Streamlined chemoenzymatic total synthesis of prioritized ganglioside cancer antigens

Article abstract of DOI:10.1039/c8ob01087k

A highly efficient streamlined chemoenzymatic strategy for total synthesis of four prioritized ganglioside cancer antigens GD2, GD3, fucosyl GM1, and GM3 from commercially available lactose and phytosphingosine is demonstrated. Lactosyl sphingosine (LacβS

Full text of DOI:10.1039/c8ob01087k

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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
benzene
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DOI: 10.1039/C8OB01087K
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Streamlined chemoenzymatic total synthesis of prioritized
ganglioside cancer antigens
Hai Yu,a Abhishek Santra,a Yanhong Li,a John B. McArthur,a Tamashree Ghosh,a Xiaoxiao Yang,a
Received 00th January 20xx,
Accepted 00th January 20xx
Peng G. Wang,b and Xi Chen*a
DOI: 10.1039/x0xx00000x
www.rsc.org/
A highly efficient streamlined chemoenzymatic strategy for total oligosaccharide with the lipid.11 Chemoenzymatic synthesis of
synthesis of four prioritized ganglioside cancer antigens GD2, GD3, gangliosides has been reported only for GM3.12, 20, 21 Therefore,
fucosyl GM1, and GM3 from commercially available lactose and efficient synthetic approaches for diverse gangliosides are
phytosphingosine is demonstrated. Lactosyl sphingosine (LacSph) lacking but are urgently needed.
was chemically synthesized (in 13-gram scale), subjected to
sequential one-pot multienzyme (OPME) glycosylation reactions
with facile C18-cartridge purification, followed by an improved
acylation condition to form target gangliosides, including fucosyl
GM1 which has never been synthesized before.
Gangliosides are sialic acid-containing glycosphingolipids
presented broadly in vertebrate cells but are particularly
abundant in the nervous cells.1, 2 They are involved in lipid raft
formation,3 viral and bacterial infection,4, 5 immune regulation,6
and axon outgrowth.2 Exogenous GM1 has shown potential in
treating central nervous system injuries and neurodegenerative
diseases.7, 8 Aberrant expressing of some gangliosides is linked
to cancer progression.9 In fact, among 75 cancer antigens
prioritized in a 2009 National Cancer Institute pilot project
report,10 four are gangliosides including GD2, GD3, fucosyl GM1,
Fig. 1 Structures of prioritized ganglioside cancer antigens GM3 (
fucosyl GM1 ( ), GD3 ( ), and GD2 ( ).
1),
and GM3 (Fig. 1).
2
3
4
Due to their important functions, gangliosides are attractive
but challenging synthetic targets.11, 12 For example, among four
prioritized ganglioside cancer antigens, only GM3 has been
synthesized chemically and chemoenzymatically.12-15 GD216 and
GD317 have been synthesized by chemical methods only.
Fucosyl GM1 has never been synthesized before and only its
oligosaccharide portion has been chemically synthesized.18, 19
Chemical synthetic procedures for gangliosides encounter
notable challenges including hard-to-control regio- and stereo-
selectivities in sialylation, and low yields for glycosylating the
Biosynthetically, gangliosides are formed in the Golgi from
lactosylceramide (Lac Cer) catalyzed by type II membrane
protein glycosyltransferases.22 These processes are not readily
duplicated in vitro as Lac Cer is not soluble in water and the
glycosyltransferases involved are immobilized on the Golgi
membrane. Recently, we showed that -Gal pentasaccharyl
ceramide, neutral glycosphingolipid, can be
chemoenzymatically synthesized from water soluble lactosyl
sphingosine (Lac Sph) with simple C18-cartridge solid phase



a

extraction (SPE) purification procedures followed by acylation.23
Herein, we develop high-yield streamlined chemoenzymatic
processes for total synthesis of synthetically challenging
complex gangliosides. A 13-gram-scale synthetic procedure is
a.Department of Chemistry, University of California, One Shields Avenue, Davis, CA
95616, USA.
E-mail: xiichen@ucdavis.edu
b.Department of Chemistry and Center of Diagnostics & Therapeutics, Georgia
State University, Atlanta, GA 30303, USA.
established for producing LacSph from commercially available
inexpensive lactose and phytosphingosine. Sequential one-pot
multienzyme (OPME) chemoenzymatic synthetic methods,
Electronic Supplementary Information (ESI) available Experimental procedures,
NMR and HRMS data, and NMR spectra of products. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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combined with facile C18-cartridge purification scheme, are States.32 Fucosyl GM1 is therefore an excellent candidate for
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successfully developed for the production of negatively charged cancer vaccine development and for DtOhIe: 10d.1e0v3e9l/oCp8mOBe0n1t087oKf
complex gangliosides from Lac
An improved acylation procedure is identified to convert sphingosine (Fuc-GM1

Sph in a highly efficient manner. antibodies for detecting SCLC. The corresponding fucosyl GM1
Sph, ) was obtained from GM3 Sph (

9

6)
glycosylsphingosines to target gangliosides in excellent 98–99% using three OPME reactions carried out in sequential (Scheme
yields. The four prioritized ganglioside cancer antigens including 2).
GM3 (1), fucosyl GM1 (2), GD3 (3), and GD2 (4) (Figure 1) are
successfully obtained in high yields.
To obtain target gangliosides, Lac
23

Sph (
5
)
was prepared
from lactose and phytosphingosine in a 13-gram scale to
demonstrate the efficiency of the streamlined chemical
synthetic procedure and to provide a sufficient amount of
starting material for glycosphingolipid synthesis. The synthesis
was achieved in 12 steps in an overall 40% yield (see ESI).
GM3 (1) is the simplest ganglioside and is a common
precursor for more complex gangliosides. It is overexpressed by
many types of tumors, including malignant melanoma,
leukemia, pulmonary cancer, neuroectodermal tumor.24, 25 The
corresponding GM3 sphingosine (GM3
acyl chain was readily obtained from Lac

Sph,
6
) lacking the fatty
Sph ( ) and

5
monosaccharide N-acetylneuraminic acid (Neu5Ac) using a one-
pot two-enzyme sialylation system containing Neisseria
meningitidis CMP-sialic acid synthetase (NmCSS)26 and
Pasteurella multocida 2–3-sialyltransferase 3 (PmST3) that can
use both oligosaccharides and glycolipids as acceptor
30
substrates27 (Scheme 1). PmST128 and its mutants29,
preferring oligosaccharides as acceptors are not suitable
sialyltransferases for the reaction. A simple C18-cartridge SPE
was used for purification by loading the sample to the C18-
cartridge, washing the cartridge with water to completely
remove excess Neu5Ac, cytidine 5’-monophosphate (CMP)-
Neu5Ac, cytidine 5’-triphosphate (CTP), by-product CMP, and
others, followed by eluting the cartridge with 60% acetonitrile
Scheme 2 Sequential one-pot multienzyme (OPME) synthesis of Fuc-
GM1 Sph ( ) from GM3 Sph ( ).

9

6
(CH3CN) in water to obtain pure GM3
(1.44 g) with an excellent 96% yield. The remaining Lac

Sph (
6
) in a gram-scale
Sph was

A one-pot four-enzyme N-acetylgalactosamine (GalNAc)-
activation and transfer system33 containing recombinant
recovered by eluting the cartridge with pure CH3CN. The whole
process took about 25 minutes in contrast to several hours
using standard silica gel chromatography.
Bifidobacterium
acetylhexosamine-1-kinase (BLNahK),34 Pasteurella multocida
N-acetylglucosamine uridylyltransferase
(PmGlmU),35
Pasteurella multocida inorganic pyrophosphatase (PmPpA),36
and Campylobacter jejuni 1–4-N–
acetylgalactosaminyltransferase (CjCgtA),33 was used to
glycosylate GM3 Sph ( ) to form GM2 Sph ( ). Although
GM3 Sph ( ) was a less favored acceptor for CjCgtA compared
to GM3 oligosaccharide,33 adding a larger amount of CjCgtA was
able to push the reaction to completion. GM2 Sph ( , 120 mg,
longum
strain
ATCC55813
N-


6

7

6

7
98% yield) was readily purified by passing the reaction mixture
through the C18 cartridge and eluting with 45% CH3CN in water.
GM1Sph (8) was then synthesized from GM2Sph (7) using
a
one-pot four-enzyme galactose-activation and transfer
system containing Escherichia coli galactokinase (EcGalK),37
Bifidobacterium longum UDP-sugar pyrophosphorylase
Scheme 1 Gram-scale (1.44 g) synthesis of GM3
multienzyme (OPME) sialylation of Lac Sph ( ).
Sph (6) by one-pot

5
(BLUSP),38 PmPpA, and Campylobacter jejuni
galactosyltransferase (CjCgtB).39 Again, GM2
efficient acceptor for CjCgtB compared to GM2
oligosaccharide,33 but adding a larger amount of CjCgtB was
able to complete the reaction without the complication of

1–3-
Fucosyl GM1 (2) has been found to be absent from normal
Sph (7) was a less
tissues but be expressed on the cell surface of small-cell lung
cancer (SCLC)31 which accounts for 10–15% of lung cancer cases
and remains as one of the leading causes of death in the United
2 | J. Name., 2012, 00, 1-4
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adding more than one galactose residue observed previously the reaction to completion. GD2
COMMUNICATION

Sph (11, 34 mVgi)ewwAartiscleeOansliinlye
for galactosylation of GM2 oligosaccharide by CjCgtB.33 purified by passing the reaction mixture DthOrI:o1u0g.1h03C91/C88cOaBr0tr10id8g7eK
GM1Sph (8, 57 mg, 99% yield) was readily purified by passing and eluting with 35% of CH3CN in water with an excellent 98%
the reaction mixture through the C18 cartridge and eluting with yield.
40% CH3CN in water.
To synthesize Fuc-GM1
suitable 1–2-fucosyltransferase was needed. Despite its
preference towards 1–3-linked galactoside acceptors,
Sph (9) from GM1Sph (8), a


Thermosynechococcus elongates

1–2-fucosyltransferase
(Te2FT)40 could not fucosylate GM1

Sph ( ). Escherichia coli
8
O126

1–2-fucosyltransferase (EcWbgL)41 that prefers


1–4-
1–3-
linked galactoside acceptors but is also active towards
linked galactosides was then cloned (see ESI) and found to be
very efficient in using GM1 Sph ( ) as the acceptor. Fuc-
GM1 Sph ( , 55 mg) was synthesized in a quantitative yield
from GM1 Sph ( ) using a one-pot three-enzyme fucose-

8

9

8
activation and transfer system containing EcWbgL and
guanosine 5'-diphosphate fucose (GDP-Fuc) biosynthetic
enzymes including Bacteroides fragilis bifunctional L-fucokinase
and guanidine 5'-diphosphate (GDP)-fucose pyrophosphorylase
(BfFKP)42 and PmPpA (Scheme 2) followed by C18 cartridge-
purification by eluting with 40% of CH3CN in water. Remarkable,
three OPME glycosylation reactions carried out in sequential led
to the formation of complex Fuc-GM1
) in an excellent 97% overall yield. The overall process for the
formation of Fuc-GM1 Sph ( ) from chemically synthesized
Lac Sph ( ) involved four sequential OPME reaction with a total
yield of 93%.
GD3 is a disialoganglioside overexpressed on melanomas,
Sph (9) from GM3Sph
(6

9
Scheme 3 Sequential one-pot multienzyme (OPME) synthesis of
GD3 Sph (10) and GD2 Sph (11) from GM3 Sph ( ).

5



6
With the desired glycosylsphingosines in hand, the
conditions for efficient acylation to form target
glycosphingolipids were explored. Similar to that reported
previously,23 palmitic acid in the presence of 1-ethyl-3-(3-
neuroectodermal tumors including neuroblastoma and glioma,
as well as cancers of lung, breast, colon, prostate, and ovary.43,
44
Therefore, GD3 has received a considerable attention as a
promising immunotherapeutic target for cancer therapy.9, 44, 45
dimethylaminopropyl)carbodiimide
hydrochloride
and
The corresponding GD3
Sph (10) was successfully synthesized
hydroxybenzotriazole (EDC-HCl/HOBt) led to successful
acylation of the amino group in glycosylsphingosines but the
reaction could not reach completion. In comparison, reactions
of glycosylsphingosines with palmitoyl chloride in a mixed
solvent of tetrahydrofuran (THF) and saturated NaHCO3
from GM3 Sph ( ) and Neu5Ac using a one-pot two-enzyme
sialylation reaction containing NmCSS and Campylobacter jejuni

6
47

2–3/8-sialyltransferase (CjCstII)46,
(Scheme 3). As CjCstII
2–8-linked Neu5Ac to
Sph was produced as a minor product and
was eluted from the C18-cartrige using 30% of CH3CN in water.
The GD3 Sph (10, 910 mg, 62% yield)was then readily obtained
could continue to add an additional
GD3 Sph (10), GT3



solution (1:1, v/v) reached completion in 2 h. GM3 (
1), GM2
(
12), GM1 (13), fucosyl GM1 ( ), GD3 ( ), and GD2 (4)
2
3

gangliosides were produced in 98–99% yields with facile C18
cartridge purification by eluting with 40–80% of CH3CN in water
(Table 1).
by eluting the cartridge with 35% of CH3CN in water.
GD2 is another disialoganglioside cancer antigen expressed
predominantly in the CD44hiCD24lo breast cancer stem cells
isolated from human breast cancer cells.48 It has also been
found in melanomas, gliomas, and neuroblastomas.49
Therapeutic effects of anti-GD2 monoclonal antibodies against
neuroblastomas have been reported.50 GD2 (#12) has the
highest priority compared to the other three prioritized
ganglioside cancer antigens GM3 (#48), fucosyl GM1 (#41), and
Table 1 Gangliosides formation by acylation of glycosylsphingosines.
Glycosylsphingosine
Ganglioside
Product
C18 cartridge
elution condition
80% CH3CN in H2O
Yield
(%)
GD3 (#40).10 Similar to the synthesis of GM2
GM3 Sph ( ), GD2 Sph (11) was readily synthesized from
GD3 Sph 10 using the one-pot multienzyme GalNAc-
activation and glycosylation system containing BLNahK,
PmGlmU, PmPpA, and CjCgtA (Scheme 3). As GD3 Sph (10) was
Sph (7) from

6

GM3 (1)
99
GM3Sph (6)
GM2Sph (7)
GM1Sph (8)
Fuc-GM1Sph (9)
GD3Sph (10)
GD2Sph (11)

(
)
GM2 (12)
GM1 (13)
50% CH3CN in H2O
50% CH3CN in H2O
50% CH3CN in H2O
50% CH3CN in H2O
40% CH3CN in H2O
98
99
99
99
98
Fucosyl GM1 (2)
GD3 (3)

not an efficient substrate for CjCgtA comparing to GD2
oligosaccharide,33 a larger amount of CjCgtA was used to push
GD2 (4)
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18 J. R. Allen and S. J. Danishefsky, J. Am. Chem. Soc., 19V9i9e,w1A2rt1ic,le1O08n7lin5e-
10882.
Conclusions
DOI: 10.1039/C8OB01087K
19 T. K.-K. Mong, H.-K. Lee, S. G. Durón and C.-H. Wong, Proc. Natl. Acad.
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20 M. D. Vaughan, K. Johnson, S. DeFrees, X. Tang, R. A. Warren and S. G.
Withers, J. Am. Chem. Soc., 2006, 128, 6300-6301.
cartridge
purification
schemes,
sequential
one-pot
multienzyme (OPME) glycosylation systems are highly efficient
in synthesizing a diverse array of gangliosides. As many bacterial
glycosyltransferases are involved in the synthesis of glycolipid
repeating units for the production of lipopolysaccharides and
capsular polysaccharides, they can use glycosylsphingosines as
acceptors for glycosylation although larger amounts of the
enzymes may be needed in some cases. The production of
gangliosides by sequential OPME glycosylation of water soluble
lactosyl sphingosine with C18 cartridge purification followed by
high-yield acylation is a highly efficient streamlined process. It
is reasonable to assume that by incorporating various
glycosyltransferases, the OPME systems can be applied to the
synthesis of other challenging complex glycosphingolipids.
21 S.-I. Nishimura and K. Yamada, J. Am. Chem. Soc., 1997, 119, 10555-
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22 S. Groux-Degroote, Y. Guerardel and P. Delannoy, Chembiochem, 2017,
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23 A. Santra, Y. Li, H. Yu, T. J. Slack, P. G. Wang and X. Chen, Chem. Commun.,
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24 X. L. Pan, T. Izumi, H. Yamada, K. Akiyoshi, S. Suenobu and S. Yokoyama,
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25 T. N. Seyfried and P. Mukherjee, J. Oncol., 2010, 2010.
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27 V. Thon, Y. Li, H. Yu, K. Lau and X. Chen, Appl. Microbiol. Biotechnol., 2012,
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Conflicts of interest
There are no conflicts to declare.
32 Q. S. C. Chu, B. Markman, N. Leighl, L. Krug, C. Rudin, D. Lathers, P.
Basciano, P. M. Fracasso, G. Kollia, P. Phillips, G. Kolaitis, D. Williams, J.
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Acknowledgements
This work was supported by the National Institutes of Health (NIH)
Common Fund Glycoscience Program grant U01GM120419. Bruker
Avance-800 NMR spectrometer was funded by NSF grant DBIO-
722538.
34 Y. Li, H. Yu, Y. Chen, K. Lau, L. Cai, H. Cao, V. K. Tiwari, J. Qu, V. Thon, P. G.
Wang and X. Chen, Molecules, 2011, 16, 6396-6407.
35 Y. Chen, V. Thon, Y. Li, H. Yu, L. Ding, K. Lau, J. Qu, L. Hie and X. Chen,
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36 K. Lau, V. Thon, H. Yu, L. Ding, Y. Chen, M. M. Muthana, D. Wong, R. Huang
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37 X. Chen, J. Fang, J. Zhang, Z. Liu, J. Shao, P. Kowal, P. Andreana and P. G.
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Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB01087K
Table of Contents
Highly efficient streamlined total synthesis of complex prioritized gangliosides was achieved
chemoenzymatically by sequential one-pot multienzyme (OPME) reactions with facile C18 cartridge
purification schemes followed by high-yield acylation.
HO
OH
O
OH
O
HO
HO
4
4
Cer
3
O
O
Sequential OPME
glycosylation
(93% yield)
3
4
AcHN
O
Acylation
(99% yield)
Sph
2
O
OH
HO
Lactosyl
sphingosine
O
OH
HO
OH
O
-O2C
O
OH
O
OH
HO
HN
O
O
HO
O
AcHN
OH
HO
OH
C18 cartridge purification
OH
Fucosyl GM1