Enzymatic synthesis of tumor-associated carbohydrate antigen globo-H hexasaccharide

Article abstract of DOI:10.1021/ol703121h

We report the enzymatic synthesis of an important tumor-associated carbohydrate antigen, Globo-H hexasaccharide. Starting with Lac-OBn as the initial acceptor, this approach employs three glycosyltransferases: LgtC, an α1,4-galactosyltransferase; LgtD, a bifunctional β1,3- galactosyl/β1,3-N-acetylgalactosaminyltransferase; and WbsJ, an α1,2-fucosyltransferase. In addition, two epimerases, GalE and WbgU, were also employed for the generation of more expensive sugar nucleotides, UDP-Gal and UDP-GalNAc, from their corresponding inexpensive C4 epimers. This study represents a facile enzymatic synthesis of the Globo-H antigen.

Full text of DOI:10.1021/ol703121h

ORGANIC
LETTERS
2008
Vol. 10, No. 5
1009-1012
Enzymatic Synthesis of
Tumor-Associated Carbohydrate Antigen
Globo-H Hexasaccharide
Doris M. Su,†,§ Hironobu Eguchi,†,§ Wen Yi,† Lei Li,‡ Peng G. Wang,*,† and
Chengfeng Xia*,†
Departments of Chemistry and Biochemistry, The Ohio State UniVersity, Columbus,
Ohio 43210, and The State Key Laboratory of Microbial Technology, School of Life
Science, Shandong UniVersity, Jinan, Shandong 250100, People’s Republic of China
wang.892@osu.edu; xia.39@osu.edu
Received December 29, 2007
ABSTRACT
We report the enzymatic synthesis of an important tumor-associated carbohydrate antigen, Globo-H hexasaccharide. Starting with Lac-OBn
as the initial acceptor, this approach employs three glycosyltransferases: LgtC, an 1,4-galactosyltransferase; LgtD, a bifunctional 1,3-
galactosyl/ 1,3-N-acetylgalactosaminyltransferase; and WbsJ, an 1,2-fucosyltransferase. In addition, two epimerases, GalE and WbgU, were
r
â
â
r
also employed for the generation of more expensive sugar nucleotides, UDP-Gal and UDP-GalNAc, from their corresponding inexpensive C4
epimers. This study represents a facile enzymatic synthesis of the Globo-H antigen.
Hexasaccharide of Globo-H (Figure 1), a cell surface
glycosphingolipid, is a member of the globo series of
cancers.1 Globo-H hexasaccharide was initially identified as
a breast cancer antigen in 1983 from human MCF-7 breast
cancer cells using the monoclonal antibody MBr1.2 High
levels of antibodies against the Globo-H epitope were found
in the serum of breast cancer patients. As a distinct tumor-
associated antigen marker, this epitope has been emerging
as an important sequence for the development of anticancer
vaccines.3 Globo-H has been conjugated to an immunogenic
protein carrier, keyhole limpet hemocyanin (KLH).4 This
Globo-H-KLH construct has shown promising results in
clinical trials as anti-breast and anti-prostate cancer vaccines.5
Figure 1. Structure of cell surface glycosphingolipid Globo-H.
(1) (a) Kannagi, R.; Levery, S. B.; Ishigami, F.; Hakomori, S.; Shevinsky,
L. H.; Knowles, B. B.; Solter, D. J. Biol. Chem. 1983, 258, 8934-42. (b)
Bremer, E. G.; Levery, S. B.; Sonnino, S.; Ghidoni, R.; Canevari, S.;
Kannagi, R.; Hakomori, S. J. Biol. Chem. 1984, 259, 14773-14777. (c)
Zhang, S.; Cordon-Cardo, C.; Zhang, H. S.; Reuter, V. E.; Adluri, S.;
Hamilton, W. B.; Lloyd, K. O.; Livingston, P. O. Int. J. Cancer 1997, 73,
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antigenic carbohydrates. This carbohydrate antigen is found
to be highly expressed on many types of human cancer cell
lines, including breast, colon, lung, ovarian and prostate
† The Ohio State University.
‡ Shandong University.
§ Contributed equally to this work.
10.1021/ol703121h CCC: $40.75
© 2008 American Chemical Society
Published on Web 02/07/2008

Scheme 1. Enzymatic Synthesis of Globo-H-OBn (5) from Lac-OBn (1) via Four Steps
Due to the difficulty of isolating such oligosaccharides
regio-/stereoselectivity and relatively lower yields. Efficient
synthesis of the Globo-H hexasaccharide in large quantities
is still in demand for evaluation of these antitumor vaccines.
Here, we report an alternative synthesis of Globo-H hexa-
saccharide using enzymes. It has been shown in literature
that enzymatic coupling has several advantages over its
chemical counterpart.13 Enzymatic glycosylation offers high
stereo- and regioselectivity under neutral aqueous conditions
without functional group protection and higher yields.
Furthermore, many enzymatic syntheses of important oligo-
saccharide antigens have been reported.14
from natural sources, synthetic approaches represent the only
feasible method to obtain homogeneous Globo-H antigen.
Danishefsky et al. reported the first total chemical synthesis
of the Globo-H hexasaccharide in 1996 using the glycal
strategy6 with later refinements.7 Many other chemical
methodologies have been explored, such as the trichloro-
acetimidate method,8 two-directional glycosylation,9 one-pot
strategies,10 linear synthesis11 and automated solid-phase
assembly.12 However, chemical synthesis often requires
multiple protection/deprotection steps and suffers from poor
In this approach (Scheme 1), we used three glycosyltrans-
ferases (LgtC, LgtD, and WbsJ) to synthesize the glycosidic
linkages. In addition, two other enzymes (GalE and WbgU)
were employed in the glycosylation reactions to convert
inexpensive donor substrates, uridine 5′-diphosphoglucose
(UDP-Glc) and uridine 5′-diphospho-N-acetylglucosamine
(UDP-GlcNAc), to expensive donors, uridine 5′-diphospho-
galactose (UDP-Gal) and uridine 5′-diphospho-N-acetyl-
galactosamine (UDP-GalNAc), respectively.
Starting from readily available acceptor 1-benzyl-lactose
(Lac-OBn) 1, globotriose (Gb3-OBn) 2 was synthesized with
an R1,4-galactosyltransferase (LgtC from Neisseria menin-
gitidis) (Scheme 1). This enzyme has been well characterized.
LgtC transfers the galactose residue from UDP-Gal onto a
variety of acceptors.15 With UDP-Gal as a sugar nucleotide
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Org. Lett., Vol. 10, No. 5, 2008

donor, the reaction yield was 95%. In another reaction, we
coupled the UDP-Glc C4 epimerase (GalE from Escherichia
coli K12) with LgtC and used UDP-Glc as the sugar
nucleotide donor. GalE has been widely employed in
oligosaccharide synthesis for in situ cofactor regeneration
to avoid both the stoichiometric use of expensive UDP-Gal
and product inhibition.16 The yield of this coupled reaction
is 90% (Table 1), which proves GalE effective in production
of Gb3-OBn.
substrate specificities of CgtB from three C. jejuni strains.
The activity of CgtB from strain HS:10 with a GA2
ganglioside mimic was significantly higher when compared
to sialylated or monosaccharide acceptors. GA2 and globo-
tetraose both have terminal GalNAc residues. However,
the GalNAcâ1,4Gal linkage of GA2 differs from the
GalNAcâ1,3Gal linkage in globotetraose.
The final step of enzymatic transformation is the addition
of a fucose residue from guanosine 5-diphosphofucose (GDP-
Fuc) via an R1,2- linkage to the galactose residue of Gb5-
OBn by an R1,2-fucosyltransferase (WbsJ from Escherichia
coli O128:B12). WbsJ is involved in the synthesis of the E.
coli O128 O-antigen.23 This enzyme was cloned and bio-
chemically characterized in our group.24 WbsJ has relaxed
substrate specificity toward Galâ1,4Fru (lactulose), Galâ1,4Glc
(lactose), Galâ1,3GalNAc (T antigen) and Galâ1,4Man. The
broad acceptor specificity of WbsJ reveals its potential
application in the synthesis of important fucosylated glyco-
conjugates. WbsJ was cloned and transformed into E. coli
BL21(DE3) and expressed as a GST-fusion protein. Using
purified WbsJ protein, we completed the transfer of a fucose
residue to Gb5-OBn with 95% yield. The structure of Globo-
H-OBn 5 was analyzed by 1H NMR, 13C NMR, H-H COSY,
HMQC and HRMS (see Supporting Information).25 It was
also confirmed by comparing with the reported hexasaccha-
rides.
Table 1. Yields of Each Enzyme-Catalyzed Glycosylation Step
step
enzyme(s)
product
yield (%)
1
2
3
4
GalE, LgtC
LgtD-WbgU
LgtD
Gb3-OBn
Gb4-OBn
Gb5-OBn
Globo-H-OBn
90
78
85
95
WbsJ
The subsequent step from Gb3-OBn to globotetraose (Gb4-
OBn) 3 was catalyzed by a â1,3-N-acetylgalactosaminyl-
transferase (LgtD from Haemophilus influenza). We have
previously reported the overexpression and biochemical
characterization of LgtD.17 Furthermore, we have also
reported the characterization of UDP-N-acetylglucosamine
C4 epimerase (WbgU from Plesiomonas shigelloides).18 The
LgtD-WbgU fusion protein was constructed and used in
coupled enzymatic reactions to synthesize a variety of
globotetraose and isoglobotetraose derivatives from corre-
sponding lactoside acceptors.19 Here, we used this fusion
protein to synthesize Gb4-OBn by epimerization of UDP-
GlcNAc to UDP-GalNAc by WbgU and subsequent transfer
of the GalNAc residue from UDP-GalNAc onto Gb3-OBn
by LgtD with 78% yield.
Synthesis of globopentaose (Gb5-OBn) 4 was also achieved
by using LgtD. Randriantsoa et al. reported a novel â1,3-
galactosyltransferase activity of LgtD.20 They demonstrated
that in the presence of globotriose, LgtD prefers UDP-
GalNAc as the donor, and acts as a GalNAc transferase. On
the other hand, donor specificity was changed to prefer UDP-
Gal in the presence of globotetraose, and LgtD acts as a Gal
transferase, resulting in the production of globopentaose.
With purified LgtD and UDP-Gal, we also demonstrated the
formation of Gb5-OBn with 85% yield.21 Another possible
candidate that has gathered much interest for the synthesis
of globopentaose is the â1,3-galactosyltransferase CgtB from
Campylobacter jejuni. Bernatchez et al.22 reported the
In this study, we report the enzymatic synthetic route to
obtain Globo-H hexasaccharide with Lac-OBn as the starting
material. The whole synthetic route contains three glycosyl-
transferases and two epimerases with an overall yield of 57%.
In views of high stereo- and regioselectivity under mild
(21) Spectroscopic data for Gb5-OBn: 1H NMR (500 MHz, D2O): δ
7.47-7.38 (m, 5H, Ph), 4.91 (d, J ) 11.7 Hz, 1H, PhCH2), 4.88 (d, J )
3.8 Hz, H-1′′′), 4.74 (d, J ) 11.8 Hz, 1H, PhCH2), 4.66 (d, J ) 8.6 Hz,
1H, H-1), 4.52 (d, J ) 8.1 Hz, 1H, H-1), 4.48 (d, J ) 7.8 Hz, 1H, H-1),
4.42 (d, J ) 7.7 Hz, 1H, H-1), 4.35 (t, J ) 6.3 Hz, 1H), 4.22 (d, J ) 1.7
Hz, 1H), 4.15 (d, J ) 2.7 Hz, 1H), 4.03 (m, 1H), 4.01 (m, 1H), 3.97 (dd,
J ) 12.5, 1.6 Hz, 1H), 3.94 (dd, J ) 10.1, 2.9 Hz, 1H), 3.91-3.85 (m,
4H), 3.82 (d, J ) 4.4 Hz, 1H), 3.79 (d, J ) 4.4 Hz, 1H), 3.78-3.69 (m,
6H), 3.68-3.65 (m, 3H), 3.64-3.61 (m, 2H), 3.60-3.53 (m, 4H), 3.50
(dd, J ) 9.9, 7.9 Hz, 1H), 3.32 (t, J ) 8.6 Hz, 1H), 1.99 (s, 3H, CH3CONH);
13C NMR (125 MHz, D2O): δ 175.2, 136.6, 128.8, 128.76, 128.5, 104.8,
103.3, 103.0, 101.0, 100.4, 79.6, 78.8, 78.7, 77.3, 75.5, 75.0, 74.9, 74.6,
73.0, 72.5, 72.2, 71.5, 70.9, 70.6, 70.3, 69.0, 68.6, 68.0, 67.6, 61.4, 61.0,
60.98, 60.4, 60.3, 60.1, 59.4, 51.5, 22.3; HRMS calcd for C39H61NO26Na
([M + Na]+) 982.3380, found 982.3405.
(22) Bernatchez, S.; Gilbert, M.; Blanchard, M.-C.; Karwaski, M.-F.;
Li, J.; DeFrees, S.; Wakarchuk, W. W. Glycobiology 2007, 17, 1333-1343.
(23) Shao, J.; Li, M.; Jia, Q.; Lu, Y.; Wang, P. G. FEBS Lett. 2003,
553, 99-103.
(24) Li, M.; Liu, X.-W.; Shao, J.; Shen, J.; Jia, Q.; Yi, W.; Song, J. K.;
Woodward, R.; Chow, C. S.; Wang, P. G. Biochemistry 2008, 47, 378-
387.
(25) Spectroscopic data for Globo-H-OBn: 1H NMR (500 MHz, D2O):
δ 7.46-7.38 (m, 5H, Ph), 5.20 (d, J ) 4.1 Hz, 1H, H-1′′′′′′), 4.91 (d, J )
11.7 Hz, 1H, PhCH2), 4.86 (d, J ) 3.9 Hz, 1H, H-1′′′), 4.74 (d, J ) 11.6
Hz, 1H, PhCH2), 4.59 (d, J ) 7.7 Hz, 1H, H-1), 4.53 (d, J ) 7.4 Hz, 1H,
H-1), 4.51 (d, J ) 6.7 Hz, 1H, H-1), 4.48 (d, J ) 7.7 Hz, 1H, H-1), 4.35
(t, J ) 6.4 Hz, 1H), 4.22-4.18 (m, 2H), 4.07 (d, J ) 2.1 Hz, 1H), 4.00 (d,
J ) 3.0 Hz, 1H), 3.98-3.94 (m, 3H), 3.91 (dd, J ) 10.6, 2.8 Hz, 1H),
3.88-3.84 (m, 3H), 3.83-3.79 (m, 4H), 3.77-3.72 (m, 7H), 3.70-3.65
(m, 5H), 3.64-3.60 (m, 5H), 3.58-3.53 (m, 3H), 3.33 (t, J ) 8.6 Hz, 1H),
2.01 (s, 3H, CH3CONH), 1.19 (d, J ) 6.6 Hz, 3H, CH3 of fucose); 13C
NMR-DEPT (125 MHz, D2O): δ 128.8, 128.77, 128.5, 104.0, 103.3, 102.1,
101.0, 100.5, 99.3, 78.9, 78.4, 77.2, 76.4, 76.2, 75.5, 75.1, 74.9, 74.7, 74.6,
73.6, 73.0, 72.7, 71.9, 71.5, 70.9, 70.7, 70.2, 69.6, 69.2, 69.17, 68.5, 68.1,
67.9, 66.8, 61.0, 61.00, 60.4, 60.37, 60.1, 51.7, 22.3, 15.4; HRMS calcd
for C45H71NO30Na ([M + Na]+) 1128.3959, found 1128.3981.
(16) (a) Chen, X.; Kowal, P.; Hamad, S.; Fan, H.; Wang, P. G.
Biotechnol. Lett. 1999, 21, 1131-1135. (b) Chen, X.; Liu, Z.; Wang, J.;
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Org. Lett., Vol. 10, No. 5, 2008
1011

conditions without protecting group manipulation, our study
paves a way for large-scale enzymatic synthesis of the
Globo-H antigen. Further studies will be geared toward
conjugation of the hexasaccharide to carrier proteins, lipids,
polymers, and nanoparticles for development of anti-cancer
vaccines.
on macromolecular structure and function in the Department
of Biochemistry at The Ohio State University.
Supporting Information Available: Full experimental
details as well as spectra for Gb5-OBn and Globo-H-OBn.
This material is available free of charge via the Internet at
http://pubs.acs.org.
Acknowledgment. P.G.W. acknowledges the support
from an endowed professorship of Ohio Eminent Scholar
OL703121H
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Org. Lett., Vol. 10, No. 5, 2008