Synthesis of a dimeric Lewis antigen and the evaluation of the epitope specificity of antibodies elicited in mice

Article abstract of DOI:10.1002/chem.200500412

The Lewis^y-Lewis^x heptasaccharide, modified by an artificial aminopropyl spacer, was synthesized by an approach that employed two orthogonally protected lactosamine building blocks. A p-(benzoyl)-benzyl glycoside was used as a novel

Full text of DOI:10.1002/chem.200500412

FULL PAPER
Synthesis of a Dimeric Lewis Antigen and the Evaluation
of the Epitope Specificity of Antibodies Elicited in Mice
Therese Buskas,[a, b] Yanhong Li,[a, b] and Geert-Jan Boons*[a]
Abstract: The Lewisy–Lewisx heptasac-
charide, modified by an artificial ami-
nopropyl spacer, was synthesized by an
approach that employed two orthogo-
nally protected lactosamine building
blocks. A p-(benzoyl)-benzyl glycoside
was used as a novel anomeric protect-
ing group, which could be selectively
removed at a late stage in the synthesis,
thus offering the benefit of enhanced
flexibility. The artificial aminopropyl
moiety was modified by a thioacetyl
group, which allowed an efficient con-
jugation to keyhole limpet hemocyanin
(KLH)that had been activated with
electrophilic 3-(bromoacetamido)-pro-
pionyl groups. Mice were immunized
with the LeyLex–BrAc–KLH antigen.
Analysis of the sera by ELISA estab-
lished that
a strong helper T-cell
immune response was raised against
the LeyLex saccharide. Further ELISA
analysis showed that the titer for mon-
omeric Ley tetrasaccharide was tenfold
lower whereas recognition of the Lex
trisaccharide was negligible.
Keywords: antigens
agents glycoconjugates
oligosaccharides · vaccines
·
antitumor
·
·
Introduction
“minimal residual disease”. In this respect, they could target
a small number of metastasized cells that may have persist-
ed after primary therapies such as surgery or chemotherapy.
This add-on immune therapy could protect cancer patients
against a relapse and, thus, enhancing survival rates.
The extreme heterogeneity of cell surface glycosylation
makes the isolation of tumor associated carbohydrate anti-
gens in well-defined forms an almost impossible task, thus,
presenting a major obstacle for the development of cancer
vaccines. This obstacle is being addressed by synthetic or-
ganic chemistry, which can provide homogeneous oligosac-
charide antigens of high purity in relatively large amounts.
However, despite recent advances in the organic synthesis
of oligosaccharides, the preparation of these large complex
antigens is by no means a trivial matter.
Another obstacle for pursuing cancer vaccines is that
tumor-associated antigens are auto-antigens and, thus, are
being tolerated by the immune system. The difficult ques-
tion thus posed is how to trick the immune system to induce
a response to these tumor-associated antigens. The inherent-
ly T-cell independent nature of oligosaccharides is an addi-
tional problem that complicates carbohydrate-based cancer
vaccine development. The inability of carbohydrates to acti-
vate T-cells results in formation of exclusively low affinity
IgM antibodies and a lack of immunological memory.[6–10]
The activation of both B-cells and T-cells and their interac-
tion with one another is necessary for an effective immuno-
logical reaction.[9,11,12] The helper T-cells are in essence the
In the event of cell malignancies, dramatic changes occur in
the nature and abundance of protein- and lipid-linked cell
surface oligosaccharides. This abnormal glycosylation is as-
sociated with tumor progression and strongly correlates with
poor survival rates. Prominent tumor associated antigens
are, for example, the Lewis antigens, Lewisy (Ley)and sialyl-
Lewisx (SLex), and KH-1. In the majority of carcinomas in-
cluding those of the breast, ovary, pancreas, prostate and
colon,[1] Ley is overexpressed. It has been established that
Ley and SLex promote metastasis by binding to endothelial
cell-surface proteins.[2,3]
The carbohydrate antigens expressed by tumor cells offer
a unique opportunity for the development of anticancer vac-
cines. Immunization with a tumor-associated antigens may
induce an immune response that is directed towards cancer
cells.[4,5] The opsonizing or cytotoxic antibodies raised in
such a response may be exploited for the treatment of a
[a] Dr. T. Buskas, Dr. Y. Li, Prof. Dr. G.-J. Boons
Complex Carbohydrate Research Center
315 Riverbend Road, Athens GA 30602-4712 (USA)
Fax : (+1)706-542-4412
E-mail: gjboons@ccrc.uga.edu
[b] Dr. T. Buskas, Dr. Y. Li
These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
Chem. Eur. J. 2005, 11, 5457 – 5467
DOI: 10.1002/chem.200500412
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5457

orchestrators of the immune response and direct the activa-
tion of cytotoxic T-cells and the antibody producing B-cells.
Fortunately, the T-cell independence and B-cell tolerance
can be overcome by conjugation of tumor-associated oligo-
saccharides to a carrier protein, such as KLH or BSA.[13,14]
In elegant studies, Danishefsky, Livingston and co-workers
have utilized this approach to develop experimental carbo-
hydrate based anticancer vaccines.[14] For example, immuni-
zations of mice with a conjugate of the tetrasaccharide
Lewisy to the carrier protein KLH in combination with the
immuno-adjuvant QS-21, resulted in good titers of both IgM
and IgG antibodies.[15] Encouraged by these results a phase I
clinical trial with patients with documented ovarian, fallopi-
an tube, or peritoneal cancer[16] was conducted. Although
the vaccine did not induce adverse effects related to auto-
immunity, the immunizations failed to induce sufficiently
strong helper T-cell responses. Obviously, there is a need for
alternative strategies for the development of vaccine candi-
dates useful for immunotherapy against cancer.
saccharide equipped with an artificial linker for selective
conjugation to a carrier protein KLH. IgM and IgG class an-
tibodies were raised when mice were immunized with a
KLH conjugate. Investigation of the epitope specificity of
the antibodies showed that they recognized the Ley–Lex as
well as the Ley antigen. However, reactivity with the Ley an-
tigen was of a much lower titer. No reactivity with Lex was
observed.
Results and Discussion
Synthesis: We required substantial quantities of the synthet-
ic Lewisy–Lewisx oligosaccharide.[21–24] Also, a reference gly-
coconjugate of the saccharide linked to a carrier protein was
needed. Furthermore, we wanted to investigate the cross-re-
activity of antibodies raised against a particular Lewis anti-
gen. Thus, we required a flexible strategy for the prepara-
tion of Lewis antigens.
As part of a program to develop fully synthetic anticancer
vaccines, we recently reported[17] a solution-phase synthesis
of the tetrasaccharide Ley. This saccharide was coupled to
the protein carrier KLH using several different linkers. The
objective was to investigate the influence of the linker on
the immunogenicity of the tetrasaccharide. It was found that
a highly immunogenic linker such as 4-(maleimidomethyl)-
cyclohexane-1-carboxylate, dramatically reduced titers of
antibodies against the weakly immunogenic Ley tetrasac-
charide antigen. The use of a less immunogenic linker such
as 3-(bromoacetamido)propionate improved the immuno-
logical response considerably.
Strategic planning is of highest importance when design-
ing a synthetic route for complex oligosaccharide. The
regio- and stereoselective outcome of glycosylations must be
taken in consideration and are in essence influenced by the
protection group pattern of the glycosyl donors and accept-
ors. In this respect, a straightforward synthesis that requires
minimal protecting group manipulations of expensive build-
ing blocks is highly desirable.
It was envisaged that the synthesis of the target heptasac-
charide 31 would require only one orthogonally protected
lactosamine building block 1 (Figure 2), which could be uti-
lized for the synthesis of both the Lex acceptor and the Ley
donor. Selective deprotection of the orthogonal protecting
groups Lev and Fmoc[25,26] of 1 would allow mono- or difu-
cosylation with 6 to obtain either properly protected Lex or
Ley. The use of the trichloroethyloxycarbonyl (Troc)group
for protection of the amino functionality would ensure com-
patibility with the removal of the Lev and Fmoc group.[27]
The implementation of the novel temporary anomeric pro-
tecting group, p-(benzoyl)benzyl, which can be removed in a
two-step fashion by using hydrogen peroxide followed by
DDQ oxidation, would allow transformation into a glycosyl
donor at a late stage in the synthesis and thus greatly en-
hance the flexibility of the synthesis. Furthermore, a silyl
protecting group at 3’-OH could be removed to furnish a
Lex acceptor, which can then be coupled with a Ley donor
providing a straightforward synthesis of heptasaccharide
Ley–Lex 31. However, attempts to implement this strategy
failed, due to an inability to difucosylate a 3,2’-diol lactosa-
mine building block obtained by removal of the Lev and
Fmoc group of 1. Probably the steric hindrance by the 3’-O-
TBDPS protecting group obstructs successful glycosylation.
Unfortunately, efforts were unsuccessful to vary the bulki-
ness of the silyl protecting group (2 and 3)without affecting
its desired stability during protecting group manipulations
and glycosylations. In addition, it was found to be difficult
to glycosylate the 3’-hydroxyl of a Lex acceptor that was car-
rying a Lev group at the 2’-position. It was thus decided to
To further investigate the ideal presentation of the Ley an-
tigen, we chose to pursue the presentation of Ley in a dimer-
ic form. Naturally occurring Lewis antigens exist not only as
positional isomers in monomeric forms, but also as homo-
and heterodimers. For example, the Ley–Lex heterodimer
(Figure 1)is part of the KH-1 antigen that was isolated from
human colonic adenocarcinoma cells.[18] This antigen has
only been found on the surface of adenocarcinomas cells,
and has never been isolated from normal colonic tissue,
thus, providing a highly specific marker for colon malignan-
cies.[19,20]
For the safe use of these antigens for active immunothera-
py, it is important to investigate the cross-reactivity of anti-
bodies raised against the KH-1 antigen with other Lewis an-
tigens, in particular Ley and Lex. In this paper, we report a
highly convergent chemical synthesis of the Ley–Lex hepta-
Figure 1. LeyLex heptasaccharide.
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Carbohydrates
FULL PAPER
abandon the initial strategy and to use the two differently
protected lactosamine derivatives, 4 and 5. As can be seen
in Figure 2, the 3’-O-silyl protecting group obstructing difu-
cosylation as to obtain a Ley derivative was replaced by a 3’-
O-benzyl ether (4). Previous, it was found that a lactosamine
building block carrying a 3’-O-benzyl ether could indeed be
difucosylated.[17] Building block 5 carrying a 2’-O-benzoyl in-
stead of a Lev group was expected to be a more appropriate
substrate for the preparation of a Lex acceptor.
Glucosamine acceptor 15, carrying the temporary anome-
ric protecting group p-(benzoyl)-benzyl was easily prepared
in high yield by coupling p-(benzoyl)-benzylalcohol with gly-
cosyl donor 14[25] by using N-iodosuccinimide/trimethylsilyl
triflate (NIS/TMSOTf)as the activator (Scheme 2.) Com-
Figure 2.
It was envisaged that key building blocks 4 and 5 could be
synthesized from monosaccharides 6, 13 and 15.
Galactose donor 13, carrying a silyl-protecting group at
C-3 and a 2-O-benzoate, was synthesized in a straightfor-
ward manner starting from tetraol 7 (Scheme 1). Selective
introduction of a butane diacetal[28] and benzylation of the
4- and 6-hydroxyl groups under standard conditions fol-
lowed by acetal cleavage proceeded smoothly to give diol 10
in good overall yield. Regioselective silylation by using di-
ethylisopropyl chloride in THF furnished the desired alcohol
11 in a yield of 78%. A small amount (15%)of the 2-O-
isomer 12 could easily be separated by silica gel column
chromatography, which could be desilyated and recycled. Fi-
nally, benzoylation of the C-2 hydroxyl gave thiogalactoside
Scheme 2. Synthesis of the Lex acceptor. a) p-benzoyl-benzyl alcohol,
NIS, TESOTf, CH2Cl2, 08C, 86%; b)CH 2Cl2/Et3N 5:1, 95%; c)NIS,
TESOTf, CH2Cl2, 08C, 74%; d)H 2O2, Et3N, THF, 80%; e)DDQ,
1
CH2Cl2/H2O 95:5, 81%; f)CCl 3CN, DBU, CH2Cl2, 90%; g) BF·Et2O,
13. The H-2 signal in H NMR spectrum of compound 13
3
CH2Cl2, 86%; h)TBAF, HOAc, THF, 82%.
was shifted down-field to 5.62 ppm, clearly demonstrating
the selectivity of the silylation and confirming the structure
of 13.
pound 15 could immediately be used as an acceptor in a gly-
cosylation with galactosyl donor 13 by using NIS/TMSOTf
as the promoter to give the first key lactosamine derivative
5 in a yield of 69%. Selective removal of the Fmoc group of
disaccharide 5 was easily achieved by treatment with 20%
triethylamine in dichloromethane to afford lactosyl acceptor
16 which was fucosylated with glycosyl donor 6[29] to afford
the fully protected Lex trisaccharide 17 in 74% yield. As de-
1
termined by the JH,H coupling (J=3.5 Hz), complete a-se-
Scheme 1. Synthesis of galactose donor 13. a)Butane-2,3-dione, HC-
(CH3)3, CSA, MeOH, reflux, 78%; b)NaH, BnBr, DMF, 85%; c)TFA/
H2O 9:1, 67%; d)DEIPSCl, imidazole, THF, 78%; e)BzCl, TEA,
DMAP, CH2Cl2, 89%.
lectivity was achieved in the glycosylation. The temporary
anomeric protecting group of 17 was then removed by a
two-step procedure without affecting any other protecting
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G.-J. Boons et al.
groups. Thus, cleavage of the phenolic benzoate using hy-
drogenperoxide in a mixture of triethylamine and THF li-
berated the p-hydroxyl 18, which was immediately subjected
to oxidation with DDQ to furnish hemiacetal 19. Conver-
sion of 19 into a trichloroacetimidate 20 was accomplished
using standard conditions[30] and the resulting 20 was glyco-
sylated with 3-[N-(benzyloxycarbonyl)amino]-propanol[31] in
the presence of boron trifluoride etherate to give the fully
protected spacer equipped Lex derivative 21 in a good over-
all yield. The 3’-O-DEIPS group (DEIPS: diethylisopropyl-
silyl)of 21 was easily removed using tetrabutylammonium
fluoride (TBAF)in THF buffered with acetic acid to give
the LewisX glycosyl acceptor 22 in a yield of 82%.
The preparation of the properly protected Lewisy donor
29 was accomplished by a similar reaction sequence as out-
lined for the synthesis LewisX acceptor 22. Thus, coupling of
galactosyl donor 23[27] with the C-3 hydroxyl of acceptor 15
gave lactosamine derivative 4 in 81% yield (Scheme 3). Re-
moval of the Fmoc group by using triethylamine in dichloro-
methane and subsequent treatment with hydrazine acetate
to remove the Lev group gave the 3,2’-diol 25. Difucosyla-
tion with glycosyl donor 6 afforded the fully protected tetra-
saccharide 26 in a yield of 86%. The fucosylations proceed-
1
ed with complete a-selectivity, as confirmed by JH,H cou-
plings (J=3.5 Hz). The phenolic ester of the anomeric pro-
tecting group was cleaved by treatment with hydrogenperox-
ide in the presence of triethylamine. The so-formed p-
hydroxybenzyl derivative 27 was oxidized with DDQ to
completely remove the temporary anomeric protecting
group to give 28. The hemiacetal 28 was converted into the
corresponding trichloroacetimidate 29 by using standard
conditions.
Scheme 3. Synthesis of the Ley donor. a)NIS, TESOTf, CH 2Cl2, 08C;
b)CH 2Cl2/Et3N 5:1, 95%; c)NH 2NH2-HOAc, MeOH, CH2Cl2, 87%;
d)H 2O2, Et3N, THF, 82%; e)DDQ, CH 2Cl2/H2O 95:5, 78%; f)CCl 3CN,
DBU, CH2Cl2, 91%.
The key glycosylation of the assembly of the fully protect-
ed heptasaccharide 30 involved a coupling of trisaccharide
acceptor 22 with tetrasaccharide donor 29 using tributylsilyl
triflate (TBSOTf)in dichloromethane at À308C. This glyco-
sylation afforded the fully protected LeyLex heptasaccharide
in a yield of 62%. The use of the common promoters[30]
such as TMSOTf or TESOTf resulted in lower yields of the
heptasaccharide (<40%). Also, the reaction temperature
was critical as it was observed that temperatures higher than
À208C gave inferior result. The heptasaccharide 30 was
then deprotected by a four-step procedure. First, the N-Troc
groups were converted into 2-acetamido-2-deoxy functional-
ities by treatment with nano-size zinc in acetic acid followed
by acetylation using acetic anhydride in pyridine. It was
found that saponification of the ester groups, in particular
the 2’-O-benzoate, required prolonged reaction times result-
ing in partial decomposition. However, improved overall
yields were achieved by performing hydrogenolysis of the
benzyl ethers prior to ester hydrolysis. Thus, catalytic hydro-
genolysis using Pd(OAc)2 to remove the benzyloxycarbonyl
moiety and the benzyl ethers followed by base mediated re-
moval of the O-acyl groups gave, after purification by Bio-
gel P2 size-exclusion column chromatography, target com-
pound 31 in an overall yield of 32%.
Preparation of carbohydrate–protein conjugates and immu-
nizations: For the immunological evaluation of heptasac-
charide 31 (Scheme 4)it was linked to a carrier protein,
KLH. In our previous studies,[17] activating KLH with a bro-
moacetyl linker and reacting it with thiolated oligosacchar-
ides had proven to result in glycoconjugates that gave an an-
tigen focused immune response with low titers of anti-linker
antibodies. To this end, the amino functionality of heptasac-
charide 31 was derivatized with an acetyl thioacetic acid
moiety by reaction with S-acetylthioglycolic acid pentafluor-
ophenyl ester to afford 32, which, after purification by size-
exclusion chromatography, was directly de-S-acetylated by
using 7% ammonia (g)in DMF just prior to conjugation.
The de-S-acylation was performed under a strict argon at-
mosphere to prevent formation of the corresponding disul-
fide. KLH was activated with succinimidyl 3-(bromoaceta-
mido)propionate (SBAP)in a sodium phosphate buffer
pH 7.2 containing 0.15m sodium chloride for 2 h and then
purified by centrifugal filters with a nominal molecular-
weight limit of 10 kDa. The bromoacetyl activated KLH was
subsequently incubated over night at room temperature
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Carbohydrates
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LeyLex conjugate raised significant IgG anti-LeyLex titers in-
dicating a proper helper T-cell response. It should be noted
that the epitope density of the glycoconjugate was high
(>1000), which may have facilitated the high titers of elicit-
ed IgG antibodies. Reports of immunizations with the KH-1
antigen show that not only the nature of the glycoconjugate,
but also the epitope density influences the immunological
response.[34] This notion is also supported by our own obser-
vations of immunizations with the Ley antigen[17] as well as
reports from other groups.[35,36]
An important aspect of active immunotherapy against
cancer is that the antibodies are specific for a particular an-
tigen. Due to the structural similarities of the Lewis anti-
gens, it is important to examine cross-reactivity with other
Lewis antigens. For example, an antibody raised against a di-
meric structure may only bind terminal saccharides and as a
result will cross react with a corresponding monomeric anti-
gen. On the other hand, an antibody raised against a dimeric
structure may recognize internal saccharides and hence be
more specific for dimeric Lewis antigens. In addition, large
oligosaccharides may have different conformational proper-
ties compared to smaller fragments and this may also affect
antibody selectivity.
Scheme 4. Synthesis of target molecule 28. a)NIS, TBSOTf, CH 2Cl2,
À308C, 62%; b)1)Zn, HOAc; 2)Ac 2O, pyridine; 3)Pd(OAc) 2, H2,
HOAc/EtOH 1:5, 4)NaOMe, MeOH, pH 10, 52% over four steps;
c)SAMA-OPfp, Et 3N, DMF.
The Ley antigen is expressed predominantly during em-
bryogenesis. In normal adult tissue expression of Ley and
Lex is mainly restricted to granulocytes and epithelial surfa-
ces.[37] The Lex antigen is, however, also expressed by neu-
trophils (PMNs).[38,39] Thus, the safe use of Lewis antigens in
vaccine development requires a detailed knowledge of the
cross-reactivity of a particular antibody with respect to
other Lewis antigens.
In order to investigate the cross-reactivity of the elicited
antibodies with other Lewis antigens, in particular Ley and
Lex, ELISA by using microtiters plates coated with Ley–
BSA and Lex–BSA was performed. As can be seen in
entry 2 of Table 1, the IgM and IgG antibodies do recognize
the Ley tetrasaccharide albeit with significantly lower titers
(ten-fold)compared with that of the heptasaccharide
LeyLex. The substantially lower reactivity clearly indicates
that the antibodies recognize an epitope spanning both the
Ley and Lex monomers. The titer of IgG antibodies against
the Lex monomer was very low (entry 3). This result is im-
portant but perhaps not surprising, since it is known that
when a large oligosaccharide is presented to the immune
system, the more accessible terminal ends become the major
epitope.[40] The finding of low cross-reactivity and thus high
specificity of the antibodies raised against the LeyLex dimer
is of great importance for the safe use of an anticancer vac-
cine, especially considering the distribution of Lex in a
healthy environment.
with thiolated heptasaccharide 32 in a 0.1mm sodium phos-
phate buffer pH 8.0 containing 0.1mm ethylenediamine tet-
raacetate (EDTA). The afforded glycoconjugate carried
1190 copies of LeyLex per KLH molecule as determined by
Lowryꢁs protein concentration test[32] and Duboisꢁ phenol
sulfuric acid assay.[33] For the ELISAs, the LeyLex–BSA,
Ley–BSA, and Lex–BSA conjugates were prepared using the
3-(bromoacetamido)propionate linker and procedures simi-
lar to the preparation of the LeyLex–KLH conjugate.
Groups of five mice were immunized with the LeyLex–
KLH conjugate together with the immunoadjuvant QS-21
(Antigenics Inc., Lexington, MA.). The mice received 24 mg
carbohydrate and 10 mg QS-21 in each boost. The immuniza-
tions were repeated three times at weekly intervals and sera
were collected seven days after the last boost. Titers of anti-
LeyLex antibodies were determined by ELISA by the addi-
tion of serial dilutions of sera to microtiter plates coating
with LeyLex–BSA. An anti-mouse IgM (m-chain specific)or
IgG (heavy chain specific)antibody labeled with alkaline
phosphatase was employed as a secondary antibody for de-
tection purposes. As can be seen in entry 1 of Table 1, the
Table 1. ELISA antibody titers[a] after four immunizations with LeyLex–
KLH.
Coating
IgM
IgG
Titers
Titers
1
2
3
LeyLex–BSA
Ley–BSA
1060
<120
n.d.
31 645
3115
500
Conclusion
Lex–BSA
[a] All titers are medians for a group of five mice. Titers were determined
by regression analysis, plotting log10 dilution vs absorbance. The titers
were calculated to be the highest dilution that gave three times the ab-
sorbance of normal saline mouse sera diluted 1:120.
We report here an efficient synthesis of the complex dimeric
Lewisy–Lewisx oligosaccharide based on two different or-
thogonally protected lactosamine building blocks. This ap-
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G.-J. Boons et al.
proach provided easy access to a Ley glycosyl donor and a
Lex glycosyl acceptor that could be coupled in one key gly-
cosylation to provide a hetero dimeric Lewis antigen. The
use of a p-(benzoyl)-benzyl glycoside as a temporary anome-
ric protecting group offered additional flexibility because it
was stable to conditions used to remove the Fmoc, Lev and
DEIPS protecting group but could be selectively cleaved at
a late stage in the synthesis without an adverse effect on the
complex structure. The anomeric aminopropyl spacer was
employed for selective conjugation to carrier proteins. Im-
munizations of the conjugate of LeyLex to KLH in combina-
tion with the immuno-adjuvant QS-21 evoked a strong
helper T-cell immune response. Further studies of antibody
cross-reactivity revealed that the antibodies recognized a
monomeric Ley tetrasaccharide, albeit with a tenfold lower
titer. This clearly demonstrated that the raised antibodies
have high specificity for the LeyLex heptasaccharide, indicat-
ing that the recognized epitope is spanning the two Lewis
antigen monomers. Importantly, it was determined that the
antibody recognition of the internal Lewisx trisaccharide was
very low. Thus, the results reported here supports the notion
that it may be possible to develop a tumor specific carbohy-
drate-based anticancer vaccine.
(dd, J=5.5, 11.8 Hz, 1H, H-6), 3.79 (dd, J=5.5, 11.8 Hz, 1H, H-6), 3.74
(dd, J=2.1, 9.6 Hz, 1H, H-3), 3.61 (t, J=5.5 Hz, 1H, H-5), 3.28 (s, 3H,
OCH3-BDA), 3.26 (s, 3H, OCH3-BDA), 2.75 (q, J=1.32, 2H, 7.42 Hz,
SCH2CH3), 1.32 (s, 3H, CH3-BDA), 1.31 (s, 3H, CH3-BDA), 1.30 ppm (t,
J=7.42 Hz, 3H, SCH2CH3); 13C NMR (100 MHz, CDCl3): d=100.51,
100.48 (2ꢂC-BDA), 83.4 (C-1), 78.9 (C-5), 71.9 (C-3), 68.2 (C-4), 66.3
(C-2), 62.3 (C-6), 48.3 (2ꢂC, OCH3-BDA), 24.7 (SCH2CH3), 17.9 (CH3-
BDA), 17.7 (CH3-BDA), 14.7 ppm (SCH2CH3); HR-MALDI-TOF MS:
m/z: calcd for C14H26O7S: 338.1399; found: 361.1307 [M+Na]+.
(2’R,3’R)-Ethyl 4,6-di-O-benzyl-2,3-O-(2’,3’-dimethoxybutane-2’,3’-diyl)-
1-thiol-b-d-galactopyranoside (9):
A mixture of compound 8 (5 g,
14.8 mmol)and sodium hydride (0.78 g, 32.5 mmol)in dry N,N-dimethyl-
formamide (30 mL)was cooled to 0 8C with an ice-bath. Benzyl bromide
(3.87 mL, 32.5 mmol)was added dropwise, the ice-bath was removed and
the reaction mixture was stirred at ambient temperature for 2 h after
which, methanol (3 mL)was added to quench the reaction. The solvent
was removed under reduced pressure and the residue was purified by
column chromatography (silica gel, hexane/EtOAc 2:3)to give com-
pound 9 (6.5 g, 85%)as a white foam. [ a]D =147.1 (c = 1.0 in CHCl3);
Rf =0.88 (hexane/EtOAc 2:3); 1H NMR (300 MHz, CDCl3): d=7.42–7.26
(m, 10H, Ar-H), 4.96 (d, J=11.6 Hz, 1H, ArCH2), 4.59 (d, J=11.6 Hz,
1H, ArCH2), 4.48 (q, J=11.8, 8.0 Hz, 2H, ArCH2), 4.26 (d, J=9.4 Hz,
1H, H-1), 4.07 (t, J=9.4 Hz, 1H, H-2), 3.81 (m, 2H, H-6), 3.75 (d, J=
2.1 Hz, 1H, H-3), 3.68–3.63 (m, 2H, H-4, H-5), 3.30 (s, 3H, OCH3-BDA),
3.27 (s, 3H, OCH3-BDA), 2.78–2.66 (m, 2H, SCH2CH3), 1.29 (t, J=
7.4 Hz, 3H, SCH2CH3), 1.28 (s, 3H, CH3-BDA), 1.27 ppm (s, 3H, CH3-
BDA); 13C NMR (CDCl3, 100 MHz): d=139.2–127.5 (12C, Ar-C), 100.2,
100.0 (2ꢂC-BDA), 83.6 (C-1), 78.2 (C-4), 74.2 (C-3), 73.9 (ArCH2), 73.8
(ArCH2), 73.5 (C-5), 69.2 (C-6), 67.0 (C-2), 48.2 (2ꢂC, OCH3-BDA),
24.7 (SCH2CH3), 17.9 (CH3-BDA), 17.7 (CH3-BDA), 15.3 ppm
(SCH2CH3); HR-MALDI-TOF MS: m/z: calcd for C28H38O7S: 518.2338;
found: 541.2315 [M+Na]+.
Experimental Section
General methods: Succinimidyl 3-(bromoacetamido)propionate (SBAP),
sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
(sulfo-SMCC), keyhole limpet hemocyanin (KLH) and bovine serum al-
bumin (BSA-MI)were purchased from Pierce Endogen, Rockford, IL.
BSA was purchased from Sigma. NIS was purchased from Fluka and re-
crystallized from dioxane/CCl4. All other chemicals were purchased from
Aldrich, Acros, and Fluka and used without further purification. Molecu-
lar sieves were activated at 1458C for 10 h. All solvents employed were
of reagent grade and dried by refluxing over appropriate drying agents.
TLC was performed by using Kieselgel 60 F254 (Merck)glass/alumiuni-
um/plastic plates, with detection by UV light (254 nm)and/or by charring
with 8% sulfuric acid in ethanol. Column chromatography was per-
formed on silica gel (Merck, mesh 70–230). Extracts were concentrated
under reduced pressure at ꢀ 408C (water bath). 1H and 13C NMR spec-
tra were recorded on a Varian Inova300 spectrometer, a Varian Inova500
spectrometer and a Varian Inova800 spectrometer equipped with Sun
workstations. 1H spectra recorded in CDCl3 were referenced to residue
CHCl3 at 7.26 ppm or TMS, and 13C spectra to the central peak of CDCl3
at 77.0 ppm. Assignments were made using standard 1D and gCOSY,
gHSQC and TOCSY 2D experiments. Positive ion matrix assisted laser
desorption ionization time of flight (MALDI-TOF)mass spectra were re-
corded using an HP-MALDI instrument by using gentisic acid as a
matrix. Centrifugal filter devices were purchased from Millipore Inc. The
immunoadjuvant QS-21 was a gift from Antigenics Inc., Lexington MA.
ELISA plates Immulon II Hb was purchased from Fisher Scientific Inc.
Ethyl 4,6-di-O-benzyl-1-thiol-b-d-galactopyranoside (10): A mixture of 9
(5 g, 9.65 mmol)and TFA/H 2O (200 mL, 9:1)was stirred for 2 min, the
solvent removed under reduced pressure and the remaining solid was pu-
rified by column chromatography (silica gel, hexane/EtOAc 2:3)to pro-
vide diol 10 (4.2 g, 10.4 mmol, 67%)as a white foam. [ a]D =147.1 (c =
1.0 in CHCl3); Rf =0.40 (hexane/EtOAc 2:3); 1H NMR (300 MHz,
CDCl3): d=7.36–7.26 (m, 10H, Ar-H), 4.71 (q, J=11.8, 12.4 Hz, 2H,
ArCH2), 4.48 (q, J=11.8, 8.0 Hz, 2H, ArCH2), 4.29 (d, J=9.4 Hz, 1H, H-
1), 3.90 (d, J=3.0 Hz, 1H, H-4), 3.74–3.58 (m, 5H, H-2, H-3, H-5, H-6),
2.78–2.66 (m, 2H, SCH2CH3), 1.29 ppm (t, J=7.4 Hz, 3H, SCH2CH3);
13C NMR (100 MHz, CDCl3): d=138.6–118.0 (12C, Ar-C), 86.4 (C-1),
77.8 (C-2), 76.4 (C-4), 75.6 (C-3), 75.4 (ArCH2), 73.8 (ArCH2), 71.1 (C-
5), 68.7 (C-6), 24.7 (SCH2CH3), 15.5 ppm (SCH2CH3); HR-MALDI-TOF
MS: m/z: calcd for C22H28O5S: 404.1657; found: 427.1620 [M+Na]+.
Ethyl 3-O-diethylisopropylsilyl-4,6-di-O-benzyl-1-thiol-b-d-galactopyra-
noside (11): DEIPSCl (2.63 mL, 9.90 mmol)was added dropwise to a so-
lution of diol 10 (4.0 g, 9.90 mmol)and imidazole (0.67 g, 100 mmol)in
dry THF (5 mL). After stirring for 4 h at room temperature, methanol
(2 mL)was added to quench the reaction. The mixture was diluted with
Et2O (30 mL), washed with water and dried over MgSO4 and concentrat-
ed. The residue was purified by column chromatography (silica gel,
hexane/EtOAc 9:1)to furnish silyl ether 11 (4.05 g, 78%)as a white
foam. [a]D =(c
= 1.0 in CHCl3); Rf =0.58 (hexane/EtOAc 11:2);
1H NMR (300 MHz, CDCl3): d=7.34–7.30 (m, 10H, Ar-H), 5.04 (d, J=
11.3 Hz, 1H, ArCH2), 4.58 (d, J=11.3 Hz, 1H, ArCH2), 4.25 (q, J=6.6,
9.8 Hz, 2H, ArCH2), 4.31 (d, J=9.4 Hz, 1H, H-1), 3.85 (dd, J=5.7,
2.0 Hz, 1H, H-3), 3.78 (d, J=2.0 Hz, 1H, H-4), 3.74 (dd, J=9.4, 5.7 Hz,
1H, H-2), 3.65 (m, 3H, H-5, H-6), 2.78 (m, 2H, SCH2CH3), 1.30 (t, J=
7.4 Hz, 3H, SCH2CH3), 1.05 (m, 12H, CH3-DEIPS), 0.75 ppm (m, 5H,
CH2, CH-DEIPS); 13C NMR (100 MHz, CDCl3): d=139.3–127.6 (12C,
Ar-C), 86.8 (C-1), 77.7 (C-3), 77.4 (C-6), 76.9 (C-2), 75.3 (CH2Ph), 73.8
(CH2Ph), 70.7 (C-5), 69.2 (C-6), 24.5 (SCH2CH3), 17.7 (SCH2CH3), 17.7,
15.6, 13.3, 7.5, 7.4 (5C, CH, CH3-DEIPS), 4.24 ppm (2C, CH2-DEIPS);
HR-MALDI-TOF MS: m/z: calcd for C29H44O5Ssi: 532.2679; found
555.2696 [M+Na]+.
(2’R,3’R)-Ethyl 2,3-O-(2’,3’-dimethoxybutane-2’,3’-diyl)-1-thiol-b-d-galac-
topyranoside (8): Galactoside 7 (14.7 g, 65.7 mmol), butane-2,3-dione
(6.9 mL, 78.8 mmol), trimethylorthoformate (23 mL, 197 mmol) and cam-
phorsulfonic acid (1.5 g, 6.5 mmol)in methanol (200 mL)were heated
under reflux for 16 h. The reaction mixture was cooled to room tempera-
ture and triethylamine (2 mL)was added to quench the reaction. After
the solution was concentrated to dryness, the residue was purified by
column chromatography (silica gel, hexane/EtOAc 2:3)to furnish diol
8
(17.2 g, 78%)as a white foam. [ a]D =147.1 (c = 1.0 in CHCl3); Rf =0.35
(hexane/EtOAc 2:3); 1H NMR (300 MHz, CDCl3): d=4.57 (d, J=9.6 Hz,
1H, H-1), 4.10 (t, J=9.6 Hz, 1H, H-2), 4.00 (d, J=2.1 Hz, 1H, H-4), 3.93
5462
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2005, 11, 5457 – 5467

Carbohydrates
FULL PAPER
Ethyl 2-O-benzoyl-3-O-diethylisopropylsilyl-4,6-di-O-benzyl-1-thiol-b-d-
galactopyranoside (13): Triethylamine (0.38 mL, 2.7 mmol), benzoylchlor-
ide (0.32 mL, 2.7 mmol), and DMAP (671 mg, 5.5 mmol) were added to a
solution of alcohol 11 (309 mg, 0.58 mmol), in dichloromethane (2 mL) at
room temperature. The mixture was stirred at room temperature for 20 h
after which it was poured into EtOAc (50 mL). The organic phase was
washed with 1m HCl, sat. aqueous sodium bicarbonate and water. The
organic phase was dried (MgSO4), filtered and concentrated in vacuo. Pu-
rification of the residue by column chromatography (silica gel, hexane/
EtOAc 2:3)yielded 13 (328 mg, 89%)as a white foam. [ a]D =(c = 1.0 in
CHCl3); Rf =0.48 (hexane/EtOAc 2:3); 1H NMR (300 MHz, CDCl3): d=
8.05 (d, J=7.2 Hz, 2H, Ar-H), 7.57–7.26 (m, 13H, Ar-H), 5.62 (t, J=
8.8 Hz, 1H, H-2), 5.04 (d, J=11.6 Hz, 1H, ArCH2), 4.86 (d, J=11.6 Hz,
1H, ArCH2), 4.25 (m, 2H, ArCH2), 4.20 (d, J=9.6 Hz, 1H, H-1), 4.01
(dd, J=5.8, 1.2 Hz, 1H, H-3), 3.83 (d, J=1.2 Hz, 1H, H-4), 3.65 (q, J=
3.2 Hz, 1H, H-5), 3.61 (m, 2H, H-6), 2.68 (m, 2H, SCH2CH3), 1.19 (t, J=
7.4 Hz, 3H, SCH2CH3), 0.95 (m, 12H, CH3-DEIPS), 0.54 ppm (m, 5H,
CH2, CH-DEIPS); 13C NMR (100 MHz, CDCl3): d=165.7 (COAr),
139.2–125.6 (18C, Ar-C), 84.0 (C-1), 77.9 (C-5), 77.8 (C-4), 76.1 (C-3),
75.5 (ArCH2), 73.8 (ArCH2), 71.5 (C-2), 68.9 (C-6), 23.7 (SCH2CH3),
17.5, 17.4, 15.1, 13.1 (4C, CH3-DEIPS), 17.7 (SCH2CH3), 7.3, 4.3, 4.0 ppm
(3C, CH2, CH-DEIPS); HR-MALDI-TOF MS: m/z: calcd for
C36H48O6SSi: 636.2941; found: 661.2965 [M+Na]+.
(t, J=9.4 Hz, 1H, H-3), 4.98 (d, J=11.6 Hz, 1H, Bn), 4.85 (d, J=
11.6 Hz, 1H, Bn), 4.68 (d, J=8.6 Hz, 1H, H-1), 4.60–4.31 (m, 8H, 2ꢂBn,
Troc, CH2PhOBz), 4.52 (d, J=8.9 Hz, 1H, H-1’), 4.35 (d, J=6.0 Hz, 2H,
Fmoc-CH2), 4.18 (t, J=6.0 Hz, 1H, Fmoc-CH), 4.10 (dd, J=5.9, 1.6 Hz,
1H, H-3’), 4.08–3.81 (m, 4H, H-2, H-4, H-6), 3.80 (d, J=1.6 Hz, 1H, H-
4’), 3.69–3.51 (m, 3H, H-5’, H-6’), 3.61 (d, J=9.1 Hz, 1H, H-5), 0.95 (m,
12H, CH3-DEIPS), 0.54 ppm (m, 5H, CH2, CH-DEIPS); 13C NMR
(CDCl3, 100 MHz): d=165.3 (COPh), 165.1 (ArCO), 154.9
(CHCH2OCO, Fmoc), 154.4 (Ar-C), 150.7 (NHCO), 143.7–12.2 (47C,
Ar-C), 101.4 (C-1’), 100.0 (C-1), 95.6 (CCl3), 77.2 (C-3’), 77.1 (C-4’), 75.7
(C-3), 75.5, 74.7, 73.8, 73.4, 73.3 (4C, 3ꢂCH2Ph, Troc, CH2PhOBz), 73.6
(2C, C-2’, C-4), 73.5 (C-5’), 70.2 (CH2-Fmoc), 68.2, 68.0 (2C, C-6, C-6’),
60.6 (C-2), 46.8 (CH-Fmoc), 17.5, 17.3, 14.5, 13.0 (4C, CH3-DEIPS), 7.3
(CH-DEIPS), 4.3, 4.0 ppm (2C, CH2-DEIPS); HR-MALDI-TOF MS m/
z: calcd for C79H86Cl3NO17Si: 1449.4418; found: 1472.4530 [M+Na]+.
p-(Benzoyl)-benzyl 6-O-benzyl-2-deoxy-2[[(2,2,2-trichloroethoxy)carbo-
nyl]amino]-4-O-(2-O-benzoyl-3-O-diethylisopropylsilyl-4,6-di-O-benzyl-
b-d-galactopyranosyl)-b-d-glucopyranoside (16): Compound 5 (100 mg,
0.07 mmol)was dissolved in dichloromethane (4 mL)and triethylamine
(1 mL)was added. The reaction mixture was stirred at room temperature
overnight and then concentrated under reduced pressure. The residue
was purified by column chromatography (silica gel, hexane/EtOAc 2:1)
to give 16 as a white powder (80 mg, 95%). [a]D =À26.4 (c
= 1.0,
p-(Benzoyl)-benzyl 6-O-benzyl-2-deoxy-2[[(2,2,2,-trichloroethoxy)carbo-
nyl]amino]-3-O-(9-fluorenylmethoxycarbonyl)-b-d-glucopyranoside (15):
A solution of thioglycoside 14 (4.77 g, 6.73 mmol)and p-benzoyl-benzyl
alcohol (3.07 g, 13.47 mmol)in dry dichloromethane (20 mL)was dried
azeotropically with toluene (Na-dried)and then subjected to high
vacuum for 2 h. The compounds were dissolved in dry dichloromethane
(30 mL)under argon atmosphere and the mixture was stirred at room
temperature in the presence of activated 3 ꢃ molecular sieves for 30 min.
The mixture was then cooled to 08C and treated with NIS (1.67 g,
7.41 mmol)and TESOTf (0.15 mL, 0.67 mmol). After stirring for 30 min
TLC showed full conversion of the donor. The solution was diluted by di-
chloromethane and the molecular sieves were removed by filtering
through Celite. The filtrate was washed with 15% aqueous sodium thio-
sulfate and brine, dried over MgSO4, filtered and concentrated. The resi-
due was purified by column chromatography (silica gel, hexane/EtOAc
2:1)to give the product 15 as a white powder (5.05 g, 86%). [a]D =À20.6
(c = 1.0, CH2Cl2); Rf =0.28 (hexane/EtOAc 2:1); 1H NMR (300 MHz,
CDCl3): d=8.20 (d, J=7.5 Hz, 2H, Ar-H), 7.76–7.09 (m, 20H, Ar-H),
5.49 (d, J=8.3 Hz, 1H, NH), 4.92 (d, J=8.8 Hz, 1H, H-1), 4.86 (t, J=
9.4 Hz, 1H, H-3), 4.66–4.54 (m, 6H, ArCH2, Troc), 4.38 (d, J=6.3 Hz,
2H, Fmoc-CH2), 4.22 (t, J=6.3 Hz, 1H, Fmoc-CH), 3.85–3.80 (m, 4H,
H-6, H-4, H-2), 3.65–3.52 ppm (m, 1H, H-5); 13C NMR (100 MHz,
CDCl3): d=165.6 (ArCO), 156.0 (CHCH2OCO, Fmoc), 154.7 (Ar-C),
150.6 (NHCO), 150.7–120.3 (29C, Ar-C), 99.2 (C-1), 95.9 (CCl3), 79.6 (C-
3), 74.7 (C-5), 74.5 (OCH2Cl3, Troc), 73.9 (OCH2PhOBz), 70.7 (C-4), 70.1
(OCH2Ph), 69.9 (CHCH2OCO, Fmoc), 64.8 (C-6), 56.1 (C-2), 46.4 ppm
(CHCH2OCO, Fmoc); HR-MALDI-TOF MS m/z: calcd for
C45H40Cl3NO11: 875.1667; found: 898.1659 [M+Na]+.
CH2Cl2); Rf =0.32 (hexane/EtOAc 2:1); 1H NMR (300 MHz, CDCl3): d=
8.21 (d, J=7.7 Hz, 2H, Ar-H), 8.07 (d, J=7.7 Hz, 2H, Ar-H), 7.67–7.03
(m, 25H, Ar-H), 5.60 (t, J=8.5 Hz, 1H, H-2’), 5.20 (d, J=9.0 Hz, 1H,
NH), 5.08 (d, J=11.6 Hz, 2H, ArCH2), 4.82–4.06 (m, 10H, 3ꢂArCH2,
CH2PhOBz, Troc), 4.38 (d, J=8.9 Hz, 1H, H-1), 4.28 (d, J=8.7 Hz, 1H,
H-1’), 4.08 (dd, J=1.6, 8.0 Hz, 1H, H-3’), 3.80 (d, J=2.0 Hz, 1H, H-4),
3.79–3.74 (m, 3H, H-2, H-6), 3.62 (m, 1H, H-3), 3.60 (d, J=1.6 Hz, 1H,
H-4’), 3.59–3.51 (m, 2H, H-5’, H-6’), 3.41–3.36 (m, 2H, H-5, H-6’), 0.98
(m, 12H, CH3-DEIPS), 0.64 ppm (m, 5H, CH2, CH-DEIPS); 13C NMR
(100 MHz, CDCl3): d=165.7 (COPh), 165.1 (ArCO), 154.4 (Ar-C), 150.7
(NHCO), 138.7–121.2 (35C, Ar-C), 101.7 (C-1’), 99.9 (C-1), 95.8 (CCl3),
81.0 (C-3’), 75.5 (C-4’), 74.7 (C-3), 74.8, 74.6, 72.6, 72.2 (4C, 3ꢂCH2Ph,
Troc, CH2PhOBz), 74.2 (C-2’), 73.3 (C-5’), 70.3 (C-4), 68.8, 68.6 (2C, C-6,
C-6’), 57.9 (C-2), 17.5, 17.4, 17.3, 14.4 (4C, CH3-DEIPS), 7.4 (CH-
DEIPS), 4.4, 4.0 ppm (2C, CH2-DEIPS); HR-MALDI-TOF MS: m/z:
calcd for C64H72Cl3NO15Si: 1227.3737; found: 1250.3685 [M+Na]+.
p-(Benzoyl)-benzyl 6-O-benzyl-2-deoxy-2[[(2,2,2-trichloroethoxy)carbo-
nyl]amino]-3-O-(3,4-di-O-acetyl-2-O-benzyl-a-l-fucopyranosyl)-4-O-(2-
O-benzoyl-3-O-diethylisopropylsilyl-4,6-di-O-benzyl-b-d-galactopyrano-
syl)-b-d-glucopyranoside (17):
A solution of the glycosyl donor 6
(53.5 mg, 0.14 mmol)and the glycosyl acceptor 16 (80 mg, 0.07 mmol)
was dissolved in dry dichloromethane (3 mL)and the mixture was stirred
at room temperature under argon in the presence of activated molecular
sieves for 30 min. The mixture was cooled to 08C and NIS (34.7 mg,
0.15 mmol)was added followed by TESOTf (3.3 mL, 0.015 mmol). After
the donor was fully converted, the reaction mixture was diluted with di-
chloromethane (50 mL)and the molecular sieves were filtered off
through a plug of Celite. The organic layer was washed with 15% aque-
ous sodium thiosulfate and brine, dried by MgSO4, filtered and concen-
trated under reduced pressure. Purification by column chromatography
(silica gel, hexane/EtOAc 2:1)furnished the fully protected trisaccharide
17 as a white powder (80 mg, 74%). [a]D =À42.6 (c = 1.0, CH2Cl2); Rf =
0.54 (hexane/EtOAc 2:1); 1H NMR (300 MHz, CDCl3): d=8.20 (d, J=
7.4 Hz, 2H, Ar-H), 8.07 (d, J=7.4 Hz, 2H, Ar-H), 7.66–7.05 (m, 30H,
Ar-H), 5.45 (t, J=8.5 Hz, 1H, H-2), 5.33 (d, J=8.9 Hz, 1H, N-H), 5.25
(dd, J=10.6, 3.3 Hz, 1H, H-3’’), 5.17 (d, J=3.6 Hz, 1-H, H-1’’), 5.14 (d,
J=3.3 Hz, 1H, H-4’’), 4.88 (q, J=6.0 Hz, 1-H, H-5’’), 4.84 (d, J=8.6 Hz,
1H, H-1), 4.76–4.37 (m, 12H, 4ꢂBn, Troc, CH2PhOBz), 4.62 (d, J=
9.0 Hz, 1H, H-1’), 4.10 (d, J=3.6 Hz, 1H, H-4’), 4.01 (t, J=9.0 Hz, 1H,
H-3), 3.85–3.67 (m, 6H, H-4, H-6a, H-3’, H-6’, H-2’’), 3.58 (d, J=8.5 Hz,
1H, H-6b), 3.44 (q, J=3.6 Hz, 1H, H-5’), 3.19–3.05 (m, 2H, H-5, H-2),
2.08 (s, 3H, COCH3), 2.00 (s, 3H, COCH3), 0.97 (d, J=6.0 Hz, 3H, H-
6’’), 0.95 (m, 12H, CH3-DEIPS), 0.54 ppm (m, 5H, CH2, CH-DEIPS);
13C NMR (100 MHz, CDCl3): d=170.6, 169.7 (2C, 2ꢂCH3CO), 165.3
(COPhOBz), 165.0 (ArCO), 153.7 (Ar-C), 150.6 (NHCO), 138.9–121.7
(41C, Ar-C), 100.1 (C-1), 99.0 (C-1), 97.5 (C-1’’), 95.6 (CCl3), 77.5 (C-3’),
p-(Benzoyl)-benzyl 6-O-benzyl-2-deoxy-2[[(2,2,2-trichloroethoxy)carbo-
nyl]amino]-3-O-(9-fluorenylmethoxycarbonyl)-4-O-(2-O-benzoyl-3-O-di-
ethylisopropylsilyl-4,6-di-O-benzyl-b-d-galactopyranosyl)-b-d-glucopyra-
noside (5): A solution of the glycosyl donor 13 (150 mg, 0.24 mmol)and
the glycosyl acceptor 15 (175 mg, 0.2 mmol)in dry dichloromethane
(2 mL)was stirred at room temperature under argon in the presence of
activated molecular sieves for 30 min. The mixture was then cooled to
08C and NIS (594 mg, 0.26 mmol)was added followed by TESOTf
(5.5 mL, 0.024 mmol). After 40 min, the reaction mixture was diluted with
dichloromethane (50 mL)and filtered through a plug of Celite. The fil-
trate was washed with 15% aqueous sodium thiosulfate and sat. aqueous
sodium bicarbonate. The organic layer was dried over MgSO4 and con-
centrated under reduced pressure. The residue was purified by column
chromatography (silica gel, hexane/EtOAc 2:1)to give the disaccharide 5
as a white powder (200 mg, 69%). [a]D =À28.9 (c = 1.0, CH2Cl2); Rf =
0.57 (hexane/EtOAc 2:1); 1H NMR (300 MHz, CDCl3): d=8.20 (d, J=
7.7 Hz, 2H, Ar-H), 8.05 (d, J=7.7 Hz, 2H, Ar-H), 7.77–7.13 (m, 33H,
Ar-H), 5.57 (t, J=8.8 Hz, 1H, H-2’), 5.19 (d, J=9.4 Hz, 1H, N-H), 5.01
Chem. Eur. J. 2005, 11, 5457 – 5467
www.chemeurj.org
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
5463

G.-J. Boons et al.
75.6 (C-5), 75.3 (C-4’), 75.1, 74.3, 73.3, 72.6, 70.7 (6C, 4ꢂCH2Ph, Troc,
CH2PhOBz), 74.6 (C-5’), 74.3 (C-3), 73.7 (C-2’’), 73.6 (C-2’), 73.5 (C-4),
73.4 (C-4’’), 70.7, 70.6 (2C, C-6, C-6’), 70.5 (C-3’’), 64.9 (C-5’’), 59.1 (C-2),
21.2, 21.0 (2C, 2ꢂCH3CO), 17.6 (C-6’’), 17.5, 17.4, 15.3, 13.0 (4C, CH3-
DEIPS), 7.3 (CH-DEIPS), 4.3, 4.0 ppm (2C, CH2-DEIPS); HR-MALDI-
TOF MS: m/z: calcd for C81H92Cl3NO21Si: 1547.4997; found: 1570.4900
[M+Na]+.
in THF (1m, 0.2 mL)were added. After stirring at room temperature for
2 d, the reaction mixture was diluted with EtOAc (50 mL)and washed
with water (10 mL), sat. aqueous sodium hydrogen carbonate and brine.
The organic phase was dried over MgSO4, filtered and then concentrated
in vacuo. The obtained residue was purified by column chromatography
(silica gel, hexane/EtOAc 1:1)to yield the alcohol 22 as a white powder
(30 mg, 82%). [a]D =À49.3 (c = 1.0, CH2Cl2); Rf =0.38 (hexane/EtOAc
2:1); 1H NMR (500 MHz, CDCl3): d=7.92 (d, J=7.8 Hz, 2H, Ar-H),
7.65–7.10 (m, 28H, Ar-H), 5.61 (d, J=9.0 Hz, 1H, N-H), 5.38 (t, J=
8.0 Hz, 1H, H-2), 5.28 (dd, J=9.6, 3.0 Hz, 1H, H-3’’), 5.18 (d, J=3.5 Hz,
1-H, H-1’’), 5.09 (d, J=3.1 Hz, 1H, H-4’’), 5.01 (s, 2H,
CH2NHCOOCH2Ph), 4.82 (q, J=6.5 Hz, 1-H, H-5’’), 4.59 (d, J=8.9 Hz,
1H, H-1), 4.76–4.37 (m, 10H, 4ꢂBn, Troc), 4.40 (d, J=9.2 Hz, 1H, H-1’),
4.10 (d, J=3.8 Hz, 1H, H-4’), 4.03 (m, 1H, H-3), 4.02–3.95 (m, 5H, H-4,
H-6’, H-3’, H-2’’), 3. 85 (m, 2H, OCH2CH2CH2N), 3.64 (d, J=3.8 Hz, 2H,
H-6), 3.58 (q, J=4.0 Hz, 1H, H-5’), 3.30 (m, 1H, OCH2CH2CH2aN),
3.19–3.10 (m, 3H, OCH2CH2CH2bN, H-5, H-2), 2.20 (s, 3H, COCH3),
2.00 (s, 3H, COCH3), 1.29 (s, 2H, OCH2CH2CH2N), 1.15 ppm (d, J=
6.5 Hz, 3H, H-6’’); 13C NMR (125 MHz, CDCl3): d=170.8, 169.8 (2C, 2ꢂ
CH3CO), 166.1 (ArCO), 156.7 (NHCOOCH2Ph), 154.0 (NHCO), 138.6–
127.9 (36C, Ar-C), 100.3 (C-1), 98.0 (C-1’), 95.7 (C-1’’), 95.4 (CCl3), 77.4
(C-3’), 76.4 (C-5), 75.9 (C-4’), 75.7, 74.8, 74.7, 72.5, 70.8 (6C, 4ꢂCH2Ph,
Troc, NHCOOCH2Ph), 74.3 (C-5’), 74.3 (C-3), 73.3 (C-2’’), 72.6 (C-2’),
73.3 (C-4), 72.6 (C-4’’), 71.7 (C-3’’), 67.8, 66.9 (2C, C-6, C-6’), 66.8
(OCH2CH2CH2NH), 65.0 (C-5’’), 59.4 (C-2), 37.8 (OCH2CH2CH2NH),
30.0 (OCH2CH2CH2NH), 21.2, 21.0 (2C, 2ꢂCH3CO), 15.8 ppm (C-6’’);
HR-MALDI-TOF MS: m/z: calcd for C71H79Cl3N2O21: 1400.4241; found:
1423.4268 [M+Na]+.
3-[(N-Benzyloxycarbonyl)amino]propyl 6-O-benzyl-2-deoxy-2[[(2,2,2-tri-
chloroethoxy)carbonyl]amino]-3-O-(3,4-di-O-acetyl-2-O-benzyl-a-l-fuco-
pyranosyl)-4-O-(2-O-benzoyl-3-O-diethylisopropylsilyl-4,6-di-O-benzyl-b-
d-galactopyranosyl)-b-d-glucopyranoside (21): Triethylamine (2.5 mL)
and H2O2 (30% in water, 0.13 mL)were added to a stirred solution of
compound 17 (80 mg, 0.05 mmol)in THF (5 mL). After stirring the reac-
tion mixture at room temperature for 30 min, it was concentrated under
reduced pressure. The obtained residue was purified by column chroma-
tography (silica gel, hexane/EtOAc 2:1)to give compound 18 (60 mg,
80%).
DDQ (9 mg, 0.04 mmol)was then added to a solution of 18 (60 mg,
0.04 mmol)in dichloromethane/water (4 mL, 95:5). The reaction mixture
was stirred in the dark at room temperature for 1 h, diluted with di-
chloromethane (50 mL)and was washed with aqueous sodium hydrogen
carbonate and brine. The organic phase was dried (MgSO4), filtered and
concentrated to dryness. Purification by column chromatography (silica
gel, hexane/EtOAc 3:2)furnished hemiacetal 19 (45 mg, 81%).
A stirred solution of 19 in dichloromethane (5 mL)was treated with tri-
chloroacetonitrile (0.5 mL)and DBU (5 mL). After stirring for 5 min
under argon atmosphere, the solution was concentrated to dryness and
the residue was purified by column chromatography (silica gel, hexane/
EtOAc/triethylamine 1:1:0.01)to give the imidate 20 as a colorless syrup
(45 mg, 90%).
p-(Benzoyl)-benzyl 6-O-benzyl-2-deoxy-2[[(2,2,2-trichloroethoxy)carbo-
nyl]amino]-3-O-(9-fluorenylmethoxycarbonyl)-4-O-(3,4,6-tri-O-benzyl-2-
O-levulinoyl-b-d-galactopyranosyl)-b-d-glucopyranoside (4): A solution
of compound 15 (4.0 g, 4.57 mmol)and donor 23 (4.06 g, 6.86 mmol)in
dry dichloromethane (20 mL)was dried azeotropically with toluene (Na-
dried)and then subjected to high vacuum for 2 h. The compounds were
dissolved in dry dichloromethane (25 mL)and the mixture was stirred at
room temperature under argon in the presence of activated 3 ꢃ molecu-
lar sieves for 30 min. The mixture was cooled to 08C and reacted with
NIS (1.69 g, 7.54 mmol)and TESOTf (0.16 mL, 0.69 mmol). After 30 min
the solution was diluted by dichloromethane and the molecular sieves
were removed by filtering through Celite. The filtrate was washed with
15% aqueous sodium thiosulfate and brine, dried over MgSO4, filtered,
and concentrated. The residue was purified by column chromatography
(silica gel, hexane/EtOAc 2:1)to give the disaccharide 4 as a white
powder (5.19 g, 81%). [a]D =À64.2 (c = 1.0, CH2Cl2); Rf =0.38 (hexane/
EtOAc 2:1); 1H NMR (300 MHz, CDCl3): d=8.20 (d, J=8.4 Hz, 2H, Ar-
H), 7.75–7.12 (m, 35H, Ar-H), 5.34 (t, J=9.2 Hz, 1H, H-2), 5.18 (d, J=
9.8 Hz, 1H, N-H), 4.96 (t, J=9.4 Hz, 1H, H-3), 4.88 (d, J=12.0 Hz, 1H,
ArCH2), 4.85 (d, J=12.0 Hz, 1H, ArCH2), 4.61 (d, J=8.2 Hz, 1H, H-1),
4.60–4.30 (m, 10H, 3ꢂArCH2, Troc, CH2PhOBz), 4.44 (d, J=7.7 Hz, 1H,
H-1’), 4.28 (d, J=6.8 Hz, 2H, Fmoc-CH2), 4.12 (t, J=6.0 Hz, 1H, Fmoc-
CH), 4.02–3.71 (m, 4H, H-4, H-4’, H-6), 3.60–3.51 (m, 3H, H-2, H-5, H-
6a’), 3.69–3.51 (m, 2H, H-5’, H-6b’), 3.44 (dd, J=9.2, 1.6 Hz, 1H, H-3’),
3.36 (d, J=9.1 Hz, 1H, H-5’), 2.69–2.36 (m, 4H, CH2CH2, Lev), 2.13 ppm
(s, 3H, COCH3, Lev); 13C NMR (100 MHz, CDCl3): d=206.6
(CH3COCH2, Lev), 171.5 (OCOCH2CH2, Lev), 165.3 (ArCO), 155.0
(CHCH2CO, Fmoc), 154.3 (Ac-C), 150.7 (NHCO), 146.0–120.1 (47C, Ar-
C), 100.5 (C-1’), 100.0 (C-1), 95.6 (CCl3), 80.5 (C-5’), 77.3 (C-3), 75.4 (C-
4), 75.0 (C-5), 74.7, 73.9, 72.0 (6C, 4ꢂOCH2Ph, OCH2CCl3, OCH2-
PhOBz), 73.5 (C-3’), 72.6 (C-4’), 72.1 (C-2’), 70.2 (CHCH2CO, Fmoc),
68.2, 67.9 (2C, C-6, C-6’), 56.5 (C-2), 46.8 (CHCH2CO, Fmoc), 38.0
(OCOCH2CH2, Lev), 30.1 (CH2COCH3, Lev), 28.1 ppm (OCOCH2CH2,
Compound 20 and 3-[(N-benzyloxycarbonyl)amino]propanol (11 mg,
0.06 mmol)was dissolved in dry dichloromethane (2 mL)and stirred
under argon atmosphere for 30 min in the presence of activated molecu-
lar sieves. BF3·Et2O (1 mL)was added and the reaction mixture was kept
at room temperature for 10 min where after the reaction was quenched
by the addition of triethylamine (20 mL), diluted with dichloromethane
(60 mL)and filtered through Celite. The filtrate was washed with brine,
dried over MgSO4 and concentrated to dryness. The residue was subject-
ed to column chromatography (silica gel, hexane/EtOAc 1:1)to give 21
as a syrup (40 mg, 86%). [a]D =À51.2 (c
= 1.0, CH2Cl2); Rf =0.49
(hexane/EtOAc 2:1); 1H NMR (500 MHz, CDCl3): d=7.88 (d, J=7.8 Hz,
2H, Ar-H), 7.50–7.12 (m, 28H, Ar-H), 5.50 (d, J=8.7 Hz, 1H, NH), 5.35
(t, J=9.3 Hz, 1H, H-2’), 5.25 (dd, J=8.8, 2.9 Hz, 1H, H-3’’), 5.10 (d, J=
3.5 Hz, 1-H, H-1’’), 5.04 (d, J=2.9 Hz, 1H, H-4’’), 4.97 (s, 2H,
CH2NHCOOCH2Ph), 4.80 (q, J=6.5 Hz, 1-H, H-5’’), 4.64 (d, J=9.0 Hz,
1H, H-1), 4.76–4.37 (m, 10H, 4ꢂBn, Troc), 4.58 (d, J=8.6 Hz, 1H, H-1’),
3.85 (d, J=3.6 Hz, 1H, H-4’), 3.83 (m, 1H, H-3), 3.80–3.65 (m, 6H, H-4,
H-6a, H-6’, H-3’, H-2’’), 3.58 (d, J=8.5 Hz, 2H, OCH2CH2CH2N), 3.44
(d, J=8.8 Hz, 1H, H-6b), 3.38 (q, J=3.8 Hz, 1H, H-5’), 3.30–3.20 (m,
1H, OCH2CH2CH2aN), 3.19–3.10 (m, 1H, OCH2CH2CH2bN), 3.09–3.01
(m, 2H, H-5, H-2), 2.00 (s, 3H, COCH3), 1.88 (s, 3H, COCH3), 1.19 (s,
2H, OCH2CH2CH2N), 0.87 (d, J=6.5 Hz, 3H, H-6’’), 0.85 (m, 12H, CH3-
DEIPS), 0.54 ppm (m, 5H, CH2, CH-DEIPS); 13C NMR (125 MHz,
CDCl3): d=170.6, 169.6 (2C, 2ꢂCH3CO), 165.0 (ArCO), 156.7
(NHCOOCH2Ph), 154.6 (NHCO), 138.9–127.7 (36C, Ar-C), 100.0 (C-1),
97.7 (C-1’), 96.5 (C-1’’), 95.4 (CCl3), 77.5 (C-3’), 75.8 (C-5), 75.5 (C-4’),
75.0, 74.3, 73.3, 72.6, 70.7 (6C, 4ꢂCH2Ph, Troc, NHCOOCH2Ph), 74.7
(C-5’), 74.6 (C-3), 73.7 (C-2’’), 73.6 (C-2’), 73.5 (C-4), 73.4 (C-4’’), 73.3
(C-3’’), 68.3, 68.2 (2C, C-6, C-6’), 66.9 (OCH2CH2CH2NH), 65.0 (C-5’’),
59.0 (C-2), 37.8 (OCH2CH2CH2NH), 30.0 (OCH2CH2CH2NH), 21.2, 21.0
(2C, 2ꢂCH3CO), 17.5 (C-6’’), 17.5, 17.3, 15.3, 13.0 (4C, CH3-DEIPS), 7.3
(CH-DEIPS), 4.3, 4.0 ppm (2C, CH2-DEIPS); HR-MALDI-TOF MS: m/
z: calcd for C78H95Cl3N2O21Si: 1528.5262; found: 1551.5194 [M+Na]+.
Lev); HR-MALDI-TOF: m/z: calcd for C74H77Cl3N2O18
: 1405.3972;
found: 1428.4265 [M+Na]+.
p-(Benzoyl)-benzyl 6-O-benzyl-2-deoxy-2[[(2,2,2-trichloroethoxy)carbo-
nyl]amino]-4-O-(3,4,6-tri-O-benzyl-2-O-levulinoyl-b-d-galactopyranosyl)-
b-d-glucopyranoside (24): Compound 4 (500 mg, 0.36 mmol)was dis-
solved in 20% triethylamine solution in dichloromethane (5 mL). The so-
lution was stirred at room temperature under argon for 18 h and concen-
trated to dryness under reduced pressure. The residue was purified by
3-[(N-Benzyloxycarbonyl)amino]propyl 6-O-benzyl-2-deoxy-2[[(2,2,2-tri-
chloroethoxy)carbonyl]amino]-3-O-(3,4-di-O-acetyl-2-O-benzyl-a-l-fuco-
pyranosyl)-4-O-(2-O-benzoyl-4,6-di-O-benzyl-b-d-galactopyranosyl)-b-d-
glucopyranoside (22): Compound 21 (40 mg, 0.03 mmol)was dissolved in
dry THF (2 mL), and then acetic acid (0.2 mL) and a solution of TBAF
5464
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2005, 11, 5457 – 5467

Carbohydrates
FULL PAPER
column chromatography (silica gel, hexane/EtOAc 2:1)to give the prod-
uct 24 as a white powder (400 mg, 95%). [a]D =À27.7 (c = 1.0, CH2Cl2);
Rf =0.21 (hexane/EtOAc 2:1); 1H NMR (300 MHz, CDCl3): d=8.21 (d,
J=7.4 Hz, 2H, Ar-H), 7.54–7.16 (m, 27H, Ar-H), 5.38 (dd, J=10.1,
8.3 Hz, 1H, H-2’), 5.08 (d, J=8.5 Hz, 1H, N-H), 4.89 (dd, J=8.8, 2.1 Hz,
2H, CH2Ar), 4.59 (d, J=8.2 Hz, 1H, H-1), 4.80–4.23 (m, 10H, 3ꢂCH2Ar,
Troc, CH2PhOBz), 4.40 (d, J=7.7 Hz, 1H, H-1’), 3.84 (d, J=2.4 Hz, 1H,
H-4’), 3.89–3.73 (m, 3H, H-3, H-6’), 3.70–3.62 (m, 2H, H-6a, H-4), 3.60
(dd, J=9.0, 5.1 Hz, H-5), 3.58–3.49 (m, 3H, H2, H-5’, H-6b), 3.46 (dd,
J=8.9, 5.2 Hz, 1H, H-3’), 2.87–2.21 (m, 4H, OCOCH2CH2, Lev),
2.10 ppm (s, 3H, CH2COCH3, Lev); 13C NMR (100 MHz, CDCl3): d=
206.4 (CH2 COCH3, Lev), 171.6 (OCOCH2CH2, Lev), 165.3 (ArCO),
154.4 (Ar-C), 150.7 (NHCO), 138.7–121.9 (35C, Ar-C), 101.7 (C-1’),
100.0 (C-1), 95.8 (CCl3), 81.2 (C-4), 80.4 (C-3’), 74.8, 74.7, 73.9, 73.8, 72.5,
72.4 (6C, 4ꢂOCH2Ph, OCH2CCl3, OCH2PhOBz), 74.5 (C-5), 74.0 (C-5’),
72.6 (C-3), 72.3 (C-4’), 71.7 (C-2’), 70.3, 68.5 (2C, C-6, C-6’), 57.9 (C-2),
37.9 (OCOCH2CH2, Lev), 30.1 (CH2COCH3, Lev), 28.1 ppm
CH3CO), 1.96 (s, 3H, CH3CO), 1.17 (d, J=6.3 Hz, 3H, H-6’’’), 0.92 ppm
(d, J=6.0 Hz, 3H, H-6’’); 13C NMR (125 MHz, CDCl3): d=170.8, 170.5,
170.4, 169.7 (4C, 4ꢂCH3CO), 165.3 (1C, Ar-C), 153.6 (NHCO), 150.7
(PhCO), 138.8–121.8 (47C, Ar-C), 99.7 (C-1’), 98.8 (C-1’’), 98.3 (C-1’’’),
97.6 (C-1), 95.6 (CCl3), 83.8 (C-3’), 75.4 (C-5), 75.2 (C-3), 74.7, 73.9, 73.7,
73.6, 73.3, 73.1 73.0, 72.9 (8C, 6ꢂOCH2Ph, OCH2PhOBz, OCH2CCl3),
73.4 (C-2’’’), 73.4 (C-2’), 72.9 (C-5’), 72.6 (C-4), 72.2 (C-2’’), 72.0 (2C, 4’’,
C-4’’’), 71.4 (C-4’), 70.9 (C-3’’’), 70.7 (C-3’’), 68.1 (2C, C-6, C-6’), 64.9 (C-
5’’), 64.8 (C-5’’’), 59.7 (C-2), 21.2, 21.1, 20.9, 20.8 (4C, 4ꢂCH3CO), 15.7
(C-6’’’), 15.6 ppm (C-6’’); HR-MALDI-TOF MS: m/z: calcd for
C91H98Cl3NO26: 1725.5443; found: 1748.6041 [M+Na]+.
p-Hydroxybenzyl 6-O-benzyl-2-deoxy-2[[(2,2,2-trichloroethoxy)carbon-
yl]amino]-3-O-(3,4-di-O-acetyl-2-O-benzyl-a-l-fucopyranosyl)-4-O-(3,4,6-
tri-O-benzyl-2-O-(3,4-di-O-acetyl-2-O-benzyl-a-l-fucopyranosyl)-b-d-gal-
actopyranosyl)-b-d-glucopyranoside (27): Triethylamine (400 mL)and
H2O2 (50% in water, 200 mL)were added to a solution of compound 26
(160 mg, 0.093 mmol)in THF (8 mL), and the mixture was stirred under
argon atmosphere for 18 h where after it was concentrated in vacuo. Puri-
fication of the residue by column chromatography (silica gel, hexane/
EtOAc 2:1)furnished 27 as a white powder (123 mg, 82%). [a]D =À83.9
(c = 1.0, CH2Cl2); Rf =0.31 (hexane/EtOAc 2:1); 1H NMR (500 MHz,
CDCl3): d=7.37–6.78 (m, Ar-H, 34H, 6ꢂOCH2Ph, OCH2PhOH), 5.68
(d, J=2.9 Hz, 1H, H-1’’’), 5.29 (dd, J=10.8, 2.9 Hz, 1H, H-3’’), 5.23 (dd,
J=11.0, 2.9 Hz, 1H, H-3’’’), 5.22–5.20 (m, 3H, H-4’’, H-4’’’, NH), 5.12 (d,
J=3.9 Hz, 1H, H-1’’), 5.03 (q, J=6.3 Hz, 1H, H-5’’), 4.76 (d, J=8.2 Hz,
1H, H-1), 4.76–4.12 (m, 20H, 6ꢂOCH2Ph, OCH2PhOH, OCH2CCl3, H-
5’’’, H-1’, H-4, H-3), 4.05 (dd, J=10.1, 9.3 Hz, 1H, H-6a), 3.96 (dd, J=
10.5, 8.2 Hz, 1H, H-2’), 3.90 (d, J=2.8 Hz, 1H, H-4’), 3.88–3.84 (m, 3H,
H-6’, H-6b), 3.84 (dd, J=11.0, 2.9 Hz, 1H, H-2’’’), 3.76 (dd, J=10.8,
3.9 Hz, 1H, H-2’’), 3.38 (dd, J=10.5, 2.8 Hz, 1H, H-3’), 3.25 (dd, J=8.6,
2.8 Hz, 1H, H-5’), 3.26 (d, J=9.3 Hz, 1H, H-5), 3.09 (ddd, 1H, H-2), 2.10
(s, 3H, CH3CO), 2.07 (s, 3H, CH3CO), 2.00 (s, 6H, 2ꢂCH3CO), 1.14 (d,
J=6.7 Hz, 3H, H-6’’’), 0.92 ppm (d, J=6.3 Hz, 3H, H-6’’); 13C NMR
(125 MHz, CDCl3): d=171.4, 170.7, 170.6, 169.6 (4C, 4ꢂCH3CO), 156.2
(1C, Ar-C), 153.5 (NHCO), 138.6–115.6 (41C, Ar-C), 99.6 (C-1’), 98.1
(C-1’’), 97.4 (C-1’’’), 97.3 (C-1), 95.5 (CCl3), 83.7 (C-3’), 75.5 (C-5), 75.3
(C-3), 74.7, 73.9, 73.7, 73.6, 73.3, 73.1 72.9, 73.6, 72.7, 72.4, 71.8, 71.1,
70.9, 70.7 (16 C, 6ꢂOCH2Ph, OCH2PhOH, OCH2CCl3, C-2’’’, C-2’, C-5’,
C-4, C-2’’, 4’’, C-4’’’, C-4’), 70.8 (C-3’’’), 70.1 (C-3’’), 68.0 (2C, C-6, C-6’),
64.7 (C-5’’), 64.6 (C-5’’’), 59.7 (C-2), 21.6, 21.2, 21.1, 20.9 (4C, 4ꢂ
CH3CO), 15.6 (C-6’’’), 15.5 ppm (C-6’’); HR-MALDI-TOF MS: m/z:
calcd for C84H94Cl3NO25: 1621.5181; found: 1644.5204 [M+Na]+.
(OCOCH2CH2, Lev); HR-MALDI-TOF: m/z: calcd for C59H67Cl3N2O16
:
1183.3291; found: 1206.3286 [M+Na]+.
p-(Benzoyl)-benzyl 6-O-benzyl-2-deoxy-2[[(2,2,2-trichloroethoxy) car-
bonyl]amino]-4-O-(3,4,6-tri-O-benzyl-b-d-galactopyranosyl)-b-d-gluco-
pyranoside (25): Methanolic hydrazine acetate (12 mL, 0.5m)was added
to a solution of compound 24 (200 mg, 0.17 mmol)in dichloromethane
(10 mL). After stirring at ambient temperature for 2 h, the reaction was
quenched by adding acetonylacetone (0.8 mL), and diluted by dichloro-
methane (40 mL). The organic phase was washed with brine, dried over
MgSO4, filtered and concentrated under reduced pressure. Purification of
the crude product by column chromatography (silica gel, hexane/EtOAc
2:1)gave diol 25 as a white powder (160 mg, 87%). [a]D =À26.5 (c =
1.0, CH2Cl2); Rf =0.30 (hexane/EtOAc 2:1); 1H NMR (300 MHz, CDCl3):
d=8.22 (d, J=7.8 Hz, 2H, Ar-H), 7.67 (d, J=1.5 Hz, Ar-H), 7.64 (d, J=
1.5 Hz, Ar-H), 7.54–7.17 (m, 25H, Ar-H), 5.28 (d, J=8.5 Hz, 1H, N-H),
4.89 (dd, J=9.8, 3.1 Hz, 2H, ArCH2), 4.85 (d, J=8.2 Hz, 1H, H-1), 4.82–
4.43 (m, 10H, 3ꢂArCH2, Troc, CH2PhOBz), 4.42 (d, J=7.7 Hz, 1H, H-
1’), 3.94 (t, J=2.4 Hz, 1H, H-4’), 3.86–3.80 (m, 3H, H-3, H-6’), 3.82 (dd,
J=10.2, 8.6 Hz, 1H, H-2’), 3.70–3.62 (m, 4H, H-6a, H-2, H-4), 3.60 (dd,
J=8.6, 5.1 Hz, 1H, H-5’), 3.58–3.49 (m, 2H, H-5, H-6b), 3.39 ppm (dd,
J=9.9, 2.2 Hz, 1H, H-3’); 13C NMR (100 MHz, CDCl3): d=167.3
(ArCO), 156.7 (Ar-C), 153.2 (NHCO), 138.2–121.8 (35C, Ar-C), 104.7
(C-1’), 100.0 (C-1), 96.8 (CCl3), 82.2 (C-4), 82.0 (C-3’), 75.2, 74.9, 73.8,
73.6, 72.4, 72.0 (6C, 4ꢂOCH2Ar, OCH2CCl3, OCH2PhOBz), 74.3 (C-5),
73.9 (C-5’), 72.8 (C-3), 71.9 (C-4’), 71.4 (C-2’), 70.3, 68.7 (2C, C-6, C-6’),
57.9 ppm (C-2); HR-MALDI-TOF: m/z: calcd for C59H67Cl3N2O16
:
1085.2923; found: 1108.2845 [M+Na]+.
Trichloroacetimidate (29): DDQ (40 mg, 0.136 mmol)was added to a stir-
red mixture of compound 27 (110 mg, 0.068 mmol)in dichloromethane
(3.8 mL)and water (0.2 mL). The mixture was stirred in the dark for 1 h,
diluted by dichloromethane, washed with brine, dried over MgSO4, fil-
tered and concentrated. Purification of the residue by column chromatog-
raphy (silica gel, hexane/EtOAc 2:1)gave hemiacetal 26 as a colorless
syrup (80 mg, 78.0%). Compound 26 (80 mg, 0.053 mmol)was dissolved
in dry dichloromethane (5 mL)and CCl 3CN (0.5 mL)and DBU (5 mL)
were added. After stirring under argon for 5 min at ambient temperature,
the solution was concentrated to dryness. Purification by column chroma-
tography (silica gel, hexane/EtOAc/triethylamine 1:1:0.01)yielded imi-
date 29 as a colorless syrup (80 mg, 91%). MALDI-TOF MS: m/z: calcd
for C79H88Cl6N2O24: 1662.29; found: 1686.10 [M+Na]+.
p-(Benzoyl)-benzyl 6-O-benzyl-2-deoxy-2[[(2,2,2-trichloroethoxy) car-
bonyl]amino]-3-O-(3,4-di-O-acetyl-2-O-benzyl-a-l-fucopyranosyl)-4-O-
(3,4,6-tri-O-benzyl-2-O-(3,4-di-O-acetyl-2-O-benzyl-a-l-fucopyranosyl)-
b-d-galactopyranosyl)-b-d-glucopyranoside (26): A solution of diol 25
(120 mg, 0.11 mmol)and thiofucoside 6 (126 mg, 0.33 mmol)in dry di-
chloromethane (2 mL)was stirred under argon with 3 ꢃ molecular sieves
for 30 min, the temperature was cooled to 08C and NIS (81.7 mg,
0.36 mmol)and TESOTf (7.5 mL, 0.03 mmol)were added. After stirring
for 30 min the solution was diluted by dichloromethane (60 mL)and the
molecular sieves were removed by filtration. The filtrate was washed
with 15% aqueous sodium thiosulfate and brine, dried over MgSO4, fil-
tered and concentrated. Purification of the crude compound by column
chromatography (silica gel, hexane/EtOAc 2:1)yielded tetrasaccharide
26 as a white powder (164 mg, 86%). [a]D =À82.7 (c = 1.0, CH2Cl2);
Rf =0.48 (hexane/EtOAc 2:1); 1H NMR (500 MHz, CDCl3): d=8.20 (d,
J=7.7 Hz, 1H, Ar-H), 7.53–6.93 (m, Ar-H, 37H, 6ꢂOCH2Ph, OCH2-
PhOBz), 5.65 (d, J=3.0 Hz, 1H, H-1’’’), 5.49 (dd, J=10.6, 2.7 Hz, 1H, H-
3’’), 5.43 (dd, J=10.8, 2.7 Hz, 1H, H-3’’’), 5.42–5.20 (m, 3H, H-4’’, H-4’’’,
NH), 5.18 (d, J=3.9 Hz, 1H, H-1’’), 5.07 (q, J=6.0 Hz, 1H, H-5’’), 4.76
(d, J=9.0 Hz, 1H, H-1), 4.80–4.15 (m, 20H, 6ꢂOCH2Ph, OCH2PhOH,
OCH2CCl3, H-5’’’, H-1’, H-4, H-3), 4.05 (m, 2H, H-6), 3.96 (t, J=8.2 Hz,
1H, H-2’), 3.95 (d, J=3.1 Hz, 1H, H-4’), 3.88–3.84 (m, 2H, H-6), 3.84
(dd, J=10.8, 3.0 Hz, 1H, H-2’’’), 3.76 (dd, J=10.6, 3.9 Hz, 1H, H-2’’),
3.38 (dd, J=8.2, 3.1 Hz, 1H, H-3’), 3.25–3.26 (m, 2H, H-5’, H-5), 3.09
(dd, 1H, H-2), 2.20 (s, 3H, CH3CO), 2.07 (s, 3H, CH3CO), 1.99 (s, 3H,
Fully protected heptasaccharide 30: Glycosyl donor 29 (45 mg,
0.027 mmol)and glycosyl acceptor 22 (35 mg, 0.025 mmol)were dissolved
in dry dichloromethane (1 mL)and activated molecular sieves were
added. After stirring at room temperature for 30 min, the mixture was
cooled to À308C and TBSOTf (1.0 mL)was added. After the donor was
fully converted, the reaction mixture was diluted with dichloromethane
(50 mL)and the molecular sieves were removed by filtration. The solu-
tion was washed with water and sat. aqueous sodium hydrogen carbonate,
dried by MgSO4, filtered and concentrated to dryness under vacuum. Pu-
rification by column chromatography (silica gel, hexane/EtOAc 1:1)af-
forded heptasaccharide 30 as a white powder (36 mg, 62%). [a]D =
À104.3 (c = 1.0, CH2Cl2); Rf =0.28 (hexane/EtOAc 1:1).
Chem. Eur. J. 2005, 11, 5457 – 5467
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ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
5465

G.-J. Boons et al.
Table 2. 1H NMR chemical shifts for heptasaccharide 30.
was incubated at room temperature
over night. Purification was achieved
as described above using centrifugal
filters. The glycoconjugate was taken
up in 10 mm Hepes buffer pH 6.5
Gal’
Fuc’’
GlcNAc’
Fuc’
Gal
GlcNAc
Fuc
H-1
H-2
H-3
H-4
H-5
H-6
4.67
5.36
3.89
4.01
3.67
4.68
5.64
3.76
5.23
5.20
4.69
1.02
5.02
3.20
4.04
3.89
3.10
3.50
5.10
3.66
4.95
4.90
4.90
0.81
4.50
5.38
3.76
4.05
3.76
4.62
4.31
2.81
4.06
3.89
3.09
3.52
5.18
3.80
5.14
5.18
4.75
0.98
(1 mL). This gave
a glycoconjugate
with 1190 copies of LeyLex per KLH
molecule as determined by phenol-sul-
furic acid total carbohydrate assay and
Lowryꢁs protein concentration test.
Immunizations: Groups of five mice
(female BALB/c, 8 weeks)were im-
The 1H NMR (800 MHz, CDCl3)spectral data for compound 30 are
listed as following in Table 2.
munized subcutaneously on days 0, 7, 14 and 21 with carbohydrate(24 mg)
and the adjuvant QS-21 (10 mg)in each boost. The mice were bled on
day 28 (leg-vein)and the sera were tested for the presence of antibodies.
HR-MALDI-TOF MS: m/z: calcd for C148H165Cl6N3O44
found: 2920.9002 [M+Na]+.
: 2897.8897;
ELISA: 96-well plates were coated over night at 48C with LeyLex–BSA,
Ley–BSA, or Lex–BSA (2.5 mgmLÀ1)in 0.2 m borate buffer (pH 8.5)con-
taining 75 mm sodium chloride (100 mL)per well.) The plates were
washed three times with 0.01m Tris buffer containing 0.5% Tween 20%
and 0.02% sodium azide. Blocking was achieved by incubating the plates
1 h at room temperature with 1% BSA in 0.01m phosphate buffer con-
taining 0.14m sodium chloride. Next, the plates were washed and then in-
cubated for 2 h at room temperature with serum dilutions in phosphate
buffered saline. Excess antibody was removed and the plates were
washed three times. The plates were incubated with rabbit anti-mouse
IgG Fcg fragment specific alkaline phosphatase conjugated antibodies
(Jackson ImmunoResearch Laboratories Inc., West Grove, PA)for 2 h at
room temperature. Then, after the plates were washed, enzyme substrate
(p-nitrophenyl phosphate)was added and allowed to react for 30 min
before the enzymatic reaction was quenched by addition of 3m aqueous
sodium hydroxide and the absorbance read at dual wavelengths of 405
and 490 nm. Antibody titers were determined by regression analysis, with
log10 dilution plotted against absorbance. The titers were calculated to be
the highest dilution that gave three times the absorbance of normal
mouse sera diluted 1:120.
Heptasaccharide 31: Zinc (10 mg, 0.15 mmol, nano-size powder)was
added to a stirred solution of heptasaccharide 30 (20 mg, 0.02 mmol)in
acetic acid (2 mL). After 20 min, the zinc dust was removed by filtration
through Celite and the filtrate was concentrated to dryness. The residue
was dissolved in pyridine (2 mL)and acetic anhydride (1 mL)and the
mixture was stirred at room temperature over night. After quenching by
addition of methanol (2 mL), the mixture was diluted by dichlorome-
thane (60 mL)and was washed successively with 1 m HCl solution, sat.
aqueous sodium hydrogen bicarbonate, and brine. The organic layer was
dried over MgSO4, filtered and concentrated. The residue was hydroge-
nolyzed over Pd(AcO)2 (20 mg)in a solution of ethanol and acetic acid
(5:1, 3 mL). After 24 h the mixture was filtered through Celite to remove
the catalyst and concentrated to dryness under reduced pressure. The ob-
tained residue was dissolved by methanol (5 mL)and sodium methoxide
(1m in methanol)was added until pH 10. The solution was stirred at
room temperature for 24 h, neutralized with Dowex 50 H+ resin, diluted
by methanol (50 mL), filtered and concentrated. The residue was purified
by size exclusion column chromatography (Biogel P2 column, eluted with
H2O containing 1% nBuOH)to give the product 31 as a white powder
(4 mg, 52%). [a]D =À99.4 (c = 1.0, MeOH); 1H NMR (800 MHz, D2O,
selected data): d=5.42 (d, J=3.5 Hz, 1H), 5.05 (d, J=4.0 Hz, 1H), 5.04
(d, J=3.7 Hz, 1H), 4.83 (q, J=6.5 Hz, 1H), 4.67 (q, J=6.5 Hz, 1H), 4.64
(d, J=7.6 Hz, 1H), 4.45 (d, J=8.0 Hz, 2H), 4.41 (d, J=7.8 Hz, 1H), 4.22
(q, J=6.5 Hz, 1H), 2.01 (s, 6H, 2ꢂNHCOCH3), 1.21, 1.22, 1.24 ppm (3d,
J=6.5 Hz, 9H, Fuc); 13C NMR (D2O, 200 MHz, selected data): d=105.1,
104.2, 103.4, 103.0, 102.5, 101.0 ppm (7C, anomeric C); HR-MALDI-
Acknowledgements
This work was supported by the National Cancer Institute of the Nation-
al Institutes of Health (Grant No. RO1 CA88986). The authors are grate-
ful for the adjuvant QS-21 that was provided by Antigenics Inc., Lexing-
ton, MA.
TOF MS: m/z: calcd for C49H85N3O33
: 1243.5065; found: 1266.5523
[M+Na]+.
Conjugation of Ley–Lex to KLH: This was accomplished as described ear-
lier. In short, compound 31 (4 mg)was slurried in dry DMF and SAMA-
OPfp (2 equiv)and triethylamine (2 equiv)were added. After stirring for
2 h, the mixture was evaporated and the residue was purified using a
Biogel P-2 column, eluted with H2O containing 1% n-butanol to give,
after lyophilization, thioacetate 32 as a white powder. MALDI-TOF MS
m/z: calcd for C49H85N3O33: 1243.5065; found: 1266.5523 [M+Na]+.
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Received: April 13, 2005
Published online: July 12, 2005
Chem. Eur. J. 2005, 11, 5457 – 5467
www.chemeurj.org
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
5467