Synthetic vaccines consisting of tumor-associated MUC1 glycopeptide antigens and bovine serum albumin

Article abstract of DOI:10.1002/anie.200501593

(Chemical Equation Presented) Sweet medicine: The mucin MUC1 is highly overexpressed on epithelial tumor cells. Glycopeptides 1 (from the repeating unit of MUC1) carrying various important sialylated tumor-associated carbohydrate antigens R were synthesiz

Full text of DOI:10.1002/anie.200501593

Communications
Antitumor Agents
DOI: 10.1002/anie.200501593
Synthetic Vaccines Consisting of Tumor-
Associated MUC1 Glycopeptide Antigens and
Bovine Serum Albumin**
Sebastian Dziadek, Danuta Kowalczyk, and
Horst Kunz*
The identification of cell-surface components that occur only
on tumor but not on healthy cells is essential for a selective
immunological attack on tumor tissue. About 30 years ago,
[
1]
Springer described membrane glycoproteins containing the
Thomsen–Friedenreich T antigen and the monosaccharide Tn
antigen side chains as tumor-associated antigens on epithelial
tumors. These T and Tn glycoproteins were described to be
structurally related to the N-terminal region of asialoglyco-
phorin. Based on this structural information, we synthesized a
vaccine consisting of N-terminal glycopeptides from asialo-
glycophorin of M blood group specificity conjugated with
[
2]
bovine serum albumin (BSA) and employed it in the
immunization of mice. Although the monoclonal antibody
(82-A6) obtained from these experiments showed affinity to
epithelial tumor cells, the selectivity for tumor cells was not
[
3]
sufficient. However, the antibody 82-A6 was capable of
distinguishing clearly between asialoglycophorin of M and N
[*] Dr. S. Dziadek, D. Kowalczyk, Prof. Dr. H. Kunz
Institut für Organische Chemie
Johannes Gutenberg-Universität Mainz
Duesbergweg 10–14, 55128 Mainz (Germany)
Fax: (+49)6131-392-4786
E-mail: hokunz@uni-mainz.de
[
**] This work was supported by the Deutsche Forschungsgemeinschaft
and the Stiftung Rheinland-Pfalz für Innovation. S.D. is grateful for a
scholarship provided by the Fonds der Chemischen Industrie and
the Bundesministerium für Bildung und Forschung.
7624
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2005, 44, 7624 –7630

Angewandte
Chemie
[
3]
blood groups, indicating that not only the saccharide, but
also the peptide sequence, contributed to the recognized
epitope. From this observation we inferred that apart from
tumor-associated saccharides, tumor-selective peptide struc-
tural elements are required to render an antigen sufficiently
tumor-selective.
Structural leads for the design of tumor-selective glyco-
peptide antigens are obtained from analyses of the tumor-
[
4]
associated epithelial mucin MUC1, which is extensively
over-expressed on tumor cells. The extracellular portion of
MUC1 contains numerous repeating units of the amino acid
[
5]
sequence HGVTSAPDTRPAPGSTAPPA. Most O-glyco-
sylation sites are located within these tandem repeats. Owing
to the down-regulation of a glucosaminyl transferase (C-
2
GnT-1) and the concomitant overexpression of sialyl trans-
[
6]
ferases, MUC1 on tumor cells carries short, prematurely
sialylated saccharide side chains. Antibodies induced with
MUC1 isolated from tumor tissues
[
4,5]
were used to identify
the peptide motif PDTRPAP as an immundominant epitope
[
7]
within the MUC1 tandem repeat. The specificity of these
anti-MUC1 antibodies was verified with synthetic Tn- and T-
[
8,9]
antigen glycopeptides.
Moreover, saturation transfer dif-
ference NMR analyses revealed the conformation of a Tn
antigen pentapeptide from MUC1 bound to a monoclonal
[
10]
antibody.
In contrast to such studies, which proved valuable for
analytical purposes, immunizations with MUC1 extracted
from tumor tissues as a vaccine appear not to be promising,
because MUC1 from biological isolates generally displays
microheterogeneity and contains structural elements that also
occur on healthy cells.
For this reason we focused our attention on the develop-
ment of anticancer vaccines based on defined synthetic Tn, T,
[
11,12]
[13]
and sialyl–Tn glycopeptides
from MUC1. A construct,
in which a glycopeptide sequence from the MUC1 tandem
repeat carrying a sialyl–Tn side chain is conjugated through a
polar spacer amino acid with a partial T-cell epitope from
tetanus toxoid, was used to induce the proliferation of
cytotoxic T cells in cell cultures of peripheral blood lympho-
Scheme 1. a) a,a-Dimethoxytoluene, cat. p-TsOH, CH CN, 3 h;
3
b) Hg(CN) , CH Cl /CH NO (2:3), 4-Š molecular sieves;
2
2
2
3
2
c) 1. NaOMe, MeOH, pH 8.5–9, 8 h; 2. Fmoc-OSu, dioxane/H O (1:1),
2
NMM; d) MeSBr, AgOTf, CH CN/CH Cl (2:1), À688C, 3-Š molecular
3
2
2
[
14]
cytes (PBLs).
This effect occurred exclusively in the
sieves; e) 80% AcOH (aq), 808C; f) CF COOH, anisole (10:1), 2 h.
3
presence of antigen-presenting cells. Apart from cytotoxic
T-cell response, strong T-helper-cell-dependent antibody
production is of crucial importance for the development of
an efficient antitumor vaccine and requires the use of
glycopeptide antigens conjugated with carrier proteins such
as BSA or KLH (keyhole limpet hemocyanine).
Herein we describe the synthesis of a series of glycopep-
tide antigens that bear the sialyl-Tn, (2,6)-sialyl-T, (2,3)-sialyl-
T, or the glycophorin (2,3-2,6-bissialyl)-Tantigens and contain
the partial or complete tandem repeat sequence of MUC1.
We also report methods to couple these tumor-selective cell
surface structures to carrier proteins or to bind them to
microtiter plates. The synthetic strategy relies on the synthesis
of the corresponding O-glycosyl amino acids, followed by
sequential solid-phase glycopeptide synthesis and subsequent
conjugation to proteins or immobilization.
complex of the tumor-associated MUC1 saccharide antigens,
the (2,3-2,6-bissialyl)-T–threonine conjugate. The N-Fmoc
[
16]
protected Tn antigen threonine tert-butyl ester 1 depro-
tected at its glycan moiety served as common starting material
for the synthesis of all tumor-associated mucin carbohydrate
antigens. The base and acid sensitivity of the Fmoc group and
the tert-butyl ester must be carefully taken into account in all
protecting-group manipulations and glycosylation reactions.
The precursor 1 was treated with a,a-dimethoxy toluene in
acetonitrile in the presence of catalytic p-toluenesulfonic acid
at pH 4 to give 4,6-benzylidene acetal 2. Although activation
[
17]
of a glycosyl trichloroacetimidate
with trimethylsilyl tri-
fluormethanesulfonate for 3-b-galactosylation is usually very
efficient, in this case the benzylidine acetal and tert-butyl ester
were cleaved or, after addition of sufficient amounts of
molecular sieves, predominantly gave the corresponding
A range O-glycosyl amino acids were synthesized accord-
[
15]
ing to a biomimetic strategy
starting from the same
[
18]
precursor. Scheme 1 illustrates the preparation of the most
orthoester. The most successful route involved activation
Angew. Chem. Int. Ed. 2005, 44, 7624 –7630
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
7625

Communications
Scheme 2. a) TFA, TIS, H O (15:0.9:0.9); b) H , Pd/C (10%), MeOH; c) NaOMe/MeOH, pH 9.5; d) NaOH (aq), pH 11.5, 1 h; then AcOH.
2
2
DIPEA=diisopropylethylamine.
[
19]
of the 6-O-benzyl protected galactosyl bromide 3
with
The selective O-deacetylation of 4 proved to be partic-
ularly challenging. On a 1-g scale, use of catalytic NaOMe in
methanol under careful adjustment of the pH value to 8.5
furnished 5 in 62% yield. On a larger scale (5 g) and at
pH 8.6–9, not only 5 (39%) but also the N-deprotected
mercury cyanide (according to the procedure of Helferich and
[
20]
Wedemeyer ) in nitromethane/dichloromethane which ster-
eoselectively furnished the T-antigen–threonine conjugate 4
in high yield.
7626 www.angewandte.org
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2005, 44, 7624 –7630

Angewandte
Chemie
conjugate (46%) were formed. Subsequent N-acylation of the
latter with 9-fluorenyl-methoxycarbonyl-N-hydroxy-succini-
mide gave more of the desired product (36%), resulting in an
overall yield of 75% of 5. Sialylation of 5 with xanthate 6 of
After completion of the peptide sequence, the trityl linker
and the acid-labile side-chain protecting groups were cleaved
simultaneously with trifluoroacetic acid (TFA), triisopropyl-
[
32]
silane (TIS), and small amounts of water. This cleavage
procedure liberated neither the expected glycoeicosapeptide
12 with unaltered carbohydrate side chain nor the completely
O-acetylated analogue 13 formed through the capping
[
14]
the peracetylated sialic acid benzyl ester
methylsulfenyl triflate gave 7 with nearly complete regio-
activated with
[
21]
and stereoselectivity. After selective cleavage of the benzyl-
[
22]
[33]
idene acetal with aqueous acetic acid (80% v/v) at 808C to
afford 8, a second sialylation step under identical conditions
gave the bissialyl-T-antigen–threonine conjugate 9. Although
the conversion remained incomplete after 3.5 h, 9 (33%) as
well as some of the corresponding b-anomer (3%) were
reactions. Instead, glycopeptide 14, which bears a lactone
structure formed between the 2,3-linked sialic acid and the 2-
OH group of the galactose moiety, was isolated in an overall
yield of 38% after approximately 40 steps. Subsequent
hydrogenolysis of the benzyl ester and benzyl ether, trans-
esterification with NaOMe/methanol (pH 9.5), opening of the
lactone with aqueous NaOH (pH 11.5), neutralization with
acetic acid, lyophilization, and purification by semiprepara-
tive HPLC furnished the desired MUC1 tandem repeat
[
23]
isolated by preparative HPLC. Acidolysis of the tert-butyl
ester with trifluoroacetic acid/anisole (10:1) furnished the
[
24]
Fmoc-protected bissialyl-T–threonine building block 10,
which was incorporated into the solid-phase synthesis without
[
25]
[34]
further O-acetylation.
Of the other tumor-associated
glycopeptide 15 in 75% yield (based on 14).
glycosyl amino acid antigens, the Tn antigen 1, T antigen 4,
and 2,3-sialyl-T antigen 7 are directly accessible by the
synthetic route shown in Scheme 1, while the sialyl-Tn
In analogous procedures, the MUC1 eicosapeptide 16 as
well as the complete tandem repeat glycopeptides from
MUC1 carrying the other important tumor-associated sialyl
saccharide antigens, sialyl-Tn (17), 2,6-sialyl-T (18), and 2,3-
sialyl-T (19), were synthesized in significantly higher overall
yields (Scheme 3). No formation of a lactone was observed
during the acidolysis of the trityl linker in any of these
syntheses.
[
14,16]
antigen is obtained by regioselective sialylation of 1
and
the 2,6-sialyl-Tantigen through regioselective sialylation after
[
15,26]
cleavage of the benzylidene acetal in 4.
These building blocks were all subsequently applied in the
solid-phase glycopeptide synthesis of various MUC1 tandem
repeat glycopeptides, as illustrated in Scheme 2 for the
bissialyl-T antigen MUC1 glycopeptide. Fmoc-proline was
According to the procedure illustrated in Scheme 2, the
dodecapeptide 20 equipped with a triethylene glycol spacer
[
27]
[14]
coupled through a trityl linker to a Rapp-Tentagel poly-
with an amino group as well as the analogous sialyl-Tn
[
28]
mer and used to assemble the protected nonapeptide 11
dodecapeptide 21 from MUC1 were synthesized to serve as
model compounds for conjugation with bovine serum albu-
min (BSA) and for immobilization on microtiter plates
[
29]
from MUC1 according to the Fmoc strategy. After cleavage
of the Fmoc group with piperidine/N-methylpyrrolidone
[
35]
(
NMP) (1:4), the O-glycosyl amino acid 10 (1.05 equiv) was
coupled through activation with O-(7-azabenzotriazol-1-yl)-
N,N,N’,N’-tetramethyluronium hexafluorophosphate
HATU), 1-hydroxy-7-azabenzotriazole (HOAt),
(Scheme 4). For this purpose, the N-terminal Fmoc group
was removed from the resin-bound peptide and all acid-labile
side-chain protecting groups were cleaved during the acid-
olysis of the trityl anchor. The synthesis of sialyl-Tn glyco-
peptide 21 required the additional removal of the carbohy-
drate-protecting groups by hydrogenolysis and transesterifi-
cation catalyzed by NaOMe after cleavage from the resin.
To conjugate the synthetic MUC1 peptides and glycopep-
tides with proteins, recourse was made to the procedure
[
30]
(
and N-
methylmorpholine (NMM) in NMP. All non-glycosylated
Fmoc-protected amino acids were coupled with O-(1H-
benzotriazole-N,N,N’,N’-tetramethyluronium
phosphate (HBTU)/1-hydroxybenzotriazole (HOBt), and
hexafluoro-
[
31]
Hünig base (iPr NEt) in DMF. The remaining free amino
2
[
36]
components were capped with acetic anhydride/HOBt/
described by Tietze et al. for the conjugation of a lactos-
[
31c]
iPr NEt after each coupling cycle.
amine phenyl glycoside. The amino groups of the spacers of 20
2
Scheme 3. Eicosapeptide 16 and the complete tandem repeat glycopeptides from MUC1 carrying sialyl-Tn (17), 2,6-sialyl-T (18), and
,3-sialyl-T (19).
2
Angew. Chem. Int. Ed. 2005, 44, 7624 –7630
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
7627

Communications
Scheme 4. a) Ethanol/water (1:1), Na CO , pH 8.0, 2 h, preparative HPLC; b) sodium borate (0.07m), NaHCO (0.035m), pH 9, 24 h, gel filtration
2
3
3
chromatography (Sephadex-G25 PD10 column), dialysis against H O.
2
and 21 were converted into the corresponding squaric
material in ELISA tests when washing with water, which had
previously caused difficulties while working with glycopep-
tide antigens.
[
37]
monoamides 22 and 23
by using diethyl squarate (3,4-
diethoxy-3-cyclobutene-1,2-dione) in ethanol/water at pH 8.
After purification by preparative HPLC, the activated
peptide structures were coupled to BSA in a sodium borate
buffer at pH 9.
The immobilized and conjugated MUC1 glycopeptide
antigens not only provide vaccines with a highly defined
structure representing tumor-selective surface antigens but
also provide tools for the verification of the structural
selectivity of an immune response. The initial results are
Conjugation of peptide 22 resulted in a coupling yield of
approximately 60% within 24 h and furnished vaccine 24 with
an average degree of incorporation of 12 peptide molecules
per molecule of protein as determined by MALDI-TOF MS.
In the case of glycopeptide 23 the coupling to furnish 25
proceeded more slowly, and led to the incorporation of only
half as many antigen molecules. Furthermore, the glycopep-
tide antigen equipped with the spacer was covalently immo-
[
40]
promising and encourage further exploration of the entire
spectrum of structures characterized by the synthetic glyco-
peptide antigens such as 17, 18, 19, 21, 23, and 25 for the
development of antitumor vaccines.
[
38]
bilized on microtiter plates functionalized with a secondary
amine 26 (Scheme 5). The covalent linkage of the MUC1
antigen to the microtiter plates (see 27) prevents the loss of
Received: May 10, 2005
Published online: October 25, 2005
[
39]
7628 www.angewandte.org
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2005, 44, 7624 –7630

Angewandte
Chemie
Scheme 5. a) sodium borate (0.07m), NaHCO (0.035m), pH 9.
3
Keywords: antigens · antitumor agents · carbohydrates ·
glycopeptides · synthetic vaccines
Price, P. M. Williams, S. B. J. Tendler, H. Kunz, Bioorg.Med.
Chem. 1998, 6, 1531.
12]B. Liebe, H. Kunz, Angew.Chem. 1997, 109, 629; Angew.Chem.
Int.Ed.Engl. 1997, 36, 618.
13]Other groups also synthesized mucin glycopeptides with Tn, T,
and sialyl-Tn antigen saccharides: a) E. Meinjohanns, M.
Meldal, A. Schleyer, H. Paulsen, K. Bock, J.Chem.Soc.
Perkin Trans. 1 1996, 985; b) S. D. Kuduk, J. B. Schwarz, X.-T.
Chen, P. W. Glunz, D. Sames, G. Ragapunti, P. O. Livingston, S. J.
.
[
[
[
[
1]G. F. Springer, Science 1984, 224, 1198.
2]H. Kunz, S. Birnbach, Angew.Chem. 1986, 98, 354; Angew.
Chem.Int.Ed.Engl. 1986, 25, 360.
[
3]a) W. Dippold, A. Steinborn, K.-H. Meyer zum Büschenfelde,
Environ.Health Perspect. 1990, 88, 255; W. Dippold, A. Stein-
born, S. Birnbach, H. Kunz, unpublished results; A. Steinborn,
PhD thesis, Universität Mainz, 1990; b) S. Dziadek, H. Kunz,
Chem.Rec. 2004, 4, 308.
Danishefsky, J.Am.Chem.Soc.
Schwarz, S. D. Kuduk, X.-T. Chen, D. Sames, P. W. Glunz, S. J.
Danishefsky, J.Am.Chem.Soc. 1999, 121, 2662; d) M. A.
1998, 120, 12474; c) J. B.
Reddish, L. Jackson, R. R. Koganti, D. Qui, W. Hong, B. M.
Longenecker, Glycoconjugate J. 1997, 14, 549.
[
4]a) S. Gendler, C. A. Lancaster, J. Taylor-Papadimitriou, T.
Duhig, N. Peal, J. Burchell, L. Pemberton, E. N. Lalani, P.
Wilson, J.Biol.Chem. 1990, 265, 15286; b) for a short overview,
see: J. Taylor-Papadimitriou, J. M. Burchell, D. W. Miles, M.
Dalziel, Biochim.Biophys.Acta 1999, 1455, 301.
[
14]S. Keil, C. Claus, W. Dippold, H. Kunz, Angew.Chem. 2001, 113,
379; Angew.Chem.Int.Ed. 2001, 40, 366.
[
15]a) C. Brocke, H. Kunz, Synlett 2003, 2052; b) S. Dziadek, H.
Kunz, Synlett 2003, 1623.
16]B. Liebe, H. Kunz, Helv.Chem.Acta. 1997, 80, 1473.
17]a) R. R. Schmidt, J. Michel, Angew.Chem. 1980, 92, 763; Angew.
[
5]D. M. Swallow, S. J. Gendler, B. Griffith, G. Corney, J. Taylor-
Papadimitriou, Nature 1987, 328, 82.
[
[
[
6]a) I. Brockhausen, Biochim.Biophys.Acta 1999, 1473, 67; I.
Brockhausen, Biochem.Soc.Trans. 2003, 31, 318; b) K. O. Lloyd,
J. Burchell, V. Kundryashov, B. W. Yin, J. Taylor-Papadimitriou,
J.Biol.Chem. 1996, 271, 33325; c) J. M. Burchell, A. Mungul, J.
Taylor-Papadimitriou, J.Mammary Gland.Biol.Neoplasia 2001,
Chem.Int.Ed.Engl.
1980, 19, 731; b) R. R. Schmidt, Angew.
Chem. 1986, 98, 213; Angew.Chem.Int.Ed.Engl. 1986, 25, 212.
18]T. Reipen, H. Kunz, Synthesis 2003, 2487.
19]M. Lergenmüller, Y. Ito, T. Ogawa, Tetrahedron 1988, 44, 1381.
20]B. Helferich, F. Wedemeyer, Justus Liebigs Ann.Chem. 1949,
[
[
[
6, 355.
563, 139.
[
[
[
7]a) J. Burchell, J. Taylor-Papadimitriou, M. Boshell, S. Gendler, T.
Duhig, Int.J.Cancer 1989, 44, 691; b) M. R. Price, F. Hudecz, C.
OꢀSullivan, R. W. Baldwin, P. M. Edwards, S. B. J. Tendler, Mol.
Immunol. 1990, 62, 795.
8]a) G. F. Hanisch, S. Müller, Glycobiology 2000, 10, 439; b) S.
Müller, F. G. Hanisch, J.Biol.Chem. 2002, 277, 26103; c) U.
Karsten, N. Serttas, H. Paulsen, A. Danielczyk, S. Goletz,
Glycobiology 2004, 14, 681.
[
[
21]F. Dasgupta, P. Garegg, Carbohydr.Res. 1988, 177, C13.
22]N. Mathieux, H. Paulsen, M. Meldal, K. Bock, J.Chem.Soc.
Perkin Trans. 1 1997, 2359.
2
2
[
23] 9: [a] = 16.6 (c = 1, CHCl ); MALDI-TOF MS (dhb, positive):
D
3
+ +
m/z: 1975.2 [M+Na] , 1991.2 [M+K] ; HR ESI-TOF MS
1
(
(
(
positive): calcd: 1973.7271; found: 1973.7257; H NMR
400 MHz, CDCl , COSY, TOSY, HMQC, HMBC): d = 4.81
J1,2 = 3.9 Hz; 1-H), 3.32 (d, 1H, J3’,4’ = 2.7 Hz; 4’-H), 2,71 (dd,
3
9]S. von Mensdorff-Pouilly, F. G. M. Snijdewint, A. A. Ver-
straeten, R. H. M. Verheijen, P. Kenemans, Int.J.Biol.Markers
1
1
H, J3eq’’,3ax’’ = 12.9 Hz, J3eq’’,4’’ = 4.7 Hz; 3’’-H ), 2.54 ppm (dd,
eq
H, J
= 12.7 Hz, J3eq’’’,4’’’ = 4.7 Hz; 3’’’-H ).
eq
2000, 15, 343.
3eq’’’, 3ax’’’
2
2
[
24] 10: [a] = 20.3 (c = 1, CHCl ); HR ESI-TOF MS (positive):
[
10]H. Möller, N. Serttas, H. Paulsen, J. M. Burchell, J. Taylor-
Papadimitriou, B. Meyer, Eur.J.Biochem. 2002, 269, 1444.
11]a) O. Seitz, H. Kunz, Angew.Chem. 1995, 107, 901; Angew.
D
3
+
calcd: 1917.6645; found: 1917.6743 [M+Na] .
[
[25]The removal of O-acetyl groups, in particular those on the 4-OH
x
Chem.Int.Ed.Engl.
1995, 34, 803; b) M. Leuck, H. Kunz, J.
groups of galactose moieties in sialyl-Lewis glycopeptides (M.
Prakt.Chem. 1997, 339, 332; c) P. Braun, G. M. Davies, M. R.
Rösch, H. Herzner, W. Dippold, M. Wild, D. Vestweber, H.
Angew. Chem. Int. Ed. 2005, 44, 7624 –7630
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 7629

Communications
Kunz, Angew.Chem. 2001, 113, 3954; Angew.Chem.Int.Ed.
[
15]
2
001, 40, 3836) and in sialyl-T glycopeptides can be difficult.
[
[
[
[
[
26]C. Brocke, H. Kunz, Synthesis 2004, 525.
27]J. M. J. FrechØt, K. E. Haque, Tetrahedron Lett. 1975, 16, 3055.
28]E. Bayer, W. Rapp, Chem.Pept.Proteins 1986, 3, 3.
29]L. A. Carpino, G. Y. Han, J.Am.Chem.Soc. 1970, 92, 5748.
30]L. A. Carpino, D. Jonescu, A. El-Falham, J.Org.Chem. 1996, 61,
2460.
[
31]a) R. Knorr, A. Trzeciak, W. Bannwarth, D. Gillessen, Tetrahe-
dron Lett. 1989, 30, 1927; b) V. Dourtoglou, B. Gross, V.
Lambropoulou, C. Ziondrou, Synthesis 1984, 572; c) W. König,
R. Geiger, Chem.Ber. 1970, 103, 788.
[
32]D. A. Pearson, M. Blanchette, M. L. Baker, C. A. Guidon,
Tetrahedron Lett. 1989, 30, 2739.
+
[
33] 14: MALDI-TOF MS (dhb, positive): m/z: 3376.0 [M+H] ,
+
+
1
3
398.0 [M+Na] , 3414.3 [M+K] ; H NMR (600 MHz,
[
D ]DMSO, COSY, TOCSY, HMQC, HMBC): d = 4.76 (2’-H),
6
4
.21 (4’-H), 4.01 ppm (3’-H).
2
2
[
34] 15: 19.4 mg; [a] = À117.4 (c = 1, H
2
O); MALDI-TOF MS (dhb,
D
+
1
positive): m/z: 2876.6 [M+H] ; H NMR (600 MHz, [D ]DMSO,
6
COSY, HMQC, HMBC): d = 4.79 (d, 1H, J1,2 = 2.1 Hz, 1-H),
2
.66 ppm (dd, 1H, J3eq’,3ax’ = 13.7 Hz, J3eq,4’ = 6.7 Hz; 3’’-H );
eq
1
3
C NMR (150.9 MHz, [D ]DMSO, HMQC, HMBC): d = 104.5
6
(
C1’), 98.8 (C1), 98.4 (C2’’), 98.1 ppm (C2’’’).
[
35]According to our previous experience, the incorporation of a
flexible, hydrophilic spacer is important for immunological
[
14]
evaluation of synthetic vaccines.
[
[
36]L. F. Tietze, C. Schröder, S. Gabius, U. Brinck, A. Goerlach-
Graw, H.-J. Gabius, Bioconjugate Chem. 1991, 2, 148.
2
6
37] 23: [a]D = À68.4 (c = 1, H O); MALDI-TOF MS (dhb, positive):
2
+
1
m/z: 1991.4 [M+H] ; H NMR (400 MHz, CD OD, COSY,
3
HMQC): d = 4.97 (d, 1H, J1,2 = 3.5 Hz; 1-H), 4.76–4.64 (m, 3H;
CH O-squarate, H -Arg), 2.66 ppm (dd, 1H, J = 12.7 Hz,
2
a
3eq’,3ax’
J3 , = 4.7 Hz; 3’-H ).
eq’ 4’
eq
[
[
[
38]A. Bergh, B.-G. Magnusson, J. Ohlsson, U. Wellmar, U. J.
Milsson, Glycoconjugate J. 2005, 18, 615.
39] 27: For carbohydrate detection with phenol/H SO , see refer-
2
4
ence [2].
40]See the following Communication in this issue: S. Dziadek, A.
Hobel, E. Schmitt, H. Kunz, Angew.Chem. 2005, 117, 7803;
Angew.Chem.Int.Ed. 2005, 44, 7630.
7630 www.angewandte.org
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2005, 44, 7624 –7630