Programmable one-pot synthesis of tumor-associated carbohydrate antigens Lewis X dimer and KH-1 epitopes

Article abstract of DOI:10.1016/j.tetlet.2010.11.055

The total synthesis of the antigenic Lewis X (Le^x) dimer and KH-1 epitopes by a reactivity-based programmable one-pot synthetic strategy is reported. This approach can minimize the protection-deprotection and purification steps. Using the react

Full text of DOI:10.1016/j.tetlet.2010.11.055

Tetrahedron Letters 52 (2011) 2132–2135
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Programmable one-pot synthesis of tumor-associated carbohydrate antigens
Lewis X dimer and KH-1 epitopes
Bin-Ling Tsai a,b, Jeng-Liang Han a, Chien-Tai Ren a, Chung-Yi Wu a, , Chi-Huey Wong a,b,
⇑
⇑
a Genomics Research Center, Academia Sinica, Taiwan
b Department of Chemistry, National Taiwan University, Taipei, Taiwan
a r t i c l e i n f o
a b s t r a c t
The total synthesis of the antigenic Lewis X (Lex) dimer and KH-1 epitopes by a reactivity-based program-
mable one-pot synthetic strategy is reported. This approach can minimize the protection–deprotection
and purification steps. Using the reactivity-based one-pot synthetic method, the fully protected Lewis
X (Lex) dimer and KH-1 epitopes were furnished in a facile manner, which were globally deprotected
to give the Lex dimer and KH-1 epitopes.
Article history:
Available online 16 November 2010
Dedicated to Professor Harry Wasserman on
the occasion of his 90th birthday
Ó 2010 Elsevier Ltd. All rights reserved.
Lewis X (Lex) dimer and KH-1, isolated by Hakomori in 1986,1
are tumor-associated carbohydrate antigens and considered as
markers of colonic adenocarcinoma.1,2 They can be used to develop
carbohydrate-based anticancer vaccines and carbohydrate micro-
array for diagnosis. Lex dimer has been synthesized via stepwise
[3+2+3] or [3+3+2] strategies,3–6 soluble polymer supported syn-
thesis,7 orthogonal, and iterative methods.8,9 Chemical synthesis
of KH-1 was first reported by both Schmidt10 and Danishefsky11
independently in 1997. In 2004, Seeberger used the automated so-
lid-phase synthesis to construct KH-1.12 Stepwise [4+3] strategy
was used by Danishefsky13 and Boons14 independently in 2005.
Our group has developed a programmable one-pot strategy for
the synthesis of complex oligosaccharides.15–21 The methodology is
based on the use of designer thioglycoside building blocks with de-
fined relative reactivity values (RRVs) that are collected in the
Optimer database.22 Herein, we report the use of this strategy to
prepare antigens Lex dimer and KH-1 epitopes.
could be assembled from building blocks 3, 10 (RRV = 115),9 and
11 via a second one-pot glycosylation.
To synthesize building blocks 4 and 5 (Scheme 2), we started by
treating thiogalactoside 7 (RRV = 5200) with 9 (RRV = 282) in the
presence of N-iodosuccinimide (NIS) and 0.1 equiv of triflic acid
(TfOH) in CH2Cl2 at À35 °C to give disaccharide 12 in 43% yield.
The levulinate (Lev) protecting group in 9 seems to be critical for
increasing the yield. Replacement of the levulinoyl group with
the benzoyl group resulted in a substantial decrease in product
yield. Selective deprotection of the Lev ester in 12 by treatment
with hydrazine hydrate in an acetic acid/pyridine mixture afforded
building block 4 in 98% yield. Likewise, building block 5 was pre-
pared from glycosylation of 8 (RRV = 4000) and 9 in a comparable
yield. The synthesis of 11 was started from the known glycosyl bro-
mide 14.23 Treatment of 4 with azido alcohol 15 in the presence of
iodine and DDQ24 gave 16 exclusively in 71% yield. Deacetylation
of 16 followed by benzylidene formation between C4–OH and
C6–OH afforded 17 (91% for two steps). Compound 17 was con-
verted into 11 by a three step sequence: protecting C3–OH in 17
as Lev ester, followed by reductive benzylidene ring cleavage and
selective Lev deprotection.
Next, we proceeded to the assembly of trisaccharide building
block 18 via a one-pot sequential glycosylation (Scheme 3). To take
advantage of the reactivity difference between C3–OH and C4–OH
in building block 1125, thiogalactoside 10 was chosen to be the first
glycosyl donor in order to introduce the galactose moiety in a de-
sired regioselective manner. In a separate experiment, treatment of
11 with glycosyl donor 10 in the presence of NIS and a catalytic
amount of TfOH in CH2Cl2 at À40 °C gave two regioisomers in a ra-
tio of 10–1, in which the C4-isomer was obtained as the major
product. Encouraged by these results, we further investigated the
one-pot synthesis of 18. Building block 11 (1.0 equiv) was coupled
From the structural point of view, the subtle difference between
the epitope structure of dimeric LewisX (1) and KH-1 (2) is that the
latter has an extra L-fucose with an a(1?2) linkage to the non-
reducing end galactose moiety (the LewisY epitope). Accordingly,
we designed a convergent synthetic approach utilizing a reactiv-
ity-based one-pot methodology (Scheme 1), in which 1 could be
derived from the building blocks 3,22, 4,18 and 6 by one-pot glyco-
sylation. In a similar manner, 2 could be prepared from the corre-
sponding building blocks 3, 5,18 and 6. The disaccharides 4 and 5
could be prepared from thiogalatosides 722 and 8,22 respectively,
with 9.22 The reducing end trisaccharide building block 6 in turn
⇑
Corresponding authors.
E-mail addresses: cyiwu@gate.sinica.edu.tw (C.-Y. Wu), chwong@gate.sinica.
edu.tw (C.-H. Wong).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.11.055

B.-L. Tsai et al. / Tetrahedron Letters 52 (2011) 2132–2135
2133
HO
HO
OH
O
HO
O
OH
O
OH
O
OH
O
O
O
O
OR1
O
O
NHAc
OH
NHAc
O
OH
O
OH
OH
HO
NH2
OH
HO
R
R
1 = H
1
2
1 = α(1-2)-L-Fucose
Ph
O
O
OBn
O
BnO
BnO
OBn
O
OBn
O
O
STol
OBn
O
O
STol
HO
O
O
HO
OR2
NHTroc
O
OBn
BnO
OH
NHTroc
O
OBn
R
2 = Bz
R2 = H
3
4
5
6
OBn
N3
BnO
Ph
O
OBn
O
BnO
BnO
OBn
O
OBn
O
O
O
HO
STol
HO
HO
STol
OBn
O
O
STol
LevO
STol
OLev
LevO
OR3
R3 = Bz
NHTroc
OBn
BnO
NHTroc
7
8
9
3
10
11
N3
R3 = Lev
Scheme 1. Retro-synthetic analysis of Lex dimer 1 and KH-1 2.
OBn
O
BnO OBn
O
BnO
OBn
O
OBn
O
+
STol
OR3
7 R3 = Bz
8 R3 = Lev
HO
STol
NIS / TfOH
O
BnO
STol
BnO
LevO
LevO
R3O
NHTroc
CH2Cl2, AW-300,
NHTroc
12 R3 = Bz
13 R3 = Lev
-45 oC to -35 o
41-43%
C
9
BnO
BnO
OBn
O
OBn
O
Hydrazine hydrate/AcOH/pyridine
98%
O
STol
NHTroc
HO
OR2
4 R2 = Bz
5 R2 = H
15
OAc
O
OAc
O
HO
N3
I2, DDQ
AcO
AcO
1) NaOMe, MeOH
AcO
AcO
O
N3
TrocHN
CH3CN, AW-300, r.t
71%
2) benzaldehyde
dimethyl acetal
TrocHN
Br
16
14
CSA, CH3CN, 40 o
C
91% two steps
Ph
O
BnO
HO
HO
O
O
O
1) Levulinic acid, EDCI, DMAP (88%)
2) Triethylsilane, TFA,TFAA (89%)
O
N3
O
N3
HO
TrocHN
TrocHN
17
11
3) Hydrazine hydrate/AcOH/pyridine (87%)
Scheme 2. Synthesis of building blocks 4, 5 and 11.
with 10 (1.4 equiv) in the presence of NIS/TfOH (0.1 equiv) at
À40 °C. After the complete consumption of 11 was confirmed by
TLC, the temperature was cooled to À50 °C, and perbenzylated
fucosyl building block 3 (1.2 equiv) was added, followed by the
addition of NIS/TfOH. After the complete consumption of 3 was
confirmed by TLC analysis, trisaccharide 18 was obtained in 40–
47% yield after standard work up and purification and no regio-
isomer of 18 was detected. Building block 6 was then prepared
by selective Lev deprotection of 18 under the conditions of hydra-
zine hydrate in an acetic acid/pyridine mixture.

2134
B.-L. Tsai et al. / Tetrahedron Letters 52 (2011) 2132–2135
BnO OBn
O
OBn
O
OBn
O
O
STol
HO
HO
O
BnO
HO
OH
5
NHTroc
RRV = 12000
NHTroc
11
Ph
O
(a)
(a)
N3
O
OBn
O
O
HO
O
O
Ph
O
O
STol
OBn
OH
O
yield: 36.2%
NHTroc
O
(b)
O
yield: 40~47%
OBn
BnO
O
STol
OBn
OBn
O
3
OBn
BnO
N3
OBn
BnO
LevO
STol
6
RRV = 0
3
OLev
(b)
10
RRV = 72000
BnO
OBn
78%
OBn
Ph
O
O
Ph
O
OBn
O
OBn
O
O
O
O
OBn
O
O
O
BnO
BnO
O
O
LevO
O
O
O
O
O
O
Hydrazine hydrate
O
O
OLev
NHTroc
OH
OBn
6
NHTroc
NHTroc
O
AcOH, Pyridine
89%
OBn
O
O
OBn
OBn
OBn
BnO
N3
OBn
BnO
OBn
BnO
N3
20
18
Scheme 5. One-pot synthesis of fully protective KH-1 epitope.
Scheme 3. One-pot synthesis of trisaccharide building block 18.
With all the desired building blocks in hand, we examined the
feasibility of one-pot synthesis of compounds 1 and 2. First, the
reaction of glycosyl donor 3 with acceptor 4 and 5, respectively,
was performed under various conditions in order to optimize the
stereochemical outcome. It was found that the best result was ob-
tained when the reaction was carried out in CH2Cl2 at À45 °C and
activated by NIS/TfOH. The reaction temperature seems to be cru-
cial for the success of this reaction; little products with poor ano-
meric selectivity and by-products derived from succinimde were
observed when the reaction was carried out at lower temperature,
such as À78 °C.
saccharide 19, a precursor of 1 in a [1+2+3] fashion (Scheme 4).
Perbenzylated fucosyl building block 3 (1.1 equiv, RRV = 7.2 Â 104)
was coupled with the less reactive disaccharide building block 4
(1.0 equiv, RRV = 5890) in the presence of NIS/TfOH at À45 °C.
After the complete consumption of 3 was confirmed by TLC analy-
sis, the reaction temperature was raised to À35 °C, and trisaccha-
ride acceptor 6 (0.8 equiv, RRV = 0) was added, followed by the
addition of NIS/TfOH. The second glycosylation was generally com-
pleted in 1 h at À35 °C as demonstrated by TLC analysis. The reac-
tion was then worked up by the standard procedures to provide 19
in 41% yield (based on 6) after flash silica gel chromatography (EA/
Hexane = 1:2). Following the same reaction sequence, heptasac-
charide 20, a precursor of 2 was prepared in a [1 Â 2+2+3] fashion
from corresponding building blocks 3 (2.2 equiv, RRV = 7.2 Â 104),
5 (1.0 equiv, RRV = 1.2 Â 104), and 6 (0.6 equiv, RRV = 0) in 36%
yield (based on 6) (Scheme 5).
For the reducing end trisaccharide 6, there was a distinct accep-
tor reactivity bias where C3–OH>>C2–OH was speculated for the
reason of their steric accessibility.9 With these preliminary results
in hand, we turned our attention to the one-pot synthesis of hexa-
To complete the synthesis, compounds 19 and 20 were sub-
jected to global deprotection (Scheme 6). The process started with
the removal of the Acyl and Troc protecting groups by alkali hydro-
lysis (1 N NaOH in THF at 50 °C for 24 h), followed by reacetylation
of the exposed hydroxyl and amine functionalities with acetic
anhydride in pyridine, and O-deacetylation with NaOMe in meth-
anol to provide intermediate 21 and 22, respectively, in 56–62%
yield. The rest of the protecting groups in 21 and 22 were removed
by catalytic hydrogenolysis (H2, 20% Pd(OH)2/C, in MeOH/H2O/
AcOH) to afford the target molecules 1 and 2, respectively, in
61–80% yield.
This approach extensively utilizes the diol functionality of the
building blocks by introducing two of the same carbohydrate
units simultaneously or taking the advantages of the distinct
reactivity order of these hydroxyl groups for attachment of
two different carbohydrates in a regioselective manner. Thus,
the protection–deprotection steps are minimized, and we pres-
ent a simple, elegant, convergent, and highly efficient reactiv-
ity-based programmable one-pot synthesis of LeX dimer 1 and
KH-1 2 epitopes.
OBn
O
BnO
BnO
OBn
O
O
STol
HO
OBz
4
NHTroc
RRV = 5890
(a)
Ph
O
O
OBn
O
O
HO
O
O
O
OH
yield: 41.1%
NHTroc
(b)
O
O
STol
OBn
OBn
N3
OBn
BnO
OBn
BnO
6
RRV = 0
3
RRV = 72000
Ph
O
BnO
BnO
OBn
O
OBn
O
O
OBn
O
O
O
O
O
O
O
O
OBz
NHTroc
OH
NHTroc
O
OBn
O
OBn
19
OBn
BnO
N3
OBn
BnO
Acknowledgment
Scheme 4. One-pot synthesis of fully protective Lex dimer epitope.
This work was supported by Academia Sinica, Taiwan.

B.-L. Tsai et al. / Tetrahedron Letters 52 (2011) 2132–2135
2135
Ph
O
BnO
BnO
OBn
O
OBn
O
O
OBn
O
1) 1N NaOH, THF, 50 o
2) Ac2O, Pyridine
3) NaOMe, MeOH
56-62%
C
O
O
O
O
O
OR4
O
O
NHTroc
OH
NHTroc
O
OBn
O
OBn
OBn
BnO
Ph
O
N3
OBn
BnO
4
BnO
BnO
OBn
O
19
20
R
= Bz
OBn
O
OBn
O
4
R
= α(1-2)-L-perbenzylated Fucosyl
O
O
O
O
O
O
OR5
O
O
NHAc
OH
NHAc
O
OBn
O
H2, 20% Pd(OH)2/carbon
MeOH/H2O/HOAc
OBn
OBn
BnO
N3
OBn
BnO
4
21
22
R
= OH
HO
HO
OH
O
61-80%
HO
O
OH
OH
O
4
OH
R
= α(1-2)-L-perbenzylated Fucosyl
O
O
O
O
O
OR1
O
O
NHAc
OH
NHAc
O
OH
O
OH
OH
HO
NH2
OH
HO
1 = H
1 = α(1-2)-L-Fucose
1
2
R
R
Scheme 6. Synthesis of Lex dimer and KH-1 epitopes.
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Supplementary data
Supplementary data associated with (full characterization of all
new compounds (1–2, 6, 19 and 20) and copies of NMR spectra)
this article can be found, in the online version, at doi:10.1016/
j.tetlet.2010.11.055.
16. Mong, K. K. T.; Wong, C. H. Angew. Chem., Int. Ed. 2002, 41, 4087–4090.
17. Mong, T. K. K.; Huang, C. Y.; Wong, C. H. J. Org. Chem. 2003, 68, 2135–
2142.
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