Influence of saccharide size on the cellular immune response to glycopeptides.

Article abstract of DOI:10.1039/b301747h

Glycopeptides that bind to MHC molecules on antigen presenting cells may elicit carbohydrate selective T cells. In order to investigate how the cellular immune response depends on the size of the carbohydrate moiety, a trigalactosylated derivative of an immunogenic peptide from hen egg-white lysozyme (HEL52-61) was prepared. Synthesis was accomplished by assembly of an alpha-1,4-linked trigalactose peracetate which was coupled to Fmoc serine. After activation as a pentafluorophenyl ester the resulting building block was used in solid-phase synthesis In contrast to the corresponding mono- and digalactosylated derivatives of HEL52-61, the trigalactosylated HEL52-61 was not immunogenic. Somewhat surprisingly, this was found to be because the trigalactosyl derivative bound approximately two orders of magnitude weaker to I-Ak MHC molecules than the mono- and digalactosyl peptides. Our observation suggests an explanation for previous findings, which show that glycopeptides isolated from MHC molecules in nature usually carry small saccharides.

Full text of DOI:10.1039/b301747h

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Influence of saccharide size on the cellular immune response to
glycopeptides
Mickael Mogemark,a Thomas P. Cirrito,b Petter Sjölin,a Emil R. Unanueb and Jan Kihlberg*a
a Organic Chemistry, Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden.
E-mail: Jan.Kihlberg@chem.umu.se
b Department of Pathology and Immunology, Washington University School of Medicine,
St. Louis, MO 63110, USA
Received 13th February 2003, Accepted 28th April 2003
First published as an Advance Article on the web 9th May 2003
Glycopeptides that bind to MHC molecules on antigen presenting cells may elicit carbohydrate selective T cells.
In order to investigate how the cellular immune response depends on the size of the carbohydrate moiety, a
trigalactosylated derivative of an immunogenic peptide from hen egg-white lysozyme (HEL52–61) was prepared.
Synthesis was accomplished by assembly of an α-1,4-linked trigalactose peracetate which was coupled to Fmoc
serine. After activation as a pentafluorophenyl ester the resulting building block was used in solid-phase synthesis. In
contrast to the corresponding mono- and digalactosylated derivatives of HEL52–61, the trigalactosylated HEL52–61
was not immunogenic. Somewhat surprisingly, this was found to be because the trigalactosyl derivative bound
approximately two orders of magnitude weaker to I-Ak MHC molecules than the mono- and digalactosyl peptides.
Our observation suggests an explanation for previous findings, which show that glycopeptides isolated from MHC
molecules in nature usually carry small saccharides.
size of the carbohydrate moiety. To avoid any structural differ-
Introduction
ences between the saccharides, apart from size, affecting the
Proper function of the immune system of higher vertebrates
immune responses the saccharides were chosen to have closely
requires processing of protein antigens into shorter peptides in
related structures, i.e. mono-, di- and trigalactosides. We
antigen presenting cells.1–3 Peptides resulting from this degrad-
decided to use a model system based on the peptide HEL52–61
ation are bound by major histocompatibility complex (MHC)
molecules and the complexes are then transported to the cell
(1, Fig. 1), which is presented to CD4 T cells by I-Ak class II
MHC molecules on antigen presenting cells after immunization
surface where they are displayed to T cells. Recognition of the
of mice with hen egg-white lysozyme (HEL).23,24 In glyco-
complexes by receptors on circulating T cells triggers immune
responses that depend on the origin of protein antigen and also
on the type of antigen presenting cell and T cell that is involved.
Most eucaryotic proteins and many viral proteins carry
peptides 2–4 the galactoside moieties are linked to a serine
which has been inserted in place of Leu56 in the T cell immuno-
genic peptide HEL52–61 (1). This position was chosen since the
crystal structure of the complex between I-Ak and 1 revealed
oligosaccharide chains; a fact which has generated interest in
that residue 56 is located in the center of the complex, with the
the role(s) of the saccharides during antigen processing, presen-
side-chain pointing away from the binding site of I-Ak.25 In this
tation and recognition by T cells.4–7 Model studies performed
location the saccharide should thus be ideally located to allow
with synthetic neoglycopeptides have revealed that mono- or
binding to I-Ak as well as carbohydrate-specific interactions
small oligosaccharides can be attached to T cell immunogenic
with the T cell receptor. In agreement with this expectation,
peptides which retain MHC binding if the position for the gly-
can is chosen carefully.8–13 These studies also demonstrated that
immunization of mice with neoglycopeptides can elicit T cells
which specifically recognize the carbohydrate moiety if it is
located in the center of the peptide. Recently, some reports have
also described that glycoproteins found in nature can be
degraded to glycopeptides in antigen presenting cells and pre-
sented to T cells as complexes with MHC molecules.14–20 In
most, but not all, of the cases that involve natural glyco-
peptides, the carbohydrate moities attached to the peptides have
been found to be small, i.e. mono- or disaccharides. This may be
considered as somewhat unexpected since both N- and O-linked
carbohydrates found on proteins in general are substantially
larger, usually consisting of at least 5–10 monosaccharide
units.21,22 Processing, i.e. degradation, of the carbohydrate
moieties in the antigen presenting cell constitutes a possible
explanation for the small size of oligosaccharides found on
natural glycopeptides presented by MHC molecules. Altern-
atively, glycopeptides that carry large oligosaccharides may
bind with low affinity to MHC molecules, or the T cell receptor
may be unable to accommodate larger oligosaccharides while
still maintaining contact with the surrounding MHC molecule.
The aim of the present study was to investigate how the cellu-
lar immune response elicited by glycopeptides depends on the
Fig. 1 Peptide 1 and neoglycopeptides 2–4 were used to study the
influence of the size of the oligsosaccharide residue for the cellular
immune response elicited on immunization of mice.
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 0 6 3 – 2 0 6 9
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Scheme 1 Reagents, conditions and yields: i) NIS, TfOH, 4 Å molecular sieves, CH2Cl2–Et2O (1 : 1, v/v), Ϫ45 ЊC (84%); ii) H2, 10% Pd/C, HOAc
8, quant.); iii) α,α-dimethoxytoluene, p-TsOH, MeCN (63%); iv) BzCl, pyridine, 0 ЊC room temp. ( 10, 96%); v) NaCNBH3, HCl (g) in Et2O,
(
4 Å molecular sieves, THF (85%); vi) NIS, TfOH, 4 Å molecular sieves, CH2Cl2–Et2O (1 : 1, v/v), Ϫ45 ЊC (77%); vii) NaOMe, MeOH; then H2, 10%
Pd/C, HOAc; then Ac2O, pyridine (72%); viii) BF3ؒEt2O, Ac2O–toluene (1 : 1, v/v), 0 ЊC (86%, α–β, 1 : 1).
synthetic neoglycopeptides26 2 and 3 have previously been
shown to bind to I-Ak class II MHC molecules with an affinity
close to that of peptide 1. Moreover, immunization of mice
with 3 elicited T cells that recognized the digalactosyl moiety
with high specificity.10,27
fluorophenyl ester 16 (Scheme 2). Boron trifluoride etherate
promoted glycosylation33,34 of 16 with 15 in acetonitrile pro-
vided building block 17 in a rather poor yield (33%). The low
yield was explained by the fact that the crude product had to be
purified three times with radial chromatography to remove the
unreacted α-anomer of 15. This lead to substantial loss of
material because the pentafluorophenyl ester in 17 was hydro-
lysed slowly on the silica gel matrix. The purification problem
could be circumvented by glycosylation of N α-Fmoc serine (18)
instead of pentafluorophenyl ester 16. When the glycosylation
was performed under the same conditions as in the synthesis
of 17, glycosylated serine 19 was obtained in 61% yield after
purification with reversed phase HPLC.
Synthesis of the glycosylated HEL-peptide 4 was performed
manually on a polystyrene resin grafted with polyethylene
glycol spacers (TentaGel S NH2). The resin was functionalized
with the Rink amide linker35,36 and DMF was used as solvent
throughout the synthesis. The N α-Fmoc amino acids (4 equiv.)
were coupled to the resin activated as the corresponding
benzotriazolyl esters.37 Surprisingly, only building block 17
(1.3 equiv.) could be used in the synthesis of 4. Attempts to
use 19, after preactivation with 1-hydroxy-7-azabenzotriazole38
(HOAt) and N,NЈ-diisopropyl carbodiimide in DMF, resulted
in migration of one of the actyl protective groups to the
N-terminus of the peptide-resin thus terminating peptide
growth. However, pentafluorophenyl esters, such as 17, may be
prepared in situ from the corresponding carboxylic acids and
used directly in solid-phase peptide synthesis thereby circum-
venting this problem.39 All couplings were monitored using
bromophenol blue as indicator of free amino groups and
removal of N α-Fmoc protecting groups was accomplished with
piperidine (20% in DMF). After completion of the solid-phase
synthesis the glycopeptide was cleaved from the solid support
and the amino acid side chains were simultaneously depro-
tected by treatment with trifluoroacetic acid containing water
(5%), thioanisole (5%) and ethanedithiol (2.5%).40 In order to
obtain complete cleavage this was performed at 40 ЊC for 2 h
instead of at room temperature.41 Finally, deacetylation with
methanolic sodium methoxide and purification with reversed
Results and discussion
Synthesis of the trigalactoside moiety of glycopeptide 4
was begun by glycosylation of acceptor 628 with donor 529
under promotion30,31 by N-iodosuccinimide and trifluoro-
methanesulfonic acid (Scheme 1). Several portions of the pro-
moters had to be added to achieve complete conversion of 6;
this was assumed to be because the molecular sieves adsorbed
the promoters. When the glycosylation was performed in a
mixture of dichloromethane and diethyl ether (1 : 1) the
α-linked disaccharide 7 was obtained in 84% yield. Hydro-
genation of 7 over Pd/C followed by protection of HO-4Ј
and HO-6Ј with a benzylidene acetal gave 9 in a modest
yield (63% over two steps). Benzoylation of 9 ( 10, 96%) and
subsequent reductive opening of the benzylidene acetal by
treatment with sodium cyanoborohydride and ethereal
hydrochloric acid liberated HO-4Ј to give acceptor 11 (85%).
Attempts to glycosylate 11 with thiocresyl galactoside 5,
using N-iodosuccinimide and trifluoromethanesulfonic acid as
promoters, were unsuccessful under a variety of conditions.
Fortunately, replacement of 5 with the more reactive thioethyl
galactoside 1232 furnished the desired α-1,4-linked trigalacto-
side 13 as the only isolated product in an excellent yield (77%).
Trisaccharide 13 was then converted into a glycosyl donor in
four steps. Debenzoylation with methanolic sodium methoxide,
followed by hydrogenolysis of the benzyl groups and acetyl-
ation with acetic anhydride in pyridine gave 14 in good overall
yield (72%). Treatment of 2-trimethylsilylethyl glycoside 14
with acetic anhydride in toluene under activation with boron
trifluoride etherate gave the anomeric mixture of acetates 15
(86%, α–β, 1 : 1).
The first attempt to convert acetates 15 into a glycosylated
amino acids involved reaction with N α-Fmoc-serine penta-
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 0 6 3 – 2 0 6 9
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Table 1 1H NMR chemical shifts (δ, ppm) for the peptide part of
glycopeptide 4.a
Residue
Proton
4
Asp52
Tyr53
α
β
NH
α
4.05
3.49, 3.45
8.61
3.33
β
2.96, 2.68
7.04, 6.64
9.20
Arom.
OH
Gly54
Ile55
NH
α
NH
α
8.33
3.79
7.86
4.28
β
1.70
γ
1.44, 1.07
0.82
β,γ-CH3
Ser56
NH
α
8.39
4.49
β
NH
3.83, 3.58
8.12
Gln57
α
4.28
β
γ
1.91, 1.71
2.09
γ-CONH2
NH
7.54, 7.03
7.91
Ile58
α
4.15
β
1.71
γ
1.41, 1.06
0.80
β,γ-CH3
Asn59
NH
α
8.28
4.59
β
2.57, 2.47
7.52, 6.98
8.05
β-CONH2
NH
Ser60
Scheme 2 Reagents, conditions and yields: i) BF3ؒEt2O, MeCN; ii)
solid-phase peptide synthesis; then NaOMe, MeOH (28%, based on
resin capacity).
α
β
NH
4.15
3.66, 3.53
8.10
Arg61
α
4.11
β
γ
δ
1.79, 1.53
1.49
3.07
7.46
7.09
phase HPLC furnished the target glycopeptide 4 (Table 1) in
28% overall yield based on the resin capacity.
δ-NH2
δ-CONH2
CBA/J mice were then immunized, in parallel and under
identical conditions, with each of glycopeptides 3 and 4 in
complete Freund’s adjuvant. Seven days later T cells were har-
vested from the draining lymph nodes and then cultured in the
presence of 1, 3 or 4 (Fig. 2). Since antigen presenting cells are
found among the T cells isolated from the lymph nodes this
allows proliferation of those T cells that recognize a specific
antigen. In agreement with previous studies,10 immunization
with digalactosylated 3 gave a T cell population that prolifer-
ated well, and in a dose-dependent manner, when incubated
with 3 (Fig. 2a). These T cells did not show any proliferative
response towards trigalactosylated peptide 4, or peptide 1, thus
revealing a high degree of specificity for glycopeptide 3 which
was used as immunogen. In addition, T cell hybridomas pro-
duced from this population were found to be highly specific for
3, in agreement with previous reports.10,27 In sharp contrast, T
cells harvested from mice immunized with trigalactosylated 4
did not react with either of 1, 3 or 4 (Fig. 2b). Moreover, T cell
hybridomas were not obtained from these mice, further
confirming the unexpected finding that 4 is not immunogenic.
The lack of immunogenicity displayed by glycopeptide 4
could, for instance, result either from poor binding to the I-Ak
class II MHC molecule or an inability of the T cell receptor to
accommodate the trisaccharide moiety while still maintaining
contact with the surrounding MHC molecule. In order to shed
light on these alternatives the binding of 1–4 to purified I-Ak
molecules was investigated. This was achieved by employing
1–4 as inhibitors of the binding of I-Ak to a 125I-radiolabelled
high-affinity peptide ligand (YEDYGILQINSR).27 It was
found that the mono- and diglycosylated peptides 2 and 3
bound almost as well as peptide 1 (Table 2). In contrast,
trigalactosylated peptide 4 bound more than 200 times weaker
a Spectra were recorded at 500 MHz for a solution of 4 in [D6]DMSO at
298 K using [D5]DMSO (δH = 2.50) as internal standard.
Table
molecules.a
2 Binding of peptide 1
and glycopeptides 2–4 to I-Ak
Peptide
IC50/µMb
1
2
3
4
0.12
n.d.c
0.55
n.d.c
0.08
n.d.c
0.75
0.08
0.27
0.43
30
24
a Peptide 1 and glycopeptides 2–4 were used as inhibitors of the binding
of an 125I-radiolabelled peptide (YEDYGILQINSR) to purified I-Ak
molecules. b The concentrations of free (glyco)peptide that inhibits 50%
of the binding of the radiolabelled peptide to I-Ak in three different
experiments are shown. c Not determined.
than peptide 1. This finding was unexpected since the side-chain
of residue 56, which carries the oligosaccharide moieties, points
away from the binding site of I-Ak in the co-crystal with 1.25
Since peptides which bind to I-Ak with an affinity equal to that
of 4 are non-immunogenic,42 we conclude that the lack of
immunogenicity displayed by 4 is explained by the low affinity it
displays for I-Ak.
Our results may also provide further understanding of previ-
ous investigations dealing with how the cellular immune system
responds to neoglycopeptides.11,43 These studies involved
immunization of mice with neoglycopeptides that carried
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 0 6 3 – 2 0 6 9
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shown that the carbohydrate moieties do not undergo process-
ing in antigen presenting cells.46,47 The extent of processing may
therefore depend on the individual glycoprotein and whether
the carbohydrates are N- or O-linked.
Experimental
General
All reactions were carried out at room temperature under an
inert nitrogen atmosphere using dry, freshly distilled solvents
under anhydrous conditions, unless otherwise stated. MeCN
and CH2Cl2 were distilled from calcium hydride. Diethyl ether
and THF were distilled from sodium benzophenone. MeOH
and pyridine were dried over 3 Å and 4 Å molecular sieves,
respectively. Organic solutions were dried over Na2SO4 before
being concentrated. TLC was performed on Silica Gel F254
(Merck) and detection was carried out by examination under
UV light and by charring with 10% sulfuric acid. Flash column
chromatography was performed on Silica Gel (Matrix, 60 Å,
35–70 µm, Grace Amicon). Preparative HPLC separations
were performed on a Beckman System Gold HPLC, using a
Kromasil C-8 column (250 × 20 mm, 5 µm, 100 Å) with a flow
rate of 11 mL minϪ1, detection at 214 nm, and the following
eluent systems: A, aq. 0.1% CF3CO2H; and B, 0.1% CF3CO2H
in MeCN. Analytical HPLC was performed on a Beckman Sys-
tem Gold HPLC, using a Kromasil C-8 column (250 × 4.6 mm,
5 µm, 100 Å) with a flow rate of 1.5 mL min Ϫ1, detection at 214
nm, and the following eluent systems: A, aq. 0.1% CF3CO2H;
1
and B, 0.1% CF3CO2H in MeCN. H and 13C NMR spectra
were recorded with a Bruker DRX-400 or ARX-500 spectro-
meter for solutions in CDCl3 [residual CHCl3 (δH 7.26 ppm),
CDCl3 (δC 77.0 ppm) as internal standard], [D6]DMSO [resi-
dual [D5]DMSO (δH 2.50 ppm), [D6]DMSO (δC 39.51 ppm) as
internal standard] or CD3OD [residual CD2HOD (δH 3.35
ppm), CD3OD (δC 49.0 ppm) as internal standard] at 298 K.
First order chemical shifts and coupling constants were deter-
mined from one-dimensional spectra and proton resonances
were assigned from COSY, TOCSY and HETCOR experi-
ments. Proton resonances that could not be assigned are not
reported. Specific optical rotations were measured in units of
10Ϫ1 deg cm2 gϪ1 using a Perkin Elmer 343 polarimeter.
Fig. 2 Proliferation of T cells obtained after immunization of mice
with (A) digalactosylated peptide 3 or (B) trigalactosylated peptide 4. T
cells obtained from each of the immunizations were incubated with
peptide 1, or glycopeptides 3 or 4 for three days, after which the amount
of 3H-thymidine incorporated in the DNA of the proliferating cells was
determined.
mono-, di- or trisaccharides. Interestingly, immune responses
were only obtained towards glycopeptides having a covalently
bound mono- or disaccharide. No clear explanation was pro-
vided for this observation, but in one case it was proposed that
the lack of immune response to a trisaccharide was due to the
presence of a terminal, non-immunogenic α2,3-linked sialic
acid residue in the trisaccharide.43 As shown in the present
study, the lack of immunogenicity of glycopeptides that carry
large saccharides may instead be explained by a weak binding
to the appropriate MHC molecule. Alternatively, the immune
system may be unable to produce T cells with receptors that
recognize glycopeptides that carry tri- or larger saccharides.4,7
Only a few glycopeptides originating from natural glyco-
proteins have been isolated from MHC molecules, or found to
be able to stimulate T cells. These glycopeptides usually carry
smaller saccharides, such as β--galactose or α--glucopyrano-
syl-(1 2)-β--galactopyranose linked to hydroxylysine,17,18,44
or β--N-acetylglucosamine linked to serine or threonine.20 It is
interesting to speculate on why glycopeptides, which carry small
saccharides, appear to be produced predominantly by the
cellular immune system. This could be a consequence of poor
binding to MHC molecules, as suggested by the model study
presented herein. Another possibility is that nature has evolved
enzymatic pathways for removal of larger oligosaccharides,
such as N-linked glycosides and O-linked saccharides of the
mucin type, during processing in the antigen presenting cell.
For N-linked glycoproteins, studies have shown that the large
N-linked carbohydrate may be processed so that only a single
N-acetylglucosamine residue remains attached to asparagine,14
or to give complete removal of the N-linked glycan.45 Other
studies dealing with O-linked glycopeptides have, however,
2-(Trimethylsilyl)ethyl 2,3-di-O-benzoyl-6-O-benzyl-4-O-
(2,3,4,6-tetra-O-benzyl-ꢀ-D-galactopyranosyl)-ꢁ-galactopyrano-
side (7)
Compound 5 (1.90 g, 2.94 mmol), 6 (1.42 g, 2.45 mmol) and
molecular sieves (1.0 g, 4 Å powder, activated) were suspended
in CH2Cl2–Et2O (35 mL, 1 : 1, v/v) in the absence of light. The
mixture was cooled to Ϫ45 ЊC (CO2–MeCN) and a red–brown
coloured solution of N-iodosuccinimide (0.75 g, 3.33 mmol)
and TfOH (56 µL, 0.63 mmol) in CH2Cl2 (25 mL) was added in
several portions over 4 h. After being stirred for 4.5 h the mix-
ture was diluted with CH2Cl2 (100 mL) and filtered (Hyflo –
Supercel). The filtrate was washed with sat. Na2S2O3–sat.
NaHCO3–H2O (100 mL, 1 : 1 : 2), water (50 mL). The organic
phase was concentrated and chromatographed (SiO2, heptane–
EtOAc 10 : 1) to give 7 (2.26 g, 84%); [α]2D5 ϩ70 (c 1.6, CHCl3);
1H NMR (400 MHz, CDCl3) δ 7.27–8.04 (m, 35 H; ArH ), 5.81
(dd, J 10.6, 7.8 Hz, 1 H; H-2), 5.27 (dd, J 10.7, 2.8 Hz, 1 H;
H-3), 4.99 (d, J 3.4 Hz, 1 H; H-1Ј), 4.92 (dd, J 11.4, 5.4 Hz, 2 H;
PhCH2O), 4.83 (s, 2 H; PhCH2O), 4.76 (d, J 7.8 Hz, 1 H; H-1),
4.70 (d, J 10.8 Hz, 1 H; PhCH2O), 4.54 (d, J 11.1 Hz, 1 H;
PhCH2O), 4.45 (d, J 2.9 Hz, 1 H; H-4), 4.42 (m, 1 H; PhCH2O),
4.34 (s, 2 H; PhCH2O), 4.22 (dd, J 10.3, 2.5 Hz, 1 H; H-3Ј), 4.16
(br s, 1 H; H-4Ј), 4.11 (br s, 3 H; OCH2, PhCH2O), 4.08 (m, 1 H;
H-2Ј), 4.03 (dd, J 9.8, 6.2 Hz, 1 H; H-6 or H-6Ј), 3.94 (t,
J 6.2 Hz, 1 H; H-5 or H-5Ј), 3.76 (dd, J 9.8, 6.2 Hz, 1 H; H-6 or
H-6Ј), 3.69 (dt, J 10.0, 6.6 Hz, 1 H; OCH2), 3.46 (t, J 9.0 Hz,
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 0 6 3 – 2 0 6 9
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1 H; H-5 or H-5Ј), 3.04 (dd, J 8.3, 5.0 Hz, 1 H; H-6 or
H-6Ј), 0.98 (m, 2 H; CH2Si), Ϫ0.01 (s, 9 H; SiMe3); 13C NMR
(100 MHz, CDCl3): δ 166.5, 165.5, 138.9, 138.8, 138.7, 138.4,
138.1, 133.1, 132.9, 129.9, 129.9, 129.6, 129.2, 128.4, 128.3,
128.2, 128.2, 128.0, 128.0, 127.9, 127.6, 127.5, 127.5, 127.4,
127.4, 127.3, 127.3, 100.9, 100.5, 78.9, 77.21, 76.4, 75.6, 74.9,
74.8, 74.5, 74.3, 73.7, 73.0, 72.8, 72.4, 69.9, 69.4, 68.4, 67.5,
67.3, 18.0, Ϫ1.5; MS (FAB) calcd for C66H72O13SiNa 1123 m/z
(M ϩ Na)ϩ, observed 1123; anal. calcd for C66H72O13Si: C, 72.0;
H, 6.6. Found: C, 72.0; H, 6.3%.
(d, J 7.8 Hz, 1 H; H-1), 4.72 (d, J 2.4 Hz, 1 H; H-4Ј), 4.47 (d,
J 2.6 Hz, 1 H; H-4), 4.06 (m, 1 H; OCH2), 3.65 (dt, J 9.9, 6.6 Hz,
1 H; OCH2), 0.97 (m, 2 H; CH2Si), Ϫ0.03 (s, 9 H; SiMe3); 13C
NMR (100 MHz, CDCl3) δ 166.2, 165.9, 165.7, 165.6, 137.5,
133.7, 133.3, 133.3, 133.2, 133.1, 129.9, 129.8, 129.8, 129.7,
129.7, 129.6, 129.6, 129.2, 129.1, 129.0, 128.8, 128.7, 128.5,
128.4, 128.3, 128.3, 128.0, 126.1, 100.7, 100.4, 100.2, 77.2, 76.4,
74.3, 74.1, 71.6, 69.6, 69.1, 68.9, 68.7, 67.4, 63.1, 60.6, 18.0,
Ϫ1.5; MS (ESI) calcd for C59H58O16Si 1050 m/z (M)ϩ, observed
1050.
2-(Trimethylsilyl)ethyl 2,3,6-tri-O-benzoyl-4-O-(2,3,di-O-
benzoyl-6-O-benzyl-ꢀ-D-galactopyranosyl)-ꢁ-galactopyranoside
(11)
2-(Trimethylsilyl)ethyl 2,3-di-O-benzoyl-4-O-(ꢀ-D-galacto-
pyranosyl)-ꢁ-galactopyranoside (8)
A solution of compound 7 (4.40 g, 4.00 mmol) in HOAc
(50 mL) was treated with 10% Pd/C (2.0 g) under hydrogen
(55 psi) for 46 h. The catalyst was removed by filtration (Hyflo –
Supercel) and co-concentrated several times with toluene to
Compound 10 (0.70 g, 0.67 mmol), NaCNBH3 (0.30 g, 4.77
mmol) and molecular sieves (0.5 g, 4 Å, activated) were sus-
pended in dry THF (15 mL). The mixture was stirred for 1 h
and saturated etheral HCl was added until the mixture became
acidic (pH 2–3), and gas evolution ceased. After 2 h the reaction
was neutralized with NaHCO3 (s). The mixture was diluted
with CH2Cl2 (100 mL), filtrated (Hyflo – Supercel), washed with
sat. NaHCO3 (40 mL) and water (50 mL). The organic phase
was concentrated and chromatographed (SiO2, heptane–EtOAc
3 : 1) to give 11 (0.60 g, 85%); [α]2D5 ϩ80 (c 2.7, CDCl3); 1H NMR
(400 MHz, CHCl3) δ 7.18–8.03 (m, 30 H; ArH ), 5.89 (dd,
J 10.8, 3.5 Hz, 1 H; H-2Ј), 5.81 (dd, J 10.8, 2.7 Hz, 1 H; H-3Ј),
5.72 (dd, J 10.6, 7.7 Hz, 1 H; H-2), 5.49 (d, J 3.6 Hz, 1 H; H-1Ј),
5.27 (dd, J 10.6, 2.7 Hz, 1 H; H-3), 4.74 (d, J 7.7 Hz, 1 H; H-1),
4.70 (m, 1 H; H-5), 4.62 (br s, 1 H; H-4Ј), 4.51 (br s, 1 H;
PhCH2O), 4.43 (d, J 2.3 Hz, 1 H; H-4), 4.37 (m, 2 H, H-6,
H-6Ј), 4.19 (d, J 11.8 Hz, 1 H; H-6Ј), 4.02 (m, 2 H; H-6, OCH2),
3.77 (d, J 1.0 Hz, 1 H; H-5Ј), 3.61 (dt, J 10.0, 6.5 Hz, 1 H;
OCH2), 3.55 (d, J 3.7 Hz, 2 H; PhCH2O), 0.95 (m, 2 H; CH2Si),
Ϫ0.04 (s, 9 H; SiMe3); 13C NMR (100 MHz, CDCl3) δ 166.3,
166.1, 165.6, 165.5, 165.2, 137.2, 133.4, 133.2, 133.1, 133.1,
133.0, 130.0, 129.9, 129.9, 129.8, 129.7, 129.7, 129.3, 128.8,
128.5, 128.4, 128.3, 128.3, 127.8, 127.6, 100.8, 99.3, 77.2, 76.1,
74.0, 73.8, 72.0, 70.9, 69.9, 69.8, 69.3, 68.7, 67.3, 61.4, 18.0,
Ϫ1.5; MS (FAB) calcd for C59H60O16SiNa 1075 m/z (M ϩ Na)ϩ,
observed 1075; anal. calcd for C59H60O16Si: C, 67.3; H, 5.7.
Found: C, 66.9; H, 5.9%.
afford 8 (2.83 g, quant.); [α]2D5 ϩ100 (c 1.0, MeOH); H NMR
1
(400 MHz, MeOD) δ 7.26–7.89 (m, 10 H; ArH ), 5.56 (dd,
J 10.7, 2.8 Hz, 1 H; H-2), 5.34 (dd, J 10.7, 2.8 Hz, 1 H; H-3),
4.92 (d, J 3.7 Hz, 1H; H-1Ј), 4.81 (d, J 7.8 Hz, 1 H; H-1), 4.39
(d, J 2.8 Hz, 1 H; H-4), 4.08 (t, J 5.7 Hz, 1 H; H-5 or H-5Ј), 4.00
(dt, J 9.8, 5.1 Hz, 1 H; OCH2), 3.92 (d, J 2.5 Hz, 1 H; H-4Ј),
3.85 (m, 1 H; H-3Ј), 3.57 (m, 1 H; H-2Ј), 0.80 (m, 2 H; CH2Si),
Ϫ0.17 (s, 9 H; SiMe3); 13C NMR (100 MHz, MeOD): 167.4,
167.1, 134.6, 134.5, 130.9, 130.6, 130.6, 129.6, 129.5, 102.5,
102.1, 76.2, 75.8, 75.4, 71.9, 71.7, 71.4, 71.2, 70.7, 68.7, 62.3,
60.5, 18.9, Ϫ1.4; MS (ESI) calcd for C31H42O13Si 650 m/z (M)ϩ,
observed 650.
2-(Trimethylsilyl)ethyl 2,3-di-O-benzoyl-4-O-(4,6-O-benzyl-
idene-ꢀ-D-galactopyranosyl)-ꢁ-D-galactopyranoside (9)
TsOH (110 mg, 0.58 mmol) was added to a solution of 8 (2.70 g,
4.33 mmol) and α,α-dimethoxytoluene (0.82 mL, 5.40 mmol) in
MeCN (20 mL). After being stirred for two days the reaction
was neutralized with Et3N (0.2 mL) and concentrated. The resi-
due was chromatographed (SiO2, heptane–EtOAc 1 : 3) to give
9 (1.94 g, 63%); [α]2D5 ϩ92 (c 1.4, CHCl3); 1H NMR (400 MHz,
CDCl3) δ 7.27–8.00 (m, 15 H; ArH ), 5.64 (dd, J 10.7, 7.9 Hz,
1 H; H-2), 5.41 (s, 1 H; PhCHO2), 5.20 (dd, J 10.8, 2.9 Hz, 1 H;
H-3), 5.02 (d, J 3.1 Hz, 1 H; H-1Ј), 4.74 (d, J 7.9 Hz, 1 H; H-1),
4.50 (d, J 2.5 Hz, 1 H; H-4), 4.24 (d, J 3.5 Hz, 1 H; H-4Ј), 4.13
(m, 1 H; H-3Ј), 4.07 (s, 1 H; H-5 or H-5Ј), 4.03 (m, 1 H; OCH2),
3.96 (m, 3 H; H-6 or H-6Ј, H-2Ј), 3.86 (t, J 6.7 Hz, 1 H; H-5 or
H-5Ј), 3.65 (br s, 1 H; OH), 3.58 (m, 2 H; H-6 or H-6Ј, OCH2),
3.43 (d, J 12.5 Hz, 1 H; H-6 or H-6Ј), 2.84 (br s, 1 H; OH), 1.85
2-(Trimethylsilyl)ethyl 2,3,6-tri-O-benzoyl-4-O-[2,3-di-O-
benzoyl-6-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-ꢀ-D-galacto-
pyranosyl)-ꢀ-D-galactopyranosyl]-ꢁ-galactopyranoside (13)
Compound 11 (0.73 g, 0.69 mmol), 12 (0.61 g, 1.04 mmol) and
molecular sieves (0.6 g, 4 Å powder, activated) were suspended
in CH2Cl2–Et2O (10 mL, 1 : 1, v/v) in the absence of light. The
mixture was cooled to Ϫ45 ЊC (CO2–MeCN) and a red–brown
coloured solution of N-iodosuccinimide (0.47 g, 2.09 mmol)
and TfOH (120 µL, 1.35 mmol) in CH2Cl2 (4 mL) was added in
several portions over 2 h. After being stirred 6 h the mixture
was diluted with CH2Cl2 (300 mL) and filtrated (Hyflo – Super-
cel). The filtrate was washed with sat. Na2S2O3–sat. NaHCO3–
H2O (120 mL, 1 : 1 : 2). The organic phase was concentrated
and chromatographed (SiO2, heptane–CH2Cl2–methyl t-butyl
ether 25 : 20 : 1) to give 13 (0.84 g, 77%); [α]2D5 ϩ92 (c 1.1,
(br s, 1 H; OH), 0.89 (m, 2 H; CH2Si), Ϫ0.11 (s, 9 H; SiMe3); 13
C
NMR (100 MHz, CDCl3): 166.1, 165.7, 137.7, 133.5, 133.2,
129.8, 129.7, 129.6, 129.2, 129.0, 128.6, 128.3, 128.1, 126.3,
102.1, 100.9, 100.9, 77.2, 76.5, 75.9, 74.3, 70.1, 69.9, 69.0, 68.9,
67.9, 63.7, 60.0, 18.0, Ϫ1.5; MS (ESI) calcd for C38H46O13Si 739
m/z (M)ϩ, observed 739. MS (ESI) calcd for C38H46O13Si 739
m/z (M)ϩ, observed 739.
2-(Trimethylsilyl)ethyl 2,3,6-tri-O-benzoyl-4-O-(2,3-di-O-
benzoyl-4,6-O-benzylidene-ꢀ-D-galactopyranosyl)-ꢁ-D-galacto-
pyranoside (10)
1
CHCl3); H NMR (CDCl3) δ 7.05–8.00 (m, 50 H; ArH ), 5.83
Benzoylchloride (1.2 mL, 10.3 mmol) was added to a solution
of compound 9 (1.91 g, 2.58 mmol) in pyridine (15 mL) at 0 ЊC.
After stirring at room temperature for 2 h the mixture was
diluted with CH2Cl2 (100 mL), washed with sat. NaHCO3
(2 × 30 mL) and water (40 mL). The organic phase was concen-
trated and chromatographed (SiO2, heptane–EtOAc 5 : 1)
(m, 2 H; H-2Ј, H-3Ј), 5.79 (dd, J 10.2, 7.4 Hz, 1 H; H-2), 5.46 (d,
J 3.0 Hz, 1 H; H-1Ј), 5.32 (dd, J 10.3, 2.7 Hz, 1 H; H-3), 5.08 (d,
J 3.5 Hz, 1 H; H-1Љ), 4.78 (m, 2 H; PhCH2O), 4.75 (d, J 7.4 Hz,
1 H; H-1), 4.73 (s, 1 H; H-6, H-6Ј or H-6Љ), 4.64 (m, 3 H; H-4Ј,
H-6, H-6Ј or H-6Љ, PhCH2O), 4.47 (d, J 3.3 Hz, 1 H; H-4), 4.42
(t, J 5.4 Hz, 1 H; H-5, H-5Ј or H-5Љ), 4.39 (s, 1 H; PhCH2O),
4.29 (m, 1 H; H-5, H-5Ј or H-5Љ), 4.16 (s, 2 H; PhCH2O), 4.13
(dd, J 10.4, 2.6 Hz, 1 H; H-3Љ), 4.03 (m, 4 H; H-2Љ, H-4Љ, H-6,
H-6Ј or H-6Љ, OCH2), 3.94 (t, J 9.3 Hz, 1 H; H-5, H-5Ј or H-5Љ),
3.75 (dd, J 24.6, 12.9 Hz, 2 H; PhCH2O), 3.60 (dt, J 10.0,
6.3 Hz, 1 H; OCH2), 3.30 (m, 2 H; H-6, H-6Ј, or H-6Љ), 2.73 (dd,
to give 10 (2.59 g, 96%); [α]2D5 ϩ177 (c 0.4, CHCl3); H NMR
1
(400 MHz, CDCl3) δ 7.27–8.04 (m, 30 H; ArH ), 5.95 (dd,
J 11.0, 3.2 Hz, 1 H; H-3Ј), 5.90 (dd, J 11.0, 3.1 Hz, 1 H; H-2Ј),
5.78 (dd, J 10.7, 7.8 Hz, 1 H; H-2), 5.48 (d, J 3.2 Hz, 1 H; H-1Ј),
5.41 (s, 1 H; PhCHO2), 5.18 (dd, J 10.7, 2.9 Hz, 1 H; H-3), 4.76
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J 8.1, 5.1 Hz, 1 H; H-6, H-6Ј, or H-6Љ), 0.93 (m, 2 H; CH2Si),
Ϫ0.07 (s, 9 H; SiMe3); δ 13C NMR (100 MHz, CDCl3) 166.5,
166.3, 166.2, 166.7, 165.0, 138.9, 138.8, 138.6, 138.1, 133.3,
133.1, 133.1, 132.9, 130.0, 129.8, 129.8, 129.7, 129.4, 129.0,
129.0, 128.4, 128.4, 128.3, 128.2, 128.2, 128.2, 128.1, 128.1,
128.1, 128.0, 127.8, 127.4, 127.4, 127.3, 127.3, 127.2, 127.2,
127.1, 127.1, 100.7, 99.6, 98.9, 78.8, 77.2, 76.3, 75.5, 75.1, 74.8,
74.8, 74.0, 73.7, 72.7, 72.5, 72.3, 72.2, 71.1, 69.8, 69.6, 69.2,
67.9, 67.5, 67.1, 66.5, 61.8, 25.6, 17.9, Ϫ1.5; MS (FAB) calcd for
C93H94O21SiNa 1598 m/z (M ϩ Na)ϩ, observed 1598; anal.
calcd for C93H94O21Si: C, 70.9; H, 6.0. Found: C, 70.5; H, 6.3%.
with CH2Cl2 (2 × 40 mL). The combined organic phases
were concentrated and purified with radical chromatography
(1 mm, heptane–EtOAc 1 : 2) to give 17 (0.10 g, 33%); [α]2D5 ϩ69
(c 1.5, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.79–7.29
(m, 8 H; ArH ), 6.13 (d, J 8.8 Hz, 1 H; Ser-NH ), 5.55 (d, J 2.2
Hz, 1 H; H-4Љ), 5.40 (dd, J 11.1, 3.3 Hz, 1 H; H-3Љ), 5.32–
5.15 (m, 4 H; H-2Ј, H-2Љ, H-3Ј, H-2), 5.00 (d, J 3.5 Hz, 1 H;
H-1Љ), 4.96 (d, J 3.3 Hz, 1 H; H-1Ј), 4.87 (d, J 8.9, 1 H; Ser-Hα),
4.81 (dd, J 10.8, 2.4 Hz, 1 H; H-3), 4.62–4.52 (m, 3H; H-5,
H-5Ј or H-5 H-6, H-6Ј or H-6Љ), 4.43 (dd, J 10.5, 6.0 Hz, 1 H;
H-6, H-6Ј or H-6Љ), 4.34 (d, J 7.6 Hz, 1 H; H-1), 4.31–4.23
(m, 3 H; H-4, Ser-Hβ), 4.17–4.00 (m, 6 H; Ser-Hβ, H-6, H-6Ј
or H-6Љ, H-4), 3.50 (t, J 6.2 Hz, 1 H; H-5, H-5Ј or H-5Љ),
2.13–1.90 (m, 30 H; OAc); 13C NMR (100 MHz, CDCl3)
δ 170.8, 170.4, 170.4, 170.3, 170.3, 170.2, 170.1, 170.0, 169.2,
166.0, 155.9, 144.8, 143.7, 141.4, 141.3, 127.8, 127.2, 127.1,
125.0, 124.9, 120.0, 120.0, 101.5, 99.4, 99.3, 77.7, 77.2, 72.4,
69.5, 69.2, 69.0, 68.4, 68.3, 68.2, 67.8, 67.4, 66.9, 62.6, 61.1,
60.4, 54.4, 47.2, 20.9, 20.7, 20.6, 20.6, 20.4, 14.2; MS (FAB)
calcd for C66H72F5NO30Na 1422 m/z (M ϩ Na)ϩ, observed
1422; anal. calcd for C66H72F5NO30: C, 53.2; H, 4.8. Found: C,
53.2; H, 5.1.
2-(Trimethylsilyl)ethyl 2,3,6-tri-O-acetyl-4-O-[2,3,6-tri-O-
acetyl-4-O-(2,3,4,6-tetra-O-acetyl-ꢀ-D-galactopyranosyl)-ꢀ-D-
galactopyranosyl]-ꢁ-galactopyranoside (14)
Sodium methoxide in methanol (6.6 mL, 2.0 M) was added to a
solution of 13 (1.04 g, 0.66 mmol) in MeOH (20 mL). The
mixture was stirred at room temperature for 3 h, then neutral-
ized with Amberlite IR-120 resin, filtered and concentrated.
Residual methanol was removed by co-concentration with tolu-
ene. A solution of the crude product in HOAc (30 mL) was
treated with 10% Pd/C (0.5 g) under hydrogen (55 psi). After
48 h, the catalyst was removed by filtration (Hyflo – Supercel)
and by co-concentration several times with toluene. The resi-
due was dissolved in pyridine (30 mL) and acetic anhydride
(30 mL). After 6 h of stirring the solution was concentrated and
residual pyridine was removed by co-concentration with tolu-
ene. The residue was chromatographed (SiO2, heptane–EtOAc
1 : 1) to give 14 (0.30 g, 72%); [α]2D5 ϩ94 (c 1.5, CHCl3); 1H NMR
(400 MHz, CDCl3) δ 5.53 (d, J 2.0 Hz, 1 H; H-4Љ), 5.36 (dd,
J 11.1, 3.2 Hz, 1 H; H-3Љ), 5.29–5.16 (m, 3 H; H-2Љ, H-2Ј, H-3Ј),
5.12 (dd, J 10.6, 7.8 Hz, 1 H; H-2), 4.96 (d, J 3.5 Hz, 1 H; H-1Љ),
4.93 (d, J 3.3 Hz, 1 H; H-1Ј), 4.80 (dd, J 10.7, 2.0 Hz, 1 H; H-3),
4.44 (m, 2 H, H-1; H-6, H-6Ј or H-6Љ), 4.29 (d, J 2.0 Hz, 1 H;
H-4Ј), 4.17 (dd, J 11.1, 6.5 Hz, 1 H H-6, H-6Ј or H-6Љ), 4.05 (d,
J 1.5 Hz, 1 H; H-4), 3.97 (m, 1 H; OCH2), 3.76 (t, J 6.5 Hz, 1 H;
H-5, H-5Ј or H-5Љ), 3.52 (dt, J 10.0, 6.7 Hz, 1 H; OCH2), 2.11–
1.96 (m, 30 H; OAc), 0.91 (m, 2 H; CH2Si), Ϫ0.02 (s, 9 H;
SiMe3); 13C NMR (100 MHz, CDCl3) δ 170.8, 170.5, 170.3,
170.3, 170.2, 170.2, 170.1, 170.0, 169.0, 100.6, 99.4, 99.2, 77.7,
77.2, 76.8, 72.7, 71.9, 69.4, 68.9, 68.8, 68.3, 68.1, 67.8, 67.4,
67.3, 66.9, 62.2, 61.0, 60.4, 21.0, 21.0, 20.8, 20.7, 20.7, 20.6,
17.9, Ϫ1.3; MS (FAB) calcd for C43H64O26SiNa 1047 m/z (M ϩ
Na)ϩ, observed 1047; anal. calcd for C43H64O26Si: C, 50.4; H,
6.3. Found: C, 50.8; H, 6.7%.
Nꢀ-(Fluoren-9-ylmethoxycarbonyl)-3-O-(2,3,6-tri-O-acetyl-4-O-
[2,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-ꢀ-D-galacto-
pyranosyl)-ꢀ-D-galactopyranosyl]-ꢁ-D-galactopyranosyl)-L-
serine (19)
BF3ؒEt2O (49 µL, 0.39 mmol) was added to a solution of 15
(0.13 g, 0.13 mmol) and 18 (64 mg, 0.19 mmol) in MeCN
(4 mL) at room temperature. After stirring for 4 h the solution
was diluted with CH2Cl2 (120 mL), washed with 1 M HCl
(30 mL) and water (40 mL). The organic phase was concen-
trated and purified with reversed phase HPLC (gradient:
0
80% B in A during 60 min) to give 19 (97 mg, 61%); [α]2D5
1
ϩ84 (c 1.7, CHCl3); H NMR (400 MHz, CDCl3) δ 7.88–7.30
(m, 8 H; ArH ), 6.39 (d, J 8.4 Hz, 1 H; Ser-NH ), 5.52 (dd, J 2.9,
1.0 Hz, 1 H; H-4Љ), 5.24–5.16 (m, 3 H; H-2Љ, H-2Ј, H-2), 5.15
(br s, 1 H; H-1Љ), 5.11 (d, J 3.5 Hz, 1 H; H-1Ј), 5.06 (dd, J 10.6,
2.9 Hz, 1 H; H-3), 4.73 (d, J 7.7 Hz, 1 H; H-1), 4.65 (t, J 6.9 Hz,
1 H; H-5, H-5Ј or H-5Љ), 4.29 (d, J 2.3 Hz, 1 H; H-4), 4.14 (d,
J 6.9 Hz, 2 H; H-6, H-6Ј or H-6Љ), 4.05 (t, J 6.5 Hz, 1 H; H-5,
H-5Ј or H-5Љ), 3.96 (dd, J 10.6, 4.0 Hz, 1 H; Ser-Hβ), 2.15–1.90
(m, 30 H; OAc); 13C NMR (400 MHz, CDCl3) δ 171.6, 171.0,
170.9, 170.7, 170.4, 170.3, 170.2, 170.2, 170.0, 169.6, 156.2,
143.9, 143.7, 141.3, 127.7, 127.1, 125.3, 125.2, 120.0, 102.5,
99.2, 99.2, 77.7, 77.2, 76.5, 73.4, 72.0, 71.2, 70.0, 69.5, 68.6,
68.5, 68.3, 67.7, 67.4, 67.1, 66.9, 63.4, 60.8, 60.4, 47.1, 21.2,
20.9, 20.9, 20.8, 20.7, 20.7, 20.6; MS (ESI) calcd for C56H67NO30
1233 m/z (M)ϩ, observed 1233.
1,2,3,6-Tetra-O-acetyl-4-O-[2,3,6-tri-O-acetyl-4-O-(2,3,4,6-
tetra-O-acetyl-ꢀ-D-galactopyranosyl)-ꢀ-D-galactopyranosyl]-
galactopyranoside (15)
Boron trifluoride etherate (103 µL, 0.82 mmol) was added
to a solution of 13 (0.27 mg, 0.26 mmol) in toluene (4 mL)
and acetic anhydride (4 mL) at 0 ЊC. The solution was
stirred for 3 h, then diluted with CH2Cl2 (100 mL), washed with
sat. NaHCO3 (40 mL) and water (40 mL). The organic
phase was concentrated and chromatographed (SiO2, heptane–
EtOAc 2 : 3) to give a 1 : 1 α–β mixture of 15 (0.22 g, 86%); MS
(FAB) calcd for C40H54O27Na 989 m/z (M ϩ Na)ϩ, observed
989.
L-ꢀ-Aspartyl-L-tyrosylglycyl-L-isoleucyl-O-{4-O-[4-O-(ꢀ-D-
galactopyranosyl)-ꢀ-D-galactopyranosyl]-ꢁ-D-galactopyrano-
syl}-L-seryl-L-glutaminyl-L-isoleucyl-L-asparaginyl-L-seryl-L-
arginine amide (4)
Synthesis was performed manually in a mechanically agitated
reactor on a TentaGel S NH2 resin (14.8 µmol), functionalized
with the the Rink amide linker p-[α-(fluoren-9-ylmethoxyform-
amido)-2,4-dimethoxybenzyl]-phenoxyacetic acid,35,36 accord-
ing to procedures that have been reported previously.48 After
cleavage from the solid phase the crude product was treated
with sodium methoxide in methanol (10 mL, 6.0 mM) during
2.5 h at room temperature. The solution was neutralized with
Amberlite IR-120 resin and filtered. The organic phase was
concentrated and the residue was purified by preparative
Nꢀ-(Fluoren-9-ylmethoxycarbonyl)-3-O-(2,3,6-tri-O-acetyl-4-O-
[2,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-ꢀ-D-galacto-
pyranosyl)-ꢀ-D-galactopyranosyl]-ꢁ-D-galactopyranosyl)-L-
serine pentafluorophenyl ester (17)
BF3ؒEt2O (70 µL, 0.55 mmol) was added to a solution of
compound 15 (0.21 g, 0.22 mmol) and 16 (0.14 g, 0.29 mmol)
in MeCN (7 mL) at room temperature. After 2 h, the solution
was diluted with CH2Cl2 (100 mL), washed with sat. NaHCO3
(60 mL) and water (60 mL). The aqeous phase was re-extracted
reversed-phase HPLC (gradient: 0
80% B in A during
60 min) to give 4 (6.7 mg, 28% based on resin capacity); MS
(ESI) calcd for C66H107N15O33 1637 m/z (M)ϩ, observed 1637;
amino acid analysis: Arg 1.00 (1), Asp 1.99 (2), Glu 1.02 (1),
Gly 1.01 (1), Ile 1.99 (2), Ser 1.96 (2), Tyr 1.00 (1).
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 0 6 3 – 2 0 6 9
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16 T. Dudler, F. Altman, J. M. Carballido and K. Blaser, Eur. J.
Immunization with glycopeptides 3 and 4
Immunol., 1995, 25, 538–542.
Five CBA/J mice were immunized with 10 nmol of each
of glycopeptides 3 and 4 emulsified in 100 µL of complete
Freund’s adjuvant. The mice were females of 10 weeks of age
purchased from Jackson Laboratories (Bar Harbor, Maine) and
the adjuvant was obtained from Difco Chemical Co. Each
mouse received approximately 50 µL of glycopeptide emulsion
in each of two footpads. Seven days later the popliteal lymph
nodes were dissected, a cell suspension was made and a
proliferative assay was carried out. In this assay 5 × 105 cells in
200 µL of Dulbecco’s minimal essential media containing 10%
fetal calf serum were added to each of the wells in 96 well
plates. Glycopeptides 3 and 4, or the parent peptide HEL52–61
(1), were added to the wells to give concentrations ranging from
30 µM to 10 nmol (cf. Fig. 2). The cells were incubated for three
17 J. Broddefalk, J. Bäcklund, F. Almqvist, M. Johansson,
R. Holmdahl and J. Kihlberg, J. Am. Chem. Soc., 1998, 120, 7676–
7683.
18 B. Holm, J. Bäcklund, M. A. F. Recio, R. Holmdahl and J. Kihlberg,
ChemBioChem, 2002, 3, 1209–1222.
19 J. Bäcklund, S. Carlsen, T. Höger, B. Holm, L. Fugger, J. Kihlberg,
H. Burkhardt and R. Holmdahl, Proc. Natl. Acad. Sci. USA, 2002,
99, 9960–9965.
20 J. S. Haurum, I. Bjerring Hoier, G. Arsequell, A. Neisig, G. Valencia,
J. Zeuthen, J. Neefjes and T. Elliott, J. Exp. Med., 1999, 190, 145–
150.
21 J. D. Marth, in Essentials of Glycobiology, eds. A. Varki,
R. Cummings, J. Esko, H. Freeze, G. Hart and J. Marth, Cold Spring
Harbor, New York, 1999, p. 85.
22 J. D. Marth, in Essentials of Glycobiology, eds. A. Varki,
R. Cummings, J. Esko, H. Freeze, G. Hart and J. Marth, Cold Spring
Harbor, New York, 1999, p. 101.
23 P. M. Allen, G. R. Matsueda, R. J. Evans, J. B. Dunbar Jr,
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cells were harvested and the amount of 3H-thymidine incorpor-
ated in the DNA was determined. The data presented in Fig. 2
is the average of three proliferative assays, having a variation of
∼10% in the cpm.
Binding of glycopeptides to I-Ak class II MHC molecules
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Acknowledgements
This work was funded by grants from the Swedish Research
Council and the Göran Gustafsson Foundation for Research in
Natural Sciences and Medicine.
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