide mixture was fermented with 2 g immobilized cells. Batch
reactions were performed in an incubator shaker at 100 rpm at
30 °C. The reaction was monitored by TLC. TLC indicated that only
trisaccharide and protected monosaccharide were inside the filtra-
tion after all natural monosaccharide was consumed. Since the
yeast cells can only digest natural monosaccharide, the monosac-
charide protected at the reduced end of the entries 3 and 4 had
to be removed by Bio-Gel P-2 gel (Bio-Rad) filtration to achieve
purified trisaccharide with water as the mobile phase. The desired
fractions were pooled and lyophilized to provide the target com-
pound. Table 1 shows the oligosaccharides synthesized and puri-
fied through this novel process.28
In this study, we report the practical enzymatic synthetic route
to obtain trisaccharide and its conjugates in large-scale, and a no-
vel efficient method using b-galactosidase and immobilized yeast
to remove lactose and monosaccharides. Further studies will be
carried out by using this process to synthesize and purify more oli-
goasaccharides for research.
Acknowledgment
P.G.W. acknowledges the support from an endowed professor-
shipof OhioEminentScholaronmacromolecularstructureandfunc-
tion in the Department of Biochemistry at The Ohio State University.

314
G. Zhou et al. / Bioorg. Med. Chem. Lett. 21 (2011) 311–314
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