Biomimetic synthesis of the tumor-associated (2,3)-sialyl-T antigen and its incorporation into glycopeptide antigens from the mucins MUC1 and MUC4

Article abstract of DOI:10.1002/chem.200400228

Glycoproteins on epithelial tumor cells often exhibit aberrant glycosylation profiles. The incomplete formation of the glycan side chains resulting from a down-regulated glucosamine transfer and a premature sialylation results in additional peptide epitop

Full text of DOI:10.1002/chem.200400228

FULL PAPER
Biomimetic Synthesis of the Tumor-Associated (2,3)-Sialyl-T Antigen and Its
Incorporation into Glycopeptide Antigens from the Mucins MUC1 and
MUC4
Sebastian Dziadek, Constanze Brocke, and Horst Kunz*[a]
Dedicated to Professor Joachim Engels on the occasion of his 60th birthday
Abstract: Glycoproteins on epithelial
tumor cells often exhibit aberrant gly-
cosylation profiles. The incomplete for-
mation of the glycan side chains result-
ing from a down-regulated glucosamine
transfer and a premature sialylation re-
sults in additional peptide epitopes,
which become accessible to the
immune system in mucin-type glyco-
proteins. These cancer-specific struc-
ture alterations are considered to be a
promising basis for selective immuno-
logical attack on tumor cells. Among
the tumor-associated saccharide anti-
gens, the (2,3)-sialyl-T antigen has been
identified as the most abundant glycan,
found in several different carcinoma
cell lines. According to a linear biomi-
metic strategy, the (2,3)-sialyl-T antigen
was synthesized by a stepwise glycan
chain extension of a protected galactos-
amine-threonine precursor. For the
construction of immunostimulating an-
tigens combining both peptide and sac-
charide motifs, this antigen was incor-
porated into glycopeptide partial struc-
tures from the mucins MUC1 and
MUC4 by sequential solid-phase syn-
thesis.
Keywords: antigens ¥ glycopeptides ¥
MUC1
¥
MUC4
¥
solid-phase
synthesis
Introduction
trisaccharide
(bGal-(1!3)-[bGlcNAc-(1!6)-]aGalNAc),
which can be further extended by the addition of polylactos-
Glycoproteins which are major constituents on epithelial
cell surfaces are decisively involved in various important
physiological processes such as cell adhesion or signal trans-
duction.[1] In a number of cases, the carbohydrate portions
of membrane glycoproteins are key elements in biological
selectivity:carbohydrates in the blood group substances, for
instance.[2] During glycoprotein biosynthesis, the post-trans-
lational O-glycosylation of the protein backbone proceeds
in different compartments of the Golgi apparatus. It is initi-
ated by the enzymatic coupling of N-acetyl-galactosamine to
serine or threonine residues (see Scheme 1). As the glyco-
protein passes through the Golgi, stepwise saccharide chain
extension leads to the formation of complex, branched
glycan structures. Galactosylation of the central N-acetyl-
galactosamine moiety furnishes the core 1 saccharide struc-
ture bGal-(1!3)-aGalNAc-O-Ser/Thr (also called T-anti-
gen), which acts as a substrate for b1,6-GlcNAc transferases.
The addition of b1,6-N-acetyl-glucosamine yields the core 2
Scheme 1. Glycopeptide biosynthesis in cancer cells. GalNAcT:UDP- N-
acetyl-a-d-galactosamine:polypeptide N-acetylgalactosaminyl transferase.
b1,3GalT: Core 1 b1,3-galactosyl transferase. C2GnT-I: Core 2 b1,6-N-
acetylglucosaminyl transferase-I. ST6GalNAc-I:CMP-Neu5AcG: alNAc-
[a] Dipl.-Chem. S. Dziadek, Dr. C. Brocke, Prof. Dr. H. Kunz
Institut f¸r Organische Chemie der Universit‰t Mainz
Duesbergweg 10 14, 55128 Mainz (Germany)
Fax :( +49)6131-39-24786
R
a2,6-sialyl transferase-I. ST6GalNAc-II:CMP-Neu5AGc:alNAc-R
a2,6-sialyl transferase-II. ST3Gal-I:CMP-Neu5AGc:al
a2,3-sialyl transferase-I.
b1,3GalNAc-R
E-mail:hokunz@uni-mainz.de
4150
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/chem.200400228
Chem. Eur. J. 2004, 10, 4150 4162

4150 4162
amine chains. Chain growth is usually terminated by the at-
tachment of sialic acid or fucose or by sulfation.[3] Unlike in
healthy cells, the activity of certain glycosyltransferases, in
particular b1,6-GlcNAc transferase, is drastically down-regu-
lated in epithelial tumor cells, whereas the activity of sialyl-
transferases is strongly increased, often resulting in a prema-
ture sialylation.[4] Consequently, glycoproteins in cancer cells
carry cryptic, incomplete glycan side chains.[5] This results in
the exposure of additional peptide epitopes, masked in
normal cells, which hence become accessible to the immune
system. The TN, T, sialyl-TN and (2,3)-sialyl-T structures con-
stitute important examples of such tumor-associated saccha-
ride antigens (Scheme 1). Among these, the (2,3)-sialyl-T an-
tigen has been identified as the most abundant glycan found
in different tumor cell lines such as the breast cancer cell
line T47D[6] and the gastric carcinoma cell lines HT-29 and
K562.[7]
Cancer immunotherapy selectively targeting the tumor-as-
sociated glycoprotein structure alterations–deficient glyco-
sylation and, thus, exposure of additional peptide epitopes–
would certainly be attractive. In this context, glycopeptides
containing partial structures of cancer-associated cell-surface
glycoproteins combining both tumor-associated saccharide
and peptide structural information are considered to be
promising candidates for the construction of tumor-selective
immunostimulating antigens. Mucin-type glycoproteins con-
stitute suitable target molecules for the induction of tumor-
selective immune responses.
the mucins MUC1 and MUC4[24] are therefore promising
candidates for the development of tumor-selective vaccines.
Results and Discussion
The tumor-associated (2,3)-sialyl-T antigen was synthesized
by an efficient linear strategy, which mimicks the enzymatic
glycan chain extension taking place in the Golgi apparatus
in cancer cells,[4] and was subsequently incorporated into
glycopeptide structures from the tandem repeat regions of
the mucins MUC1 and MUC4. For this purpose, a suitably
protected threonine derivative was first coupled with N-
acetyl galactosamine. From this TN antigen-threonine conju-
gate, the (2,3)-sialyl-T building block was assembled by step-
wise saccharide chain extension and utilized in solid-phase
27]
glycopeptide syntheses.[25
In contrast to other strategies
reported earlier,[28 the complex sialic acid was introduced,
30]
as in nature, at a late stage of the synthesis.
Synthesis of the (2,3)-sialyl-T threonine building block:Ac-
cording to this biomimetic strategy, the (2,3)-sialyl-T threo-
nine building block for solid-phase glycopeptide synthesis
was prepared by starting from the preformed N-acetylgalac-
tosamine threonine conjugate 3 (Scheme 2).[31,32] To provide
a glycosyl acceptor suitable for galactosylation at the 3-OH
position, 3 was converted into the corresponding benzyli-
dene derivative 4 by de-O-acetylation and subsequent for-
mation of the 4,6-benzylidene acetal. The de-O-acetylation
was carefully carried out under Zemplÿn conditions[33] at
pH 8.5,[31] followed by protection of the 4-OH and 6-OH
groups with a,a-dimethoxytoluene and a catalytic amount of
p-toluenesulfonic acid as reagents. The benzylidene deriva-
tive 4 was isolated in 44% yield (over two steps) when N,N-
dimethyl formamide was used as the solvent in the second
step and methanol was removed continuously from the reac-
tion mixture at 508C and reduced pressure (15 mbar). For
the same reaction, the yield was increased to 75% (two
steps) in acetonitrile as the solvent and at ambient tempera-
ture.[34]
Mucins are heavily O-glycosylated glycoproteins ex-
pressed on the surfaces of many types of epithelial cells.[8,9]
Their main function is to provide lubrication, as well as pro-
tection from proteolytic degradation and invasion of micro-
organisms. Of particular interest are the mucins MUC1 and
MUC4, which both exist in membrane-bound forms. The
14]
mucin MUC1,[10 which was the first mucin-type glycopro-
tein to be defined by murine monoclonal antibodies, is a
ubiquitously distributed integral membrane glycoprotein
found on epithelial cells in a variety of tissues. The extracel-
lular domain consists of tandem repeats comprising 20
amino acids of the sequence HGVTSAPDTRPAPGSTAP-
PA (1), including five potential O-glycosylation sites. First
isolated from tracheobronchial tissue in 1991,[15] the human
mucin MUC4 contains the largest apoprotein of all known
mucins.[16] Its heterodimeric structure consists of a mucin-
characteristic a-chain and a membrane-associated b-chain,
which are connected through the proteolytic cleavage region
The galactosylation of TN-threonine conjugate 4 was one
of the key steps in the synthesis of the sialyl-T threonine an-
tigen. The reaction was optimized by comparing different
glycosyl donors derived from 1,2,3,4-tetra-O-acetyl-6-O-
benzyl galactopyranose 5[35] (Table 1). First, 5 was converted
38]
into the anomeric trichloroacetimidate a-6,[36
which was
GDPH.[17] The extracellular a-chain, which contains
region rich in proline, threonine, and serine, is composed of
repetitive sequence of 16 amino acids (TSSAST-
a
subjected to glycosylation by Schmidt×s procedure.[39,40] The
galactosylation of 4 with trichloroacetimidate a-6 was car-
ried out at ꢀ158C by the use of catalytic amounts of trime-
thylsilyl triflate in 1,2-dichloroethane as the promoter. After
purification by flash chromatography, a mixture of the de-
sired product 7 and the corresponding orthoester 8 was iso-
lated in a ratio of 1:2.7. Separation of the two components
was achieved by semi-preparative RP-HPLC and gave the
b-glycosylation product 7 in a yield of 21%, together with
58% of the orthoester 8.
a
GHATPLPVTD, 2). Both MUC1 and MUC4 adopt extend-
ed structures, and so are major components of the glycoca-
lyx.
Abnormal expression of the mucins MUC1 and MUC4
has been observed in tumor cells of various tissues, including
lung,[18] colon,[19] pancreatic,[20,21] ovarian, and breast can-
cers.[22,23] In addition, MUC4 is the only tumor-associated
mucin exclusively occurring on pancreatic adenocarcinoma
cells. Tumor-associated glycopeptide partial structures from
Second, treatment of galactose derivative 5 with a mixture
of ethanethiol and boron trifluoride etherate in dichlorome-
thane afforded ethylthio glycoside 9 in 63% yield (a/b ratio
Chem. Eur. J. 2004, 10, 4150 4162
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4151

H. Kunz et al.
FULL PAPER
Scheme 2. Reagents and conditions:a) i) MeOH, NaOMe, pH 8.5, 3 h; ii) DMF, a,a-dimethoxytoluene, PTSA, 15 mbar, 508C, 2 h, 44%. b) i) MeOH,
NaOMe, pH 8.5, 3 h; ii) MeCN, a,a-dimethoxytoluene, PTSA, room temperature, 15 h, 75%. c) MeOH, NaOMe, pH 8.5, 12 h, 62%. d) AcOH (80%),
808C, 1 h, 82%. e) pyridine/Ac2O (2.3:1), DMAP, 6 h, 87%. f) TFA, anisole, quant. DMAP = N,N-dimethylaminopyridine; DMF = dimethylforma-
mide; DTBP = di-tert-butylpyridine; PTSA = p-toluenesulfonic acid; TFA = trifluoroacetic acid.
Table 1. Synthesis of glycosyl donors 6, 9, and 10 and subsequent galactosylation of TN-threonine conjugate 4.
1.2:1). The glycosylation of TN-
threonine conjugate 4 with b-
thio glycoside b-9 was promot-
ed with N-iodosuccinimide and
catalytic amounts of trifluoro-
methanesulfonic acid in di-
chloromethane at 08C and re-
sulted in the formation of 52%
of the b-glycosylation product 7
(Scheme 2).
Compound
R
Conditions A
Conditions B
Yield of 7 [%]
a-6
1. H2NNH2¥AcOH, DMF, 83% cat. TMSOTf, MS
MS 4 ä, ClCH2CH2Cl
2. Cl3CCN, DBU,
CH2Cl2, 53%
21[a]
Finally, different activation
procedures for the glycosylation
a,b-9
SEt
EtSH, BF3¥Et2O,
CH2Cl2, 63%
cat. TfOH, NIS,
MS 4 ä, CH2Cl2,
52
of TN-threonine conjugate
4
(a/b=1.2:1)
08C to 208C
a-10
a-10
Br
Br
33% HBr in glacial acid,
CH2Cl2, 08C, 49%
33% HBr in glacial acid,
CH2Cl2, 08C, 49%
AgOTf, MS 4 ä,
66
93
were explored with galactosyl
bromide a-10 as donor, this
compound also being obtained
from galactose derivative 5.[41]
According to the classical pro-
cedure developed by Koenigs
and Knorr,[42] the 3-b-galactosy-
CH2Cl2, ꢀ408C to +208C
Hg(CN)2, MS 4 ä,
CH3NO2/CH2Cl2, 08C to 208C
[a] In addition, 58% of the corresponding orthoester 8 were formed.
lation of 4 with a-10 was carried out with two equivalents of
silver triflate as the promoter at ꢀ408C in dichloromethane
and gave the product 7 of the b-glycosylation reaction in
66% yield.
Alternatively, the same glycosylation reaction was per-
formed by Helferich×s procedure[43] with mercury(ii) cyanide
for activation at ambient temperature and a mixture of ni-
tromethane and dichloromethane as solvents. Under these
4152
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 4150 4162

Tumor-Associated (2,3)-Sialyl-T Antigen
4150 4162
conditions, the desired b-product 7 was formed selectively in
93% yield, due to the neighboring group effect of the 2-ace-
toxy function of the galactosyl donor.
selective a-sialylation was achieved in a yield of 58% when
methylsulfenyl triflate, produced in situ from methylsulfenyl
bromide and silver triflate,[49] was used as reagent for activa-
tion of xanthate 15.[50]
The de-O-acetylation of the disaccharide derivative 7,
without the Fmoc group being affected or b-elimination of
the saccharide promoted, was achieved by careful, pro-
longed treatment with sodium methoxide in methanol at
pH 8.5 and resulted in the formation of a 62% yield of the
disaccharide acceptor 11. The regio- and stereoselective gly-
cosylation at the 3’-OH position of the galactose moiety of
this compound with a sialic acid donor comprises another
pivotal step in the synthesis of the sialyl-T threonine build-
ing block. For this type of sialylation, sialic acid thio donors
have usually been employed in the Lewis antigen series.[44]
Therefore, N-acetylneuraminic acid benzyl ester 12 was first
converted into the corresponding ethylthio sialoside 13 in
85% yield. The regioselective 3’-sialylation of the disaccha-
ride 11 with ethylthio donor 13 was carried out in acetoni-
trile at ꢀ378C in the presence of N-iodosuccinimide and cat-
alytic amounts of trifluoromethanesulfonic acid, furnishing
the trisaccharide derivative 14 as an anomeric mixture. Sep-
aration of the anomers by flash chromatography gave a
38% yield of the desired a product and a 24% yield of the
corresponding b anomer (Table 2).
For the conversion of trisaccharide 14 into a (2,3)-sialyl-T
threonine conjugate suitable as a building block for sequen-
tial solid-phase glycopeptide synthesis, a number of protect-
ing group manipulations were required. Acidolytic cleavage
of the benzylidene acetal of 14 with 80% aqueous acetic
acid proceeded at 808C in a yield of 82% without affecting
the acid-labile tert-butyl ester to give 16. The ensuing O-ace-
tylation of the remaining hydroxy groups, including the ste-
rically hindered 4’-OH, was performed with a mixture of
pyridine and acetic acid catalyzed by N,N-(dimethylamino)-
pyridine in a yield of 87%. Subsequent acidolysis of the tert-
butyl ester of 17 with trifluoroacetic acid in dichlorome-
thane and anisole as a cation scavenger quantitatively pro-
vided the Fmoc-(2,3)-sialyl-T threonine building block 18.
Solid-phase synthesis of a MUC1 glycopeptide containing
the tumor-associated (2,3)-sialyl-T antigen:The Fmoc-(2,3)-
sialyl-T threonine building block 18 was incorporated in the
sequential solid-phase synthesis of a glycododecapeptide de-
rived from the tandem repeat sequence 1 of the epithelial
mucin MUC1. The glycopeptide
was assembled in an automated
synthesizer by the Fmoc strat-
Table 2. Glycosylation of 11 with sialosyl donors 13 and 15.
egy, with side chain O-tert-
butyl-protected
threonine/
serine and N-Pmc arginine
building blocks. NovaSyn Ten-
tagel coupled with the novel
Compound
R’
Conditions A
Conditions B
Yield of 14 [%]
fluoride-labile
PTMSEL
13
SEt
1) EtSH, BF3¥Et2O,
CH2Cl2, 85%
cat. TfOH, NIS, MS 3 ä,
CH3CN, ꢀ378C to ꢀ238C
38[a]
linker[51,52] loaded with the start
amino acid Fmoc-proline 19
(Scheme 3) was employed as
the resin. The PTMSEL anchor
can be cleaved by tetrabutylam-
monium fluoride trihydrate in
dichloromethane under almost
neutral conditions, thereby
avoiding side reactions such as
aspartimide rearrangements.
15
1) CH3COCl
2) KS(CS)OEt, EtOH, 49%
PhSCl, AgOTf, DTBP,
MS 3 ä, CH3CN/CH2Cl2, ꢀ688C
49[b]
58
15
1) CH3COCl
2) KS(CS)OEt, EtOH, 49%
MeSBr, AgOTf, MS 3 ä,
CH3CN/CH2Cl2, ꢀ688C
[a] Plus 24% of the corresponding b anomer. [b] Plus 2% of the corresponding b anomer.
The first eight amino acids of
Besides thio sialosides, sialosyl xanthates[45] have also
proven to be efficient glycosyl donors for sialylation reac-
tions. Xanthate 15[45,46] of the N-acetylneuraminic acid
benzyl ester was prepared in a yield of 49% from 12 by in-
termediate formation of the anomeric chloride. The glycosy-
lation of disaccharide conjugate 11 with sialosyl xanthate 15
was performed at low temperature (ꢀ688C) in a mixture of
acetonitrile/dichloromethane (2:1) in order to favor the ki-
netically controlled stereoselective formation of the a-sialo-
side 14. Activation of the xanthate with phenylsulfenyl tri-
flate, which was generated in situ from phenylsulfenyl chlo-
ride and silver triflate,[47] and the use of di-tert-butylpyridine
as a proton scavenger gave a 49% yield of the desired a2,3-
sialyl-T threonine conjugate 14.[48] Only a 2% yield of the
corresponding b anomer was produced. Completely stereo-
the MUC1 sequence were coupled by the standard proce-
dure. Piperidine in N-methylpyrrolidone (NMP) was used
for the removal of the temporary Fmoc protecting group in
each coupling cycle, followed by coupling with an excess
(20 equiv) of the next Fmoc-amino acid activated by
HBTU[53]/HOBt[54] and diisopropylethylamine (DIPEA) in
N,N-dimethylformamide (DMF). Unreacted amino groups
were capped after each cycle with acetic anhydride in the
presence of DIPEA and catalytic amounts of HOBt in
NMP. Subsequent coupling of only 1.4 equivalents of the
valuable Fmoc-(2,3)-sialyl-T threonine building block 18
was carried out manually over a period of 4 h with use of
HATU/HOAt[55] and N-methylmorpholine (NMM) in NMP
for activation. The final two Fmoc-amino acids were again
attached by the standard procedure, and the N-terminal
Chem. Eur. J. 2004, 10, 4150 4162
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4153

H. Kunz et al.
FULL PAPER
Scheme 3. Reagents and conditions:a) i) solid-phase glycopeptide synthesis (SPGS):Fmoc-removal (piperidine/NMP (20%)), coupling (1st 8th, 10t
h
11th:Fmoc-AA-OH (20 equiv), HBTU/HOBt/DIPEA, DMF, 9th: 18 (1.4 equiv), HATU/HOAt/NMM, DMF, 4 h), capping:Ac 2O, DIPEA, HOBt
(4:1:0.12). ii) TBAF¥3H2O (2î2.5 equiv), CH2Cl2, 45 min (each time), 39% (with respect to 19). b) TFA/TIS/H2O (15:0.9:0.9), 2 h. c) H2, Pd/C (10%),
MeOH, 23 h. d) NaOHaq (5 mm), 59 h (56% over three steps). HATU = O-(7-aza-benzotriazole-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophos-
phate, HBTU = O-(1H-benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate, HOAt = N-hydroxy-7-azabenzotriazole, HOBt = N-hy-
droxybenzotriazole; NMM = N-methylmorpholine; TBAF = tetrabutylammonium fluoride; TIS = triisopropylsilane; Pmc = 2,2,5,7,8-pentamethyl-
chroman-6-sulfonyl-; PTMSEL = (2-phenyl-2-trimethylsilyl)ethyl-linker.
Fmoc group was exchanged for an acetyl group. Treatment
with tetrabutylammonium fluoride in dichloromethane re-
leased the glycopeptide 20 from the resin without affecting
the permanent protecting groups of the amino acid side
chains and the glycan portion. After purification by semi-
preparative RP-HPLC, the protected glycododecapeptide 20
was isolated in a yield of 39% (based on the loaded resin
19).
Cleavage of the acid-labile protecting groups of the amino
acid side chain functionalities with a mixture of trifluoroace-
tic acid, triisopropylsilane, and water furnished the partially
deblocked glycopeptide 21. For the simultaneous removal of
the sialic acid benzyl ester and the galactose benzyl ether,
21 was dissolved in methanol and subjected to hydrogenoly-
sis, with 10% palladium on activated charcoal as the cata-
lyst. The final de-O-acetylation of the saccharide moiety was
accomplished by prolonged treatment with 5 mm aqueous
sodium hydroxide solution and carefully monitored by ana-
lytical RP-HPLC. Under these basic conditions (pH 11.5),
neither b-elimination of the glycan nor epimerization of the
amino acids were observed. The completely deblocked
MUC1 glycododecapeptide 22 was obtained after purifica-
tion by semi-preparative RP-HPLC in a yield of 56% (over
three steps).
Solid-phase synthesis of a (2,3)-sialyl-T glycopeptide derived
from the tandem repeat sequence of the epithelial mucin
MUC4:The automated solid-phase synthesis of the MUC4
glycohexadecapeptide containing the (2,3)-sialyl-T antigen
was performed starting from commercially available Tenta-
gel S resin[56] coupled with the acid-labile Wang linker[57]
loaded with Fmoc-aspartic acid b-tert-butyl ester 23
(Scheme 4). All Fmoc-amino acids were coupled in excess
(20 equiv) by the standard Fmoc strategy procedure descri-
bed above. N-Tritylimidazole was used for protection of the
histidine side chain. The manual coupling of 1.3 equivalents
of the Fmoc-(2,3)-sialyl-T threonine building block 18, dis-
solved in NMP, was again activated by HATU/HOAt and
NMM. After completion of the 16-amino acid MUC4
tandem repeat sequence 2, the N-terminus of the glycopep-
tide was acetylated. Simultaneous detachment of the glyco-
hexadecapeptide from the resin and cleavage of the acid-
labile protecting groups on the aspartic acid, serine, threo-
nine, and histidine side chains was achieved by treatment of
the resin with a mixture of trifluoroacetic acid, triisopropyl-
silane, and water to give a 46% yield of the partially depro-
tected MUC4 glycopeptide 24 (yield based on loaded resin
23, after semipreparative RP-HPLC).
4154
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 4150 4162

Tumor-Associated (2,3)-Sialyl-T Antigen
4150 4162
Scheme 4. Reagents and conditions:a) i) solid-phase glycopeptide synthesis (SPGS):Fmoc-removal (piperidine/NMP (20%)), coupling (1st 5th, 7th
15th.:Fmoc-AA-OH (20 equiv), HBTU/HOBt/DIPEA, DMF; 6th 18 (1.3 equiv), HATU/HOAt/NMM, DMF, 4 h), capping:Ac 2O, DIPEA, HOBt
(4:1:0.12). ii) TFA/TIS/H2O (15:0.9:0.9), 2 h, 46% (with respect to 23). b) H2, Pd/C (10%), MeOH, 23 h. c) i) MeOH, NaOMe, pH 10, 48 h. ii) NaOHaq
(5 mm), 36 h (64% over three steps).
Complete deprotection of the MUC4 glycopeptide 24 was
accomplished by concomitant hydrogenolysis of the sialic
acid benzyl ester and the galactose benzyl ether, followed
by de-O-acetylation of the glycan moiety. The deacetylation
was first carried out under Zemplÿn conditions[33] at pH 10.
In this case, one O-acetyl group, presumably at the hindered
4’-position of the galactose portion, remained resistant to
saponification, so additional treatment with aqueous sodium
hydroxide at pH 11.5 as described above was necessary.
After purification by semi-preparative RP-HPLC, the
MUC4 glycohexadecapeptide 25 was isolated in a yield of
64% (over three steps).
nine derivative in a yield of 93% and the regio- and stereo-
selective a-sialylation of a disaccharide threonine precursor,
which proceeded in yields of up to 58%. The obtained
Fmoc-(2,3)-sialyl-T threonine building block 18 proved to
be stable under the conditions applied during the standard
Fmoc procedure for glycopeptide assembly, therefore consti-
tuting a versatile synthetic conjugate for the solid-phase syn-
thesis of (2,3)-sialyl-T glycopeptides. By the use of different
anchoring systems in automated solid-phase syntheses, the
glycopeptides were assembled either in their completely
protected or in partially deblocked forms, as is demonstrat-
ed for glycopeptides of the tandem repeat regions of MUC1
and MUC4.
Investigations on the immunological activity of the tumor-
associated (2,3)-sialyl-T MUC1 and MUC4 glycopeptides 22
and 25 concerning their influence on the proliferation of pe-
ripheric blood lymphocytes are currently underway.[46] In ad-
dition, detailed conformational studies by NMR spectrosco-
py in aqueous solution are being undertaken.
Conclusion
The sequential solid-phase synthesis of two different mucin-
type glycopeptides has been accomplished with the use of a
preformed Fmoc-(2,3)-sialyl-T threonine conjugate as a
building block. For the preparation of the crucial glycosyl
amino acid conjugate 18, a novel, linear synthetic strategy
based on the biosynthesis of the (2,3)-sialyl-T antigen has
been established. In addition to an efficient selective pro-
tecting group strategy compatible with the sensitive struc-
ture of the O-glycosyl amino acid derivative, the key steps
included the stereoselective b-galactosylation of a TN threo-
Experimental Section
General:Solvents for moisture-sensitive reactions (acetonitrile, nitrome-
thane, methanol, dichloromethane) were distilled and dried by standard
procedures. DMF (amine-free, for peptide synthesis) was purchased from
Chem. Eur. J. 2004, 10, 4150 4162
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4155

H. Kunz et al.
FULL PAPER
Roth, acetic anhydride and pyridine in p.a. quality from ACROS. Re-
agents were purchased at the highest available commercial quality and
were used without further purification unless outlined otherwise. Fmoc-
protected amino acids were purchased from Novabiochem. As resins for
solid-phase synthesis, aminomethylated TentaGel (NovaSyn Tg amino
resin, Novabiochem) and Rapp TentaGel were used. Reactions were
monitored by thin-layer chromatography with pre-coated silica gel 60 F254
aluminium plates (Merck KGaA, Darmstadt). Flash column chromatog-
raphy was performed with silica gel (40 63 mm) purchased from
Merck KGaA, Darmstadt. Optical rotations [a]D were measured with a
Perkin-Elmer 241 polarimeter. RP-HPLC analyses were carried out on a
Knauer HPLC system with Phenomenex Luna C18(2) (250î4.6 mm, 5 m)
and Phenomenex Jupiter C18 columns (250î4.6 mm, 5 m) at a pump rate
of 1 mLminꢀ1. Preparative HPLC separations were performed on a
Knauer HPLC system with a Phenomenex Luna C18(2) column (250î
50 mm, 10 mm) and a pump rate of 20 mLminꢀ1. Semi-preparative HPLC
separations were carried out on a Knauer HPLC system with Phenomen-
ex Luna C18(2) (250î21.20 mm, 10 m) and Phenomenex Jupiter (250î
21.20 mm, 5 m) columns at a flow rate of 10 mLminꢀ1. Water and CH3CN
were used as solvents. 1H, 13C, and 2D NMR spectra were recorded on
Bruker AC 200, AM 400, ARX 400, or DRX 600 spectrometers. Proton
(m, 1H; H3), 3.70 (s, 1H; H4), 2.44 (d, 1H; OH, JOH,H3 = 9.4 Hz), 2.07
g
ꢀ
(s, 3H; CH3-Ac), 1.45 (s, 9H; CH3 tBu), 1.27 (d, 3H; T , JTb,Tg
=
6.3 Hz) ppm; 13C NMR (100.6 MHz, BB, DEPT, CDCl3): d
= 172.5,
170.7 (C=O), 156.5 (C=O-urethane), 143.7 (C1a-, C8a-Fmoc), 141.3
(C4a-, C5a-Fmoc), 137.5 (Cq-Bzn), 129.1, 128.2, 126.3 (Carom-Bzn), 127.8
(C3-, C6-Fmoc), 127.1 (C-2, C7-Fmoc), 125.0 (C1-, C8-Fmoc), 120.0 (C4-,
C-5-Fmoc), 101.2 (CH-Bzn), 100.0 (C1), 83.4 (Cq-tBu), 76.7 (Tb), 75.5
(C3), 69.6 (C4), 69.2 (CH2-Fmoc), 67.2 (C6), 63.7 (C5), 58.9 (Ta), 50.4
(C2), 47.2 (C9-Fmoc), 28.1 (CH3-tBu), 23.1 (CH3-Ac), 19.1 (Tg) ppm;
ESI-MS: m/z:found:711.3 [ M+Na]+; C38H44N2O10Na calcd 711.3; ele-
mental analysis calcd (%):C 66.26, H 6.44, N 4.07; found:C 66.32, H
6.35, N 3.90.
Ethyl 2,3,4-tri-O-acetyl-6-O-benzyl-a,b-d-thiogalactopyranoside (6-O-Bn-
Ac3Gal-Set) (9):Ethanethiol (169 mL, 2.29 mmol) was added under argon
to a solution of 1,2,3,4-tetra-O-acetyl-6-O-benzyl-a,b-d-galactopyranose
5[35] (911 mg, 2.08 mmol) in dry dichloromethane (40 mL). The mixture
was treated with boron trifluoride etherate (48%, 650 mL, 5.20 mmol) at
08C and stirred for 2 days at ambient temperature. The reaction mixture
was then diluted with dichloromethane (100 mL), washed with saturated
aqueous NaHCO3 (3î70 mL), dried over MgSO4, and filtered, and the
solvent was removed at reduced pressure. The residue was purified by
flash chromatography (silica gel, petroleum ether/ethyl acetate 6:1) to
give the anomeric products a-9 and b-9 as colorless oils in a ratio of a/b
= 1.2:1 (overall yield: 573 mg, 63%).
a Anomer: Rf = 0.63 (petroleum ether/ethyl acetate 3:1); [a]2D3 = 148.2
(c = 1, CH2Cl2); 1H NMR (400 MHz, CDCl3): d = 7.31 7.23 (m, 5H;
Har-Bn), 5.71 (d, JH1,H2 = 5.5 Hz, 1H; H1), 5.47 (d, JH3,H4 = 2.0 Hz, 1H;
H4), 5.26 5.17 (m, 2H; H2, H3), 4.56 4.50 (m, 2H; H5, CH2a-Bn), 4.39
(d, 1H; CH2b-Bn), Ja,b = 12.1 Hz), 3.48 3.45 (m, 2H; H6a,b), 2.58 2.46
(m, 2H; CH2-Et), 2.03 (s, 3H; CH3-Ac), 2.02 (s, 3H; CH3-Ac), 1.95 (s,
3H; CH3-Ac), 1.21 (t, JCH2,CH3 = 7.4 Hz, 3H; CH3-Et) ppm; 13C NMR
(100.6 MHz, BB, CDCl3): d = 170.1, 169.7 (C=O), 137.6 (Cq-Bn), 128.3,
127.7, 127.6 (Car-Bn), 81.6 (C1), 73.3 (CH2-Bn), 68.4, 68.3, 68.1, 67.4 (C2,
C3, C4, C5), 67.8 (C6), 23.8 (CH2-Et), 20.8, 20.6, 20.5 (CH3-Ac), 14.5
(CH3-Et) ppm; MS (ESI): m/z:found:463.3 [ M+Na]+; C21H28NaO8S
calcd 463.1.
chemical shifts are reported in ppm relative to residual CHCl3 (d
=
7.24), DMSO (d = 2.49), methanol (d = 3.31), or water (d = 4.76).
Multiplicities are given as:s (singlet), d (doublet), t (triplet), q (quartet),
m (multiplet). 13C chemical shifts are reported relative to CDCl3 (d =
77.0), DMSO (d
= 39.5), or methanol (d = 49.00). Assignment of
proton and carbon signals was achieved by COSY, TOCSY, HMQC, and
1
HMBC experiments when noted. For H and 13C signals of the saccharide
portions the following denominations were used: N-acetyl-d-galactosa-
mine (no prime), d-galactose (’), and N-acetyl-neuraminic acid (’’).
MALDI-TOF mass spectra were acquired on a Micromass Tofspec E
spectrometer while ESI-mass spectra were obtained on a ThemoQuest-
Navigator spectrometer. HR-ESI mass spectra were recorded on a Mi-
cromass Q-TOF Ultima spectrometer.
N-Fluorenylmethoxycarbonyl-O-(2-acetamido-4,6-O-benzylidene-2-
deoxy-a-d-galactopyranosyl)-l-threonine tert-butyl ester (Fmoc-Thr(a-
4,6-O-Bzn-GalNAc)-OtBu) (4): Procedure A:A solution of sodium
methoxide in methanol (5%) was added dropwise to Fmoc-Thr(a-Ac3-
GalNAc)-OtBu 3[31,32] (186 mg, 0.256 mmol) dissolved in dry methanol
(6 mL) until pH 8.5 was reached. The reaction mixture was stirred for 3 h
and was subsequently neutralized by addition of acidic ion-exchange
resin (Amberlyst 15). After filtration, the solvent was removed in vacuo.
The resulting solid was dissolved in N,N-dimethylformamide (10 mL),
and a,a-dimethoxytoluene (77 mL) was added. The pH was adjusted to
3.5 with a catalytic amount of p-toluenesulfonic acid (10 mg). The reac-
tion was carried out under reduced pressure (15 mbar) at 508C in a
rotary evaporator. After 2 h the mixture was neutralized with ethyldiiso-
propylamine (4 drops), and the solvent was removed in vacuo. The result-
ing residue was co-evaporated with toluene (3î20 mL) and purified by
flash chromatography (silica gel, petroleum ether/ethyl acetate 1:2) to
afford compound 4 (78 mg, 44%) as a colorless, amorphous solid.
b Anomer: Rf = 0.53 (petroleum ether/ethyl acetate 3/1); [a]2D3 = ꢀ20.3
(c = 1, CH2Cl2); 1H NMR (400 MHz, CDCl3): d = 7.31 7.22 (m, 5H;
Har-Bn), 5.46 (d, JH3,H4 = 3.1 Hz, 1H; H4), 5.17 (t, JH1,H2 = 9.8 Hz, JH2,H3
= 10.1 Hz, 1H; H2), 5.00 (dd, JH2,H3 = 10.1 Hz, JH3,H4 = 3.1 Hz, 1H;
H3), 4.50 (d, Ja,b = 12.1 Hz, 1H; CH2a-Bn), 4.45 (d, JH1,H2 = 9.8 Hz, 1H;
H1), 4.36 (d, Ja,b = 11.7 Hz, 1H; CH2b-Bn), 3.83 (t, JH5,H6a = 6.3 Hz,
JH5,H6b = 6.7 Hz, 1H; H5), 3.52 (dd, JH5,H6a = 6.3 Hz, JH6a,b = 9.4 Hz,
1H; H6a), 3.52 (dd, JH5,H6b = 7.1 Hz, JH6a,b = 9.4 Hz, 1H; H6b), 2.73
2.62 (m, 2H; CH2-Et), 2.01 (s, 3H; CH3-Ac), 2.00 (s, 3H; CH3-Ac), 1.93
(s, 3H; CH3-Ac), 1.23 (t, JCH2,CH3 = 7.4 Hz, 3H; CH3-Et) ppm; 13C NMR
(100.6 MHz, BB, CDCl3): d = 170.1, 170.0, 169.6 (C=O), 137.5 (Cq-Bn),
128.4, 127.9, 127.8 (Car-Bn), 83.9 (C1), 75.8 (C2), 73.5 (CH2-Bn), 72.1
(C3), 67.7 (C4), 67.4 (C5, C6), 24.3 (CH2-Et), 20.8, 20.6 (CH3-Ac (3î)),
14.8 (CH3-Et) ppm; MS (ESI): m/z:found:463.1
[
M+Na]+;
C21H28NaO8S calcd 463.1.
Procedure B:A solution of sodium methoxide in methanol (5%) was
N-Fluorenylmethoxycarbonyl-O-(2-acetamido-4,6-O-benzylidene-2-
desoxy-3-O-[2,3,4-tri-O-acetyl-6-O-benzyl-b-d-galactopyranosyl]-a-d-gal-
actopyranosyl)-l-threonine tert-butyl ester (Fmoc-Thr(b-6-O-Bn-Ac3Gal-
(1 3)-a-4,6-O-Bzn-GalNAc)-OtBu) (7): Procedure A:Fmoc-Thr( a-4,6-
O-Bzn-GalNAc)-OtBu 4 (1.00 g, 1.45 mmol) and 6-O-Bn-Ac3Gal-TCI a-
added dropwise to
a solution of Fmoc-Thr(a-Ac3GalNAc)-OtBu 3
(1.65 g, 2.27 mmol) in dry methanol (50 mL) until pH 8.5 was reached.
The reaction mixture was stirred for 3 h and was subsequently neutral-
ized by addition of acidic ion-exchange resin (Amberlyst 15). After filtra-
tion, the solvent was removed in vacuo. The resulting solid was dissolved
in dry acetonitrile (300 mL), and a,a-dimethoxytoluene (684 mL) was
added. The pH was adjusted to 4 with a catalytic amount of p-toluenesul-
fonic acid. The reaction mixture was stirred for 15 h at room temperature
and was neutralized with triethylamine (4 5 drops). After removal of the
solvent in vacuo, purification of the resulting residue by flash chromatog-
raphy (silica gel, petroleum ether/ethyl acetate 1:2) gave compound 4
(1.18 g, 75%) as a colorless amorphous solid. Rf = 0.60 (toluene/ethanol
38]
6[36 (979 mg, 1.81 mmol) were each separately co-evaporated with tolu-
ene (3î50 mL) and dried under high vacuum. The glycosyl acceptor 4
and the glycosyl donor a-6 were dissolved in dry dichloromethane
(25 mL), and activated molecular sieves (1 g, 4 ä) were added. The sus-
pension was stirred for 1 h at ambient temperature under argon. After
the system had been cooled to ꢀ158C, a solution of trimethylsilyl triflate
(29 mL) in dry dichloromethane (2.5 mL) was added. The reaction mix-
ture was allowed to warm up to room temperature over 1 h, diluted with
dichloromethane (50 mL), and neutralized with triethylamine. The mix-
1
4:1); [a]2D3 = 64.4 (c = 1, CHCl3); H NMR (400 MHz, CDCl3): d = 7.76
¾
(d, JH3,H4 = JH5,H6 = 7.4 Hz, 2H; H4-, H5-Fmoc), 7.61 (d, JH1,H2 = JH7,H8
=
ture was filtered through Hyflo Super Cel , and the solvent was removed
7.4 Hz, 2H; H1-, H8-Fmoc), 7.51 7.49 (m, 2H; H3-, H6-Fmoc), 7.42
7.30 (m, 7H; H2-, H7-Fmoc, Harom-Bzn), 6.49 (d, JNH,H2 = 8.6 Hz, 1H;
NH (GalNAcNH)), 5.56 (s, 1H; CH-Bzn), 5.49 (d, JNH,Ta = 9.4 Hz, 1H;
NH-T), 4.93 (d, JH1,H2 = 3.1 Hz, 1H; H1), 4.50 4.44 (m, 3H; H2, CH2-
Fmoc), 4.25 4.04 (m, 6H; H5, H6a, H6b, H9- Fmoc, Ta, Tb), 3.86 3.82
from the filtrate in vacuo. Purification of the residue by flash chromatog-
raphy (silica gel, ethyl acetate/petroleum ether 2:1) resulted in a mixture
of the desired b-glycosylation product 7 and the orthoester 8. The two
components were separated by preparative RP-HPLC (column:Phenom-
enex Luna, isocratic:CH 3CN/H2O 70:30) to give the product 7 (Rt
=
4156
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemeurj.org Chem. Eur. J. 2004, 10, 4150 4162

Tumor-Associated (2,3)-Sialyl-T Antigen
4150 4162
96 min) and the orthoester 8 (Rt = 108 min) as colorless, amorphous
solids in a ratio of 7/8 = 1:2.7 (overall yield: 1.22 g, 79%).
(C1a-, C8a-Fmoc), 141.3 (C4a-, C5a-Fmoc), 137.6, 137.4 (Cq-Bn, Cq-
Bzn), 128.7, 128.1, 126.2 (Car-Bzn), 128.4, 127.9 (Car-Bn), 127.8 (C3-, C6-
Fmoc), 127.1 (C2-, C7-Fmoc), 124.9 (C1-, C8-Fmoc), 120.1 (C4-, C5-
Fmoc), 101.7 (C1’), 100.5 (CH-Bzn), 100.3 (C1), 83.1 (Cq-tBu), 76.1 (Tb),
75.5 (C4), 74.9 (C3), 73.5 (CH2-Bn), 72.2 (C5’), 71.2 (C3’), 67.0 (C6), 68.9
(C2’), 67.9 (C6’), 67.6 (C4’), 66.9 (CH2-Fmoc), 63.6 (C-5), 59.0 (Ta), 47.9
Procedure B:Fmoc-Thr(
a-4,6-O-Bzn-GalNAc)-OtBu
4
(100 mg,
0.145 mmol) and 6-O-Bn-Ac3Gal-SEt b-9 (80 mg, 0.182 mmol) were each
co-evaporated with toluene (3î10 mL), and dried under high vacuum.
The glycosyl acceptor 4 and the glycosyl donor b-9 were dissolved in dry
dichloromethane (3 mL). Flame-dried molecular sieves (300 mg, 4 ä)
were added to this solution, and the mixture was stirred for 20 min at am-
bient temperature under argon. The suspension was cooled to 08C, N-io-
dosuccinimide (82 mg, 0.363 mmol) was added, and the mixture was stir-
red for an additional 20 min. Subsequently, trifluoromethanesulfonic acid
(6.3 mL) was added dropwise, and the reaction mixture was stirred for
20 h at ambient temperature. It was diluted with dichloromethane
(25 mL) and filtered through Hyflo Super Cel. The filtrate was washed
with saturated aqueous NaHCO3 (3î15 mL) and saturated aqueous
Na2S2O3 (2î15 mL), dried over MgSO4, and filtered, and the solvent was
removed in vacuo. The crude product was purified by flash chromatogra-
phy (silica gel, petroleum ether/ethyl acetate 2:3) to give 7 as a colorless,
amorphous solid (81 mg, 52%).
ꢀ
(C2), 47.2 (C9-Fmoc), 28.0 (CH3 tBu), 23.4 (CH3-NHAc), 20.7, 20.6
(CH3-OAc (3î)), 18.9 (Tg) ppm; HR-MS (ESI-TOF): m/z:found:
1089.4196 [M+Na]+; C57H66N2O18Na calcd 1089.4208.
N-Fluorenylmethoxycarbonyl-O-(2-acetamido-2-deoxy-4,6-O-benzyli-
dene-3-O-[6-O-benzyl-b-d-galactopyranosyl]-a-d-galactopyranosyl)-l-
threonine tert-butyl ester (11):A solution of sodium methoxide in metha-
nol (0.1m) was added dropwise to a solution of Fmoc-Thr(b-6-O-Bn-
Ac3Gal-(1 3)-a-4,6-O-Bzn-GalNAc)-OtBu
7 (1.03 g, 0.965 mmol) in
methanol (25 mL) until pH 8.5 was reached. The reaction mixture was
stirred for 12 h, during which the pH was carefully monitored and read-
justed when necessary. Neutralization with acetic acid (0.05 mL), removal
of the solvent in vacuo, and purification by flash chromatography (silica
gel, ethyl acetate/ethanol 50:1!25:1) afforded compound 11 (560 mg,
62%) as a colorless, amorphous solid. Rf = 0.35 (toluene/ethanol 4:1);
[a]2D4 = 62.4 (c = 1, CHCl3); 1H NMR (400 MHz, CDCl3): d = 7.76 (d,
Procedure C:Fmoc-Thr(
a-4,6-O-Bzn-GalNAc)-OtBu
4
(100 mg,
0.145 mmol) and 6-O-Bn-Ac3Gal-Br a-10[41] (100 mg, 0.218 mmol) were
separately co-evaporated with toluene (3î10 mL) and dried under high
vacuum. Molecular sieves (200 mg, 4 ä) and silver triflate (75 mg,
0.290 mmol) were added to a solution of glycosyl acceptor 4 in dry di-
chloromethane (2 mL). After the suspension had been stirred for 30 min
at ambient temperature under an argon atmosphere with exclusion of
light, the glycosyl donor a-10, dissolved in dry dichloromethane (2 mL),
was added at ꢀ408C. The reaction mixture was allowed to warm up to
room temperature over 20 h. Since the reaction was not complete at this
stage (monitoring by TLC), 6-O-Bn-Ac3Gal-Br a-10 (100 mg,
0.218 mmol) was again added to the reaction mixture, which was stirred
for an additional two days at ambient temperature with protection from
light. The mixture was diluted with dichloromethane (100 mL) and fil-
tered through Hyflo Super Cel. The filtrate was washed with saturated
aqueous NaHCO3 (3î50 mL), dried over MgSO4, and filtered, and the
solvent was removed in vacuo. Purification of the residue by flash chro-
matography (silica gel, petroleum ether/ethyl acetate 2:3) afforded the b-
glycosylation product 7 as a colorless, amorphous solid (102 mg, 66%).
JH4,H3 = JH5,H6 = 7.8 Hz, 2H; H4-, H5-Fmoc), 7.60 (dd, JH1,H2 = JH8,H7
=
7.0 Hz, 2H; H1-, H8-Fmoc), 7.49 (d, JHa,Hb = 7.4 Hz, 2H; Har-Bzn), 7.41
7.35 (m, 2H; H3-, H6-Fmoc), 7.35 7.25 (m, 10H; H2-, H7-Fmoc, Har-Bzn
(3H), Har-Bn (5H), 6.31 (d, JNH,H2 = 9.1 Hz, 1H; NH-GalNAc), 5.58 (d,
JNH,Ta = 9.4 Hz, 1H; NH-T), 5.45 (s, 1H; CH-Bzn), 4.92 (d, JH1,H2
=
3.1 Hz, 1H; H1), 4.65 4.43 (m, 5H; H2, CH2-Bn, CH2-Fmoc), 4.31 4.08
(m, 6H; H4, Ta, Tb, H9-Fmoc, H1’, H6a), 3.90 3.86 (m, 2H; H3, H6b),
3.73 3.68 (m, 3H; H3’, H4’, H5’), 3.62 3.55 (m, 3H; H6a’, H6b’, H5),
3.42 3.37 (m, 1H; H2’), 2,99 (sb, 1H; OH), 2.02 (s, 3H; CH3-NHAc), 1.42
(s, 9H; CH3-tBu), 1.26 (d, 3H; Tg, JTg,Tb
=
5.9 Hz) ppm; 13C NMR
(100.6 MHz, CDCl3, BB, HMQC): d = 172.2 (2îC=O), 156.4 (C=O-ure-
thane), 143.7 (C1a-, C8a-Fmoc), 141.3 (C4a-, C5a-Fmoc), 138.1, 137.5
(Cq-Bn, Cq-Bzn), 128.9, 128.1, 126.1 (Car-Bzn), 128.4, 127.8 (Car-Bzn),
127.6 (C3-, C6-Fmoc), 127.1 (C2-, C7-Fmoc), 125.0 (C1-, C8-Fmoc), 120.1
(C4-, C5-Fmoc), 105.5 (CH-Bzn), 101.1 (C1’), 100.6 (C1), 83.4 (Cq-tBu),
77.2 (Tb) 76.7 (C4), 75.8 (C3), 73.7 (C5’), 73.6 (CH2-Bn), 73.4 (C3’), 70.9
(C2’), 69.8 (C6), 68.9 (C6’), 68.7 (C4), 67.1 (CH2-Fmoc), 63.7 (C5), 59.0
a
ꢀ
(T ), 48.3 (C2), 47.2 (C9-Fmoc), 28.1 (CH3 tBu), 23.5 (CH3-NHAc), 18.9
Procedure D:Fmoc-Thr(
a-4,6-O-Bzn-GalNAc)-OtBu
4
(890 mg,
(Tg) ppm; HR-MS (ESI-TOF): m/z:found:963.3879
[
M+Na]+;
1.29 mmol) and 6-O-Bn-Ac3Gal-Br a-10 (1.187 g, 2.58 mmol) were each
separately co-evaporated with toluene (3î50 mL) and dried under high
vacuum. The glycosyl acceptor 4 was dissolved in a mixture of dry nitro-
methane (25 mL) and dry dichloromethane (5 mL), was treated with mo-
lecular sieves (3 g, 3 ä) and Hg(CN)2 (652 mg, 2.58 mmol), and was stir-
red for 30 min at ambient temperature under argon. The glycosyl donor
a-10, dissolved in dry dichloromethane (10 mL), was subsequently added
at 08C. The reaction mixture was stirred for four days at room tempera-
ture, was diluted with dichloromethane (150 mL), and was filtered
through Hyflo Super Cel. The filtrate was washed with saturated aqueous
NaHCO3 (3î100 mL) and saturated aqueous NaI (2î100 mL), dried
over MgSO4, and filtered, and the solvents were removed under reduced
pressure. After purification of the crude product by flash chromatogra-
phy (silica gel, petroleum ether/ethyl acetate 2:3), the b-glycosylation
product 7 was isolated as a colorless, amorphous solid (1.288 g, 93%).
C51H60N2O15Na calcd 963.3891.
N-Fluorenylmethoxycarbonyl-O-(2-acetamido-2-deoxy-4,6-O-benzyli-
dene-3-O-[6-O-benzyl-3-O-{benzyl-(5-acetamido-4,7,8,9-tetra-O-acetyl-
3,5-dideoxy-a-d-galacto-2-nonulopyranosyl)onat}-b-d-galactopyranosyl]-
a-d-galactopyranosyl)-l-threonine tert-butyl ester (Fmoc-Thr(a-Ac4Neu-
NAcCOOBn-(2 3)-b-6-O-Bn-Gal-(1 3)-a-4,6-O-Bzn-GalNAc)-OtBu)
(14) Procedure A:Fmoc-Thr( b-6-O-Bn-Gal-(1 3)-a-4,6-O-Bzn-GalNAc)-
OtBu 11 (110 mg, 0.177 mmol) and Ac4NeuNAcCOOBn-SEt 13 (147 mg,
0.240 mmol) were separately co-evaporated with toluene (3î20 mL) and
dried under high vacuum. The glycosyl acceptor 11 and the glycosyl
donor 13 were dissolved in anhydrous acetonitrile (10 mL), treated with
molecular sieves (350 mg, 3 ä), and stirred for 30 min at ambient temper-
ature under argon. The suspension was cooled to ꢀ378C, and N-iodosuc-
cinimide (108 mg, 0.480 mmol), dissolved in dry acetonitrile (1 mL), and
trifluoromethanesulfonic acid (125 mL of a 0.4m solution in dry acetoni-
trile) were added slowly. The reaction mixture was stirred for an addi-
tional 30 min at ꢀ378C and then for 20 h at ꢀ238C. It was neutralized
with pulverized NaHCO3 and allowed to warm to ambient temperature.
The suspension was diluted with dichloromethane (100 mL) and filtered
through Hyflo Super Cel. The filtrate was washed with saturated aqueous
NaHCO3 (2î50 mL) and saturated aqueous Na2S2O3 (1î50 mL), and the
combined aqueous layers were re-extracted with dichloromethane. The
combined organic phases were dried over MgSO4, and filtered, and the
solvents were removed at reduced pressure. Flash chromatography of the
residue (silica gel, ethyl acetate/ethanol 10:1) gave one fraction of the
pure a-glycosylation product a-14 as a colorless, amorphous solid (66 mg,
38%), together with a second fraction containing the b-glycosylation
product b-14 and the sialic acid glycal. Separation of the b-product and
the glycal was achieved by further flash chromatography on silica gel
(ethyl acetate/cyclohexane 10:1) and afforded b-14 (42 mg, 24%) as a
colorless, amorphous solid. The sialic acid glycal (62 mg, 47% yield
Rf
CH2Cl2); 1H NMR (400 MHz, COSY, HMQC, CDCl3): d
JH3,H4 = JH5,H6 = 7.4 Hz, 2H; H4-, H5-Fmoc), 7.61 (d, JH1,H2 = JH7,H8
=
0.63 (ethyl acetate/petroleum ether 3:1); [a]D23
=
74.4 (c
= 1,
=
7.76 (d,
=
7.8 Hz, 2H; H1-, H8-Fmoc), 7.52 (d, Ja,b = 6.3 Hz, 2H; Har-Bzn), 7.43
7.20 (m, 12H; H2-, H3-, H6-, H7-Fmoc, Har-Bzn (3H), Har-Bn (5H)),
5.81 (d, JNH,H2 = 9.0 Hz, 1H; NH-GalNAc), 5.50 (s, 1H; CH-Bzn), 5.46
(d, JNH,Ta = 9.4 Hz, 1H; NH-T), 5.41 (d, JH3’,H4’ = 3.1 Hz, 1H; H4’), 5.19
(dd, JH1’,H2’ = 7.8 Hz, JH2’,H3’ = 10.2 Hz, 1H; H2’), 5.00 4.90 (m, 2H; H3’
{4.97, dd, JH2’,H3’ = 10.2 Hz, JH3’,H4’ = 3.5 Hz}, H1 {4.93}), 4.68 (d, JH1’,H2’
=
8.2 Hz, 1H; H1’), 4.66 4.59 (m, 1H; H2), 4.52 4.37 (m, 4H; CH2-Fmoc
{4.47}, CH2-Bn {4.39}), 4.30 4.10 (m, 5H; H4 {4.26}, H9-Fmoc {4.22},
Ta {4.18}, Tb {4.16}, H6a {4.12}), 3.93 3.79 (m, 3H; H6b {3.88}, H5’
{3.85}, H3 {3.81}), 3.60 (s, 1H; H5), 3.55 (t, JH5’,H6’a = 6.7 Hz, 1H; H6a’),
3.51 3.45 (m, 1H; H6b’), 2.05 (s, 3H; CH3-Ac), 2.04 (s, 3H; CH3-Ac),
1.98 (s, 3H; CH3-Ac), 1.95 (s, 3H; CH3-Ac), 1.43 (s, 9H; CH3-tBu), 1.24
(sb, 3H; Tg) ppm; 13C NMR (100.6 MHz, BB, HMQC, CDCl3): d
170.3, 170.2, 169.8, 169.7, 169.4 (C=O), 156.4 (C=O-urethane), 143.6
=
Chem. Eur. J. 2004, 10, 4150 4162
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4157

H. Kunz et al.
FULL PAPER
based on the sialic acid donor 13) was formed as a by-product, and a
small amount of the glycosyl acceptor 11 (17 mg, 15%) was re-isolated.
83.13 (Cq-tBu), 78.15 (C3), 75.94, 75.15 (Tb, C4, C3’), 73.60 (CH2-Bn),
73.43, 72.92 (C6’’,C5’), 69.72 (C8’’, C6’), 69.07 (C6), 68.61 (C4’’), 68.22,
68.11 (CH2-Bn, C2’,C4’,C7’’), 67.25 (CH2-Fmoc), 63.64 (C9’’), 63.52 (C5),
59.24 (Ta), 49.08 (C5’’), 47.79 (C2), 47.09 (C9-Fmoc), 37.42 (C3’’), 28.06
(CH3-tBu), 23.16, 23.07 (CH3-NHAc), 21.33, 21.21, 21.01, 20.78, 20.64
(CH3-OAc), 19.47 (Tg) ppm; MS (HR-ESI-TOF): m/z:found:1512.5729
[M+Na]+; C77H91N3O27Na calcd 1512.5737.
Procedure B:Fmoc-Thr( b-6-O-Bn-Gal(1 3)-a-4,6-O-Bzn-GalNAc)-OtBu
11 (338 mg, 0.359 mmol) and a-Ac4NeuNAcCOOBnXan 15 (482 mg,
0.718 mmol, 2 equiv) were dissolved in a mixture of acetonitrile and di-
chloromethane (55 mL, 2:1). The solution was stirred for 1 h in a Schlenk
flask (brown glass) in the presence of flame-dried molecular sieves (2.1 g,
powder, 3 ä) under an argon atmosphere and with exclusion of moisture.
After the reaction mixture had been cooled to ꢀ688C, silver triflate
(200 mg, 0.78 mmol) and di-tert-butylpyridine (186 mL, 213.8 mg,
1.12 mmol) were added. A pre-cooled (ꢀ158C) solution of phenylsulfenyl
chloride[47] (90 mL, 0.78 mmol) in dry dichloromethane (0.5 mL) was
added dropwise, and stirring was continued at ꢀ688C for 3.5 h. The reac-
tion mixture was diluted with a suspension of silica gel (0.7 g) in ethyl
acetate (10 mL), filtered through Hyflo Super Cel, and washed with satu-
rated aqueous NaHCO3 (2î75 mL) and brine (75 mL). The organic layer
was dried over MgSO4 and concentrated in vacuo. Purification of the
crude product by flash chromatography (silica gel, ethyl acetate!ethyl
acetate/ethanol 65:1) yielded 14 (262 mg, 50%) as a colorless, amorphous
solid (a/b, 97:3).
b Anomer: Rf = 0.43 (ethyl acetate/ethanol 10/1); [a]2D3 = 30.9 (c = 1,
CH2Cl2); 1H NMR (400 MHz, COSY, HMQC, CDCl3): d
=
7.75 (d,
JH3,H4 = JH5,H6 = 6.3 Hz, 2H; H4-, H5-Fmoc), 7.61 (dd, JH1,H2 = JH7,H8
=
7.1 Hz, 2H; H1-, H8-Fmoc), 7.52 7.48 (m, 2H; Har-Bzn), 7.41 7.24 (m,
17H; H2-, H3-, H6-, H7-Fmoc, Har-Bzn (3H), Har-Bn (2î, 10H)), 6.40
(d, JNH,2H = 9.0 Hz, 1H; NH-GalNAc), 5.86 (d, JNH,H5’’ = 9.8 Hz, 1H;
NH-NeuNAc), 5.63 (d, JNH,Ta = 9.4 Hz, 1H; NH-T), 5.49 (s, 1H; CH-
Bzn), 5.43 (dt, JH3’’ax,H4’’ = JH4’’,H5’’ = 10.6 Hz, JH3’’eq,H4’’ = 4.7 Hz, 1H;
H4’’), 5.34 (dd, JH6’’,H7’’ = 2.0 Hz, JH7’’,H8’’ = 6.3 Hz, 1H; H7’’), 5.26 5.22
(m, 1H; H8’’), 5.21 (d, Ja,b = 12.1 Hz, 1H; CH2a-Bn), 5.12 (d, Ja,b
=
11.7 Hz, 1H; CH2a’-Bn), 4.94 (d, JH1,H2 = 3.1 Hz, 1H; H1), 4.66 3.86 (m,
16H; H2 {4.62}, H9a,b’’ {4.59, 3.95}, CH2-Bn {4.55}, CH2-Fmoc {4.45},
H6’’ {4.37, dd, JH5’’,H6’’ = 10.6 Hz, JH6’’,H7’’ = 2.0 Hz}, H1’ {4.31, d, JH1’,H2’
=
7.4 Hz}, Ta {4.27}, H4 {4.26}, H9-Fmoc {4.26}, Tb {4.18}, H6a,b {4.15,
3.94}, H5’’ {4.14}), 3.82 3.58 (m, 8H; H3 {3.78}, H3’ {3.78}, H2’ {3.70}, H4’
{3.66}, H6’a,b {3.66}, H5’ {3.65}, H5 {3.61}), 2.55 (dd, JH3’’ax,eq’’ = 13.7 Hz,
JH3’’eq,H4’’ = 4.3 Hz, 1H; H3’’eq), 2.10 (s, 3H; CH3-Ac), 2.01 (s, 3H; CH3-
Ac), 2.06 2.03 (m, 1H; H3’’ax), 1.99 (s, 3H; CH3-Ac), 1.96 (s, 3H; CH3-
Procedure C:Fmoc-Thr(
b-6-O-Bn-Gal-(1 3)-a-4,6-O-Bzn-GalNAc)-
OtBu 11 (210 mg, 0.223 mmol) and Ac4NeuNAcCOOBn-Xan 15 (375 mg,
0.558 mmol) were each separately co-evaporated with toluene (3î
10 mL) and dried under high vacuum. The glycosyl acceptor 11 and the
glycosyl donor 15 were dissolved in a mixture of dry dichloromethane
(3 mL) and dry acetonitrile (7 mL), powdered molecular sieves (600 mg,
3 ä) were added, and the reaction mixture was stirred for 1 h at ambient
temperature under argon. The suspension was cooled to ꢀ688C, and
silver triflate (143 mg, 0.558 mmol) and a solution of methylsulfenyl bro-
mide in dry 1,2-dichloroethane (0.35 mL of a 1.6m solution; the methyl-
sulfenyl bromide solution in 1,2-dichloroethane was prepared by addition
ꢀ
Ac), 1.95 (s, 3H; CH3-Ac)), 1.86 (s, 3H; CH3-Ac), 1.44 (s, 9H; CH3
tBu), 1.28 (d, JTb,g = 6.3 Hz, 3H; Tg) ppm; 13C NMR (100.6 MHz, BB,
HMQC, CDCl3): d = 172.6, 170.9, 170.7, 170.4, 170.2, 170.1, 167.7 (C=
O), 156.5 (C=O-urethane), 143.7 (C1a-, C8a-Fmoc), 141.3 (C4a-, C5a-
Fmoc), 138.1, 137.6 (Cq-Bn, Cq-Bzn), 134.7 (Cq-Bn), 128.9, 128.5, 128.4,
127.6 (Car-Bn (2î)), 128.6, 128.1, 126.4 (Car-Bzn), 127.8 (C3-, C6-Fmoc),
127.1 (C2-, C7-Fmoc), 125.0 (C1-, C8-Fmoc), 120.0 (C4-, C5-Fmoc), 105.3
(C1’), 100.9 (CH-Bzn), 100.5 (C1), 98.9 (C2’’), 83.4 (Cq-tBu), 76.8 (C3,
C3’), 75.7 (C4, Tb), 73.5 (CH2-Bn), 73.4 (C5’), 71.2 (C6’’), 70.0 (C8’’), 69.5
(C6’), 69.1 (C4’’), 68.9 (C6), 68.0 (CH2-Bn), 67.8 (C2’, C4’, C7’’), 67.3
(CH2-Fmoc), 63.7 (C5), 62.4 (C9’’), 59.0 (Ta), 48.5 (C2, C5’’), 47.2 (C9-
of bromine (410 mL, 7.99 mmol) to
a solution of dimethyl disulfide
(709 mL, 7.99 mmol) in dry 1,2-dichloroethane (10 mL) and stirring at
ambient temperature with exclusion of light for 15 h) were added slowly.
The reaction mixture was stirred for 4 h at ꢀ688C under an argon atmos-
phere with exclusion of light. It was then neutralized with Huenig×s base
(115 mL, 3 equiv) and allowed to warmed to room temperature. The sus-
pension was diluted with dichloromethane (100 mL) and filtered through
Hyflo Super Cel, and the solvents were removed from the filtrate in
vacuo. The residue was purified by flash chromatography (silica gel;
100% ethyl acetate) to afford the desired a-glycosylation product a-14
(194 mg, 58%) as a colorless, amorphous solid. In addition, a small
amount of the unreacted glycosyl acceptor 11 (17 mg, 8%) and the sialic
acid glycal (235 mg, 77% yield based on the sialic acid donor 15) formed
as a by-product were isolated.
ꢀ
Fmoc), 34.2 (C3’’), 28.1 (CH3 tBu), 23.3, 23.1 (CH3-NHAc), 21.0, 20.9,
20.8 (CH3-OAc (4î)), 19.4 (Tg) ppm; MS (MALDI-TOF, dhb, positive
ion mode):m/z:found:1513.3 [ M+Na]+; C77H91N3NaO27 calcd 1513.5;
found:1529.4 [ M+K]+; C77H91KN3O27 calcd:1529.5; MS (HR-ESI-TOF):
m/z:found:1512.5740 [ M+Na]+; C77H91N3O27Na calcd 1512.5737.
N-Fluorenylmethoxycarbonyl-O-(2-acetamido-2-deoxy-3-O-[6-O-benzyl-
3-O-{benzyl-(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-a-d-galacto-
2-nonulopyranosyl)onat}-b-d-galactopyranosyl]-a-d-galactopyranosyl)-l-
threonine tert-butyl ester (16):A solution of trisaccharide 14 (359 mg,
0.241 mmol) in aqueous acetic acid (80%, 15 mL) was stirred at 808C for
1 h. Toluene (15 mL) was added, and the reaction mixture was concen-
trated in vacuo and subsequently co-evaporated with toluene (3î15 mL).
The resulting crude product was purified by flash chromatography (silica
gel, ethyl acetate/ethanol 15:1) to give the title compound as a colorless,
amorphous solid (308 mg, 82%). Rf = 0.12 (ethyl acetate/ethanol 10:1);
a Anomer: Rf = 0.28 (ethyl acetate/ethanol 10:1); Rt = 30.1 min (Phe-
nomenex Luna C18(2), grad.:CH 3CN/H2O (60:40)!(90:10), 40 min);
[a]2D7
= 32.0 (c =
1, CHCl3); 1H NMR (400 MHz, CDCl3, COSY,
HMQC): d = 7.74 (d, JH4,H3 = JH5,H6 = 7.8 Hz, 2H; H4-, H5-Fmoc), 7.58
(dd, JH1,H2 JH8,H7 = 7.8 Hz, 2H; H1-, H8-Fmoc), 7.49 (d, JHa,Hb
5.8 Hz, 2H; Har-Bzn), 7.42 7.35 (m, 2H; H3-, H6-Fmoc), 7.35 7.24 (m,
15H; H2-, H7-Fmoc, Har-Bzn (3H), Har-Bn (10H)), 6.59 (d, JNH,H2
=
=
[a]2D8
= 12.0 (c =
1, CHCl3); 1H NMR (400 MHz, CDCl3, COSY,
=
HMQC): d = 7.73 (d, JH4,H3 = JH5,H6 = 7.4 Hz, 2H; H4-, H5-Fmoc), 7.56
(d, JH1,H2 = JH8,H7 = 7.8 Hz, 2H; H1-, H8-Fmoc), 7.42 7.23 (m, 14H;
H2-, H3-, H6-, H7-Fmoc, Har-Bn (10H)), 6.61 (d, JNH,H2 = 9.4 Hz, 1H;
9.4 Hz, 1H; NH-GalNAc), 6.12 (d, JNH,Ta = 9.7 Hz, 1H; NH-T), 5.45 (s,
1H; CH-Bzn), 5.40 (t, JH8’’,H9’’ = 6.6 Hz, 1H; H8’’), 5.28 5.13 (m, 2H;
H7’’, NH-NeuNAc), 5.16 (s, 2H; CH2-Bn), 4.98 (d, JH1,H2 = 3.5 Hz, 1H;
H1), 4.92 4.81 (m, 1H; H4’’), 4.74 4.63 (m, 1H; H2), 4.55 (s, 2H; CH2-
Bn), 4.52 4.03 (m, 10H; H9a’’{4.46}, CH2-Fmoc {4.39, 4.34}, Tb {4.33}, H4
{4.26}, Ta {4.25}, H9-Fmoc {4.19}, H1’ {4.15}, H6a {4.13}, H5’’ {4.06}),
3.99 3.84 (m, 4H; H6’’ {3.93}, H9b’’ {3.92}, H3’ {3.90}, H6b {3.87}), 3.72
3.55 (m, 4H; H3 {3.67}, H2’ {3.61}, H6a’ {3.59}, H5 {3.57}), 3.54 3.39 (m,
2H; H6b’{3.87}, H5’{3.41}), 3.24 (s, 1H; H4’), 2,78 (sb, 1H; OH), 2.71
(dd, JH3eq’’,H3ax’’ = 12.7 Hz, JH3eq’’,H2’’ = 4.3 Hz, 1H; H3eq’’), 2.36 (s, 1H;
OH), 2.03 2.00 (m, 1H; H3ax’’), 2.09, 2.07, 2.02, 2.00, 1.92, 1.82 (6îs,
18H; 6îCH3-Ac), 1.42 (s, 9H; CH3-tBu), 1.26 (d, JTg,Tb = 5.9 Hz, 3H;
ꢀ
NH GalNAc), 5.99 (d, JNH,Ta = 9.8 Hz, 1H; NH-T), 5.49 5.41 (m, 1H;
H8), 5.23 5.07 (m, 4H; CH2-Bn1 {5.16, 5.14} H7’’ {5.10}, NH-NeuNAc),
4.94 4.88 (d, JH1,H2 = 1.6 Hz, 1H; H1), 4.87 4.77 (m, 1H; H4’’), 4.59 4.44
(m, 4H; H9a’’ {4.52}, H2 {4.52}, CH2-Bn {4.50}), 4.43 4.35 (m, 2H; CH2-
Fmoc), 4.34 4.26 (m, 1H; Tb), 4.26 4.12 (m, 4H; Ta {4.23}, H1’ {4.19},
H9-Fmoc {4.18}, H4 {4.16}), 4.11 4.01 (m, 1H; H5’’), 3.95 (dd, JH6’’,H5’’
=
10.8 Hz, JH6’’,H7’’ = 1.6 Hz, 1H; H6’’), 3.91 3.83 (m, 2H; H6a {3.87}, H3’
{3.86}), 3.82 3.63 (m, 4H; H5 {3.78}, H9b’’ {3.74}, H6b {3.73}, H2’ {3.67}),
3.61 3.51 (m, 2H; H3 {3.57}, H6a’ {3.54}), 3.49 3.35 (m, 2H; H6b’ {3.44},
Tg) ppm; 13C NMR (100.6 MHz, CDCl3, BB, HMQC):
d = 171.53,
H5’ {3.39}), 3.26 3.20 (m, 1H; H4’), 2.72 (dd, JH3eq’’,H3ax’’
= 13.1 Hz,
JH3eq’’,H4’’ 4.7 Hz, 1H; H3eq’’), 2.15 1.90 (m, 16H; 4îCH3OAc, 1î
=
170.91, 170.82, 170.24, 170.09, 169.76, 167.77 (C=O), 156.83 (C=O-ure-
thane), 143.71 (C1a-, C8a-Fmoc), 141.29, 141.15 (C4a-, C5a-Fmoc),
138.07, 137.51 (Cq-Bn), 134.21 (Cq-Bn), 128.97, 128.84, 128.78, 128.43,
127.51 (Car-Bn), 128.78, 128.04, 126.44 (Car-Bzn), 127.79 (C3-, C6-Fmoc),
127.09, 126.97 (C2-, C7-Fmoc), 125.17, 124.94 (C1-, C8-Fmoc), 120.03
(C4-, C5-Fmoc), 106.55 (C1’), 100.96 (CH-Bzn), 100.45 (C1), 97.47 (C2’’),
CH3NAc, H3ax’’ {2.02}), 1.83 (s, 3H; CH3NAc), 1.42 (s, 9H; CH3-tBu),
1.26 (d, JTg,Tb = 6.26 Hz, 3H; Tg) ppm; 13C NMR (100.6 MHz, CDCl3,
BB, HMQC): d = 171.77, 171.38, 170.86, 170.57, 170.06, 169.62 (C=O),
167.59 (C1’’), 156.73 (C=O-urethane), 144.64, 143.66 (C1a-, C8a-Fmoc),
141.24 (C4a-, C5a-Fmoc), 137.89, 134.16 (Cq (2îBn)), 128.94, 128.82,
4158
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 4150 4162

Tumor-Associated (2,3)-Sialyl-T Antigen
4150 4162
128.73, 128.73, 128.39, 128.12, 127.76, 127.69, 127.54 (Carom (2îBn), C3-,
C6-Fmoc), 127.03, 126.92 (C2-, C7-Fmoc), 125.20, 125.09 (C1-, C8-Fmoc),
124.86 (C4-, C5-Fmoc), 105.90 (C1’), 100.07 (C1), 97.44 (C2’’), 80.68 (C3),
76.13 (C3’), 75.87 (Tb), 73.48 (CH2-Bn), 73.40 (C5’), 73.10 (C6’’), 69.69
(C6’), 69.60 (C4), 69.51 (C5), 68.79 (C8’’), 68.49 (C4’’), 68.40 (C4’), 68.27
(C2’), 68.17 (CH2-Bn), 67.89 (C7), 67.11 (CH2-Fmoc), 63.52 (C9’’), 62.94
(C6), 59.15 (Ta), 49.01 (C5’’), 47.43 (C2), 47.06 (C9-Fmoc), 37.41 (C3’’),
23.16, 23.01 (CH3-NHAc), 21.08, 21.02, 20.72, 20.57, (CH3 (4îOAc)),
19.22 (Tg) ppm; MS (HR-ESI-TOF): m/z:found:1402.5597; C 70H87N3O27
calcd 1402.5606.
JH1’,H2’ = 7.8 Hz, 1H; H1’), 4.66 4.40 (m, 6H; H3’ {4.64}, CH2-Bn {4.61,
4.54}, CH2-Fmoc {4.60, 4.48}, Tb {4.44}), 4.38 4.29 (m, 2H; H2 {4.33},
H9a’’ {4.31}), 4.28 4.21 (m, 2H; H9-Fmoc {4.26}, H5 {4.23}), 4.16 4.11
(m, 2H; Ta {4.14}, H6a {4.12}), 4.08 3.94 (m, 4H; H3 {4.05}, H9b’’ {4.01},
H6b {4.00}, H5’’ {3.96}), 3.92 3.86 (m, 1H; H5’), 3.82 3.77 (m, 1H; H6’’),
3.63 (dd, JH5’,H6’a = 6.3 Hz, JH6’a,b = 9.4 Hz, 1H; H6a’), 3.49 (dd, JH5’,H6’b
=
6.3 Hz, J6’a,6’b = 9.4 Hz, 1H; H6b’), 2.65 (dd, JH3’’ax,eq = 12.5 Hz,
JH3’’eq,H4’’ = 4.7 Hz, 1H; H3eq’’), 2.29 (s, 3H; CH3-Ac), 2.19 (s, 3H; CH3-
Ac), 2.13 (s, 3H; CH3-Ac), 2.10 (s, 3H; CH3-Ac), 2.08 (s, 3H; CH3-Ac),
2.05 (s, 6H; CH3 (2îAc)), 2.03 (s, 3H; CH3-Ac), 1.99 (s, 3H; CH3-Ac),
1.84 (s, 3H; CH3-Ac), 1.55 (dd, JH3’’ax,H3’’eq = 12.5 Hz, JH3’’ax,H4’’ = 12.1 Hz,
1H; H3ax’’), 1.25 (d, JTb,g = 5.5 Hz, 3H; Tg) ppm; 13C NMR (100.6 MHz,
CD3OD, BB, HMQC): d = 173.5, 172.7, 172.5, 172.2, 172.1, 171.8, 171.5,
171.0, 168.7 (C=O), 159.0 (C=O-urethane), 145.4, 145.2 (C1a-, C8a-
Fmoc), 142.7 (C4a-, C5a-Fmoc), 139.4, 136.1 (Cq (2îBn)), 129.8, 129.7,
129.6, 129.5, 128.9, 128.8, 128.2 (Carom (2îBn)), C2-, C3-, C6-, C7-Fmoc),
126.1, 126.0 (C1-, C8-Fmoc), 121.0 (C4-, C5-Fmoc), 102.7 (C1’), 100.3
(C1), 98.1 (C2’’), 77.5 (Tb), 74.9 (C3), 74.5 (CH2-Bn), 73.2 (C5’), 73.0
(C6’’), 72.8 (C3’), 71.4 (C4, C2’), 70.9 (C4’’), 69.8 (CH2-Bn), 69.4 (C4’),
69.3 (C6’), 69.0 (C8’’), 68.8 (C5), 68.5 (C7’’), 67.5 (CH2-Fmoc), 64.3 (C6),
63.5 (C9’’), 61.2 (Ta), 50.5 (C2), 50.0 (C5’’), 48.7 (C9-Fmoc), 38.7 (C3’’),
23.6, 22.7 (CH3-NHAc), 21.7, 21.0, 20.8, 20.7, 20.6, 20.5 (CH3 (8îOAc)),
19.5 (Tg) ppm; MS (HR-ESI-TOF): m/z:found:1558.5040 [ M+2NaꢀH]+
; C74H86N3NaO31 calcd 1558.5041.
N-Fluorenylmethoxycarbonyl-O-(2-acetamido-4,6-di-O-acetyl-2-deoxy-3-
O-[2,4-di-O-acetyl-6-O-benzyl-3-O-{benzyl-(5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-a-d-galacto-2-nonulopyranosyl)onat}-b-d-galactopyra-
nosyl]-a-d-galactopyranosyl)-l-threonine tert-butyl ester (17):Conjugate
16 (286 mg, 0.204 mmol) was dissolved in pyridine (5 mL) and cooled to
08C. After the addition of catalytic N,N-dimethylaminopyridine, acetic
anhydride (2.2 mL) was added dropwise. The solution was stirred at am-
bient temperature for 6 h, diluted with toluene (10 mL), and concentrat-
ed in vacuo. Purification of the residue by flash chromatography on silica
gel (ethyl acetate/ethanol 20:1) afforded the protected compound 17 as a
colorless, amorphous solid (279 mg, 87%). Rf
= 0.13 (ethyl acetate);
[a]2D8 1, CHCl3); 1H NMR (400 MHz, CDCl3, COSY,
=
36.0 (c
=
HMQC): d = 7.74 (d, JH4,H3 = JH5,H6 = 7.4 Hz, 2H; H4-, H5-Fmoc), 7.57
(t, JH1,H2 = JH8,H7 = 7.8 Hz, 2H; H1-, H8-Fmoc), 7.43 7.19 (m, 14H; H2-
ꢀ
, H3-, H6-, H7-Fmoc, Har-Bn (10H)), 6.36 (d, JNH,H2 = 9.4 Hz, 1H; NH
GalNAc), 5.85 (d, JNH,Ta = 9.8 Hz, 1H; NH-T), 5.50 5.43 (m, 1H; H8’’),
5.41 (s, 1H; CH2a-Bn), 5.40 5.33 (m, 1H; H4); 5.22 (dd, JH6’’,H7’’
General procedure for the automated solid-phase glycopeptide synthesis:
Peptide syntheses were carried out in a Perkin Elmer ABI 433A peptide
synthesizer by use of Fmoc-Pro-PTMSEL[52] and Fmoc-Asp(OtBu)-PHB
preloaded Tentagel resins.[56] In iterative coupling cycles, the subsequent
amino acids were attached sequentially. In every coupling step, the N-ter-
minal Fmoc group was removed by three 2.5 min treatments with 20%
piperidine in NMP. Amino acid couplings were performed with Fmoc-
protected amino acids (1 mmol) activated by HBTU/HOBt[53] (1 mmol
each) and DIPEA (2 mmol) in DMF (20 30 min vortex). After every
coupling step, unreacted amino groups were capped by treatment with a
mixture of Ac2O (0.5m), DIPEA (0.125m), and HOBt (0.015m) in NMP
(10 min vortex). Attachment of the glycosylated amino acids was per-
formed manually as described in the procedures for the corresponding
glycopeptides.
=
2.3 Hz, JH7’’,H8’’ = 7.6 Hz, 1H; H7’’), 5.08 (d, JH4’,H3’ = 3.12 Hz, 1H; H4’),
5.04 (s, 1H; CH2b-Bn), 4.99 4.90 (m, 2H; H2’ {4.94}, NH NeuNAc),
ꢀ
4.89 4.74 (m, 2H; H1 {4.86}, H4’’ {4.81}), 4.58 4.30 (m, 8H; H2 {4.52},
CH2a-Bn {4.51}, H1 {4.50}, CH2b-Bn {4.44}, H3’ {4.41}, CH2-Fmoc {4.36},
H9a’’ {4.35}), 4.28 4.06 (m, 5H; Tb {4.25}, Ta {4.22}, H9-Fmoc {4.20}, H6a
{4.11}, H5 {4.08}), 4.01 (d, JH4’’,H5’’ = JH5’’,H6’’ = 10.6 Hz, 1H; H5’’), 3.94
(dd, JH5,H6b = 7.4 Hz, JH6a,H6b = 11.3 Hz, 1H; H6b), 3.90 3.76 (m, 2H;
H9b’’ {3.89}, H3 {3.81}) 3.75 3.68 (m, 1H; H5’), 3.53 (dd, JH5’,H6a’
6.24 Hz, JH6a’,H6b’ 9.40 Hz, 1H; H6a’), 3.47 (dd, JH5’’,H6’’ 10.9 Hz,
JH6’’,H7’’ 2.4 Hz, 1H; H6’’), 3.40 (dd, JH5’,H6b’ 6.7 Hz, JH6a’,H6b’ =
=
=
=
=
=
9.6 Hz, 1H; H6b’), 2.59 (dd, JH3eq’’,H3ax’’ = 12.7 Hz, JH3eq’’,H4’’ = 4.3 Hz,
1H; H3eq’’), 2.24 (s, 3H; CH3-Ac), 2.03 2.00 (m, 1H; H3ax’’), 2.09, 2.07,
2.03, 2.02, 2.00, 1.92, 1.82 (7îs, 27H; 9îCH3-Ac), 1.42 (s, 9H; CH3-tBu),
1.26 (d, JTg,Tb = 5.92 Hz, 3H; Tg) ppm; 13C NMR (100.6 MHz, CDCl3,
BB, HMQC): d = 171.6, 171.0, 170.6, 170.3, 170.2, 169.9, 169.7, 167.2 (C=
O), 156.7 (C=O (urethane)), 143.7, 143.6 (C1a-, C8a-Fmoc), 141.2, 141.3
(C4a-, C5a-Fmoc), 137.9, 134.7 (Cq-Bn), 128.9, 128.6, 128.3, 127.6, 127.5
(Carom (2îBn), 127.8 (C3-, C6-Fmoc), 127.0 (C2-, C7-Fmoc), 125.1, 125.0
(C1-, C8-Fmoc), 120.0 (C4-, C5-Fmoc), 101.6 (C1’), 99.8 (C1), 96.8 (C2’’),
83.0 (Cq-tBu), 76.2 (Tb), 74.1 (C3), 73.4 (CH2-Bn), 72.5 (C6’’), 72.2 (C5’),
71.6 (C3’), 69.5 (C4), 69.2 (C2’), 69.1 (C4’’), 68.5 (C8’’, CH2-Bn), 68.1
(C5), 68.0 (C6’), 67.8 (C4’), 67.7 (C7’’), 67.4 (CH2-Fmoc), 63.3 (C6), 63.1
(C9’’), 59.2 (Ta), 48.9 (C5’’), 48.5 (C2), 47.0 (C9-Fmoc), 37.4 (C3’’), 28.1
(CH3-tBu)), 23.3, 23.1 (CH3-NHAc), 21.2, 21.1, 21.0, 20.8, 20.7, 20.6 (CH3
(8îOAc), 19.0 (Tg) ppm; HR-MS (ESI-TOF): m/z:found:1592.5853
[M+Na]+; C78H95N3O31Na calcd 1592.5847.
Ac-Gly-Val-Thr(a-Ac4NeuNAcCOOBn-(2 3)-b-6-O-Bn-Ac2Gal-(1 3)-a-
Ac2GalNAc)-Ser(tBu)-Ala-Pro-Asp(OtBu)-Thr(tBu)-Arg(Pmc)-Pro-
Ala-Pro-OH (20):Starting from Fmoc-Pro-PTMSEL-preloaded Nova-
Syn Tg resin 19[52] (182 mg, 0.05 mmol, loading:0.275 mmolg ꢀ1), the cou-
pling of the first eight amino acids was performed by the automated stan-
dard procedure. The glycosylated amino acid 18 (110 mg, 0.073 mmol,
1.45 equiv) was coupled manually with the aid of HATU (31.5 mg,
0.082 mmol, 1.64 equiv), HOAt (11.2 mg, 0.082 mmol, 1.64 equiv), and
NMM (18.2 mL, 16.7 mg, 0.165 mmol, 3.32 equiv) for activation (coupling
time 6 h). Then, in a resumption of the standard procedure, the final two
amino acids were attached to the polymer-bound glycopeptide, and the
N-terminal Fmoc-group was exchanged for an acetyl group. For the
cleavage procedure the resin was placed in a Merrifield glass reactor,
washed with dichloromethane (3î20 mL), treated with a solution of tet-
rabutylammonium fluoride trihydrate (38 mg, 0.12 mmol, 2.4 equiv) in
dry dichloromethane (8 mL), and shaken for 35 min at room tempera-
ture. The mixture was filtered, and the resin was washed with CH2Cl2
N-Fluorenylmethoxycarbonyl-O-(2-acetamido-4,6-di-O-acetyl-2-deoxy-3-
O-[2,4-di-O-acetyl-6-O-benzyl-3-O-{benzyl-(5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-a-d-galacto-2-nonulopyranosyl)onat}-b-d-galactopyra-
nosyl]-a-d-galactopyranosyl)-l-threonine (18):A solution of protected
trisaccharide 17 (250 mg, 0.159 mmol) in a mixture of TFA (5 mL) and
anisole (0.5 mL) was stirred at room temperature for 2 h. The reaction
mixture was diluted with toluene (25 mL) and the solvent was removed
in vacuo. The resulting residue was co-evaporated with toluene (4î
25 mL) and purified by flash chromatography (silica gel, ethyl acetate/
methanol 2:1) to yield compound 18 (242 mg, quant.) as a colorless,
amorphous solid. Rf = 0.27 (ethyl acetate/ethanol 1:1); [a]2D8 = 24.8 (c =
1, CHCl3); 1H NMR (400 MHz, CD3OD, COSY, HMQC): d = 7.82 (d,
JH3,H4 = JH5,H6 = 7.4 Hz, 2H; H4-, H5-Fmoc), 7.72 7.68 (m, 2H; H1-,
H8-Fmoc), [7.52 7.48 (m, 2H), 7.44 7.27 (m, 12H)] (H2-, H3-, H6-, H7-
Fmoc, Harom (2îBn, 10H)), 5.60 5.54 (m, 1H; H8’’), 5.46 5.39 (m, 3H;
(4î10 mL). The cleavage process was repeated with
a solution of
TBAF¥3H2O (35 mg, 0.11 mmol) in dichloromethane (8 mL). Filtrates
and washing solutions of both cleavage procedures were combined,
washed with water (3î20 mL), dried over anhydrous MgSO4, and liberat-
ed from the solvents in vacuo to yield 100 mg of a colorless solid. After
purification by preparative RP-HPLC (Rt = 55.7 min, column:Phenom-
enex Jupiter, grad:CH 3CN/H2O+0.1% TFA (10:90)!(100:0), 80 min, Rt
= 56.3 min) and lyophilization, the protected glycopeptide 20 was ob-
tained as a colorless solid (55.0 mg, 39%). Rt = 29.0 min (Phenomenex
Luna C18(2), grad.:CH CN/H2O + 0.1% TFA (10:90)!(0:90), 40 min);
3
[a]2D2
=
4.58 (c
=
1, CHCl3); 1H NMR (400 MHz, CDCl3, COSY,
HMQC): d = 7.66 7.44 (m, 2H; DNH {7.65}, VNH {7.45}) 7.41 7.25 (m,
12H; AN1 H {7.37}, 2î5 Har-Bn), 6.96 6.88 (m, 1H; GNH), 6.76 6.68 (m,
=
2
H4 {5.44}, H7’’ {5.41}, CH2a-Bn {d, Ja,b = 12.1 Hz}), 5.20 (d, JH3’,H4’
3.1 Hz, 1H; H4’), 5.15 (d, Ja,b = 12.1 Hz, 1H; CH2b-Bn), 5.02 4.95 (m,
2H; H1 {5.00}, H4’’ {4.96}), 4.87 (d, JH2’,H3’ = 10.2 Hz, 2H; H2’), 4.76 (d,
=
1H; NH-GalNAc), 5.49 5.41 (m, 1H; H8’’), 5.40 5.30 (m, 3H; CH2a-Bn1
{5.37}, H4 {5.33}, H7’’ {5.28}), 5.29 5.26 (m, 1H; H7’’), 5.12 4.94 (m, 4H;
ꢀ
NH NeuAc {5.08}, H4’ (5.06), CH2b-Bn1 {5.02}, H1 {4.99}), 4.93 4.78 (m,
Chem. Eur. J. 2004, 10, 4150 4162
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4159

H. Kunz et al.
FULL PAPER
2H; H4’’ {4.84}, H2’ {4.90}), 4.72 4.19 (m, 18H; Da {4.62}, H1’ {4.61}, Aa1
(Rb),26.16 (Pg), 25.90 (Rg), 23.76, 22.69, 22.55 (3îNHAc), 22.06 21.79,
21.04, 20.96, 20.88, 20.74, 20.62 (8îOAc), 20.54 (Tg1), 19.94 (Vga), 19.41
(TgST), 19.23 (Vgb), 16.67 (Ab); MALDI-TOF-MS (dhb, positive-ion
=
2
{4.60}, CH2a-Bn2 {4.52}, Ra {4.49}, H3’ {4.44}, CH2b-Bn2 {4.43}, 3îPa
{4.43}, H9a’’ {4.40}, Sa {4.40}, Va {4.32}, T1a {4.31}, TSbT {4.29}, H2 {4.29},
TbST), 4.15 3.88 (m, 9H; H5 {4.10}, Tb1 {4.05}, Sb {4.08}, H3 {3.99}, H5’’
{3.99}, Ga {3.97}, H9b {3.94}), 3.76 3.68 (2H; H5’ {3.72}, H6a {3.72}),
3.66 3.42 (m, 11H; 3îPd {3.59, 3.50}, H6a’ {3.53}, H6’’ {3.47}, H6 {3.47},
Rd), 3.37 (dd, JH6a’,H6b’ = 6.8 Hz, JH6b’,H5’ = 9.2 Hz, 1H; H6b’), 3.17 (sb,
1H; OH), 2.88 2.71 (m, 1H; Db {2.84, 2.75}), 2.64 2.45 (m, 9H; CH42-
mode):found:2383.7
[
M+H]+; C106H152N17O45 calcd 2383.0; found:
2405.7 [M+Na]+; calcd 2405.0; found:2421.8 [ M+K]+; calcd 2421.1;
found:2443.8 [ MꢀH+Na+K]+; calcd 2443.1.
Ac-Gly-Val-Thr(a-NeuNAcCOOH-(2 3)-b-6-O-Bn-Gal-(1 3)-a-
GalNAc)-Ser-Ala-Pro-Asp-Thr-Arg-Pro-Ala-Pro-OH (22):Catalytic pal-
ladium on activated charcoal (10%; 0.1 mg) was added under argon to a
solution of 21 (41.0 mg, max. 0.017 mmol) in methanol (35 mL). The re-
action flask was purged with hydrogen, and the solution was stirred
under a hydrogen atmosphere for two days. The mixture was filtered
through Hyflo Super Cel, which was afterwards washed with methanol
(3î25 mL). The filtrate and washing solutions were combined. After re-
moval of the solvent in vacuo, the resulting crude product (33.6 mg) was
of sufficient purity to be employed for the final deprotection without ad-
2
8
ꢀ
Pmc {2.60}, CH3 Pmc, CH3-Pmc {2.52, 2.51}}, H3eq’’ {2.56}), 2.21 (s, 3H;
CH3-Ac), 2.13 1.86 (m, 18H; 3îPb {2.12, 2.02}, Vb {2.08}, 3îPg {2.04,
1.93}, CH73-Pmc {2.08}, Rb), 2.12, 2.08, 2.05, 2.04, 2.00, 1.95 (6 s, 27H; 7î
ꢀ
ꢀ
CH3-Ac, 2îCH3 NAc {2.00, 1.94}), 1.82 1.75 (m, 6H; CH3 NAc {s,
3
g
ꢀ
1.79}, CH2 Pmc {1.78}), 1.68 1.56 (m, 3H; R {1.67}, H3ax’’ {t, 1.65,
JH3’’ax,eq = 12.5 Hz, JH3’’ax,H4’’ = 12.2 Hz}), 1.39 (s, 9H; tBuD), 1.32 1.25 (m,
12H; 2îAb, 2îCH35-Pmc {s, 1.28}), 1.24 1.15 (m, 12H; TSgT {1.19}, tBuS {s,
1.18}), 1.15 0.99 (m, 12H; tBuT {s, 1.07}, Tg1 {1.03}), 0.88 (dd, JVag,Vbg
=
17.2 Hz, JVg,Vb = 6.2 Hz, 6H; Vg) ppm; 13C NMR (CDCl3, d obtained
from HMQC): d = 129.0, 128.7, 128.4, 127.9, 126.0 (Car-Bn), 100.9 (C1’),
100.3 (C1), 78.4 (TbST), 73.7 (C3), 73.6 (CH2-Bn2), 72.1 (C6’’), 71.8 (C5’),
71.6 (C3’), 69.8 (C2’), 69.4 (C4, C4’’), 68.6 (CH2-Bn1), 68.4 (C8’’), 68.0
(C5), 67.9 (C6’), 67.7 (C4’), 67.3 (C7’’), 66.2 (Tb1), 63.1 (Sb), 62.6 (C9’’),
61.6 (C6), 60.9 (Pa), 59.4 (Sa), 58.4 (Va), 58.2 (Ta1), 50.3 (Da), 50.0 (Aa),
48.8 (C2), 48.7 (C5’’), 47.4 (Pd), 43.1 (Ga), 37.4 (Db), 37.2 (C3’’), 32.8
ditional purification. Rt
= 16.0 min (Phenomenex Jupiter C18, grad.:
CH3CN/H2O + 0.1% TFA (10:90)!(60:40), 40 min); MALDI-TOF-MS
(dhb, positive-ion mode): m/z:found:2204.0 [ M+H]+; C92H140N17O45
calcd. 2204.2; found:2226.0 [ M+Na]+; calcd 2226.2.
For the concluding deacetylation, one part of the crude glycopeptide
(16.0 mg, max. 0.0072 mmol) was dissolved in a 5 mm aqueous solution of
sodium hydroxide (5 mL, pH 11.5). The mixture was stirred for 59 h,
during which the course of the reaction was continuously monitored by
analytical RP-HPLC. After 89% conversion, the solution was neutralized
by the addition of acetic acid and lyophilized, and the resulting crude
product was purified by semi-preparative RP-HPLC (Phenomenex Jupi-
3
b
b
S
g
D
ꢀ
(CH2 Pmc), 32.7 (R ), 28.9, 28.6 (P ), 28.4 (tBu ) 28.2 (R ), 28.0 (tBu ),
27.4 (tBuT), 26.8 (2îCH53-Pmc), 25.1 (Pg), 23.2, 22.9, 22.8 (3îNHAc),
21.4 (CH42-Pmc), 21.6, 21.0, 20.9 (8îOAc), 20.9 (Vb), 19.5, 18.2 (Vg), 18.4
(T1g), 18.8 (TSgT), 18.5 (CH38-Pmc), 17.7 (Ab), 17.5 (CH32-Pmc), 12.2 (CH73-
Pmc); MALDI-TOF-MS (dhb, positive-ion mode):found:2818.4
[M+H]+; C132H194N17O48S calcd 2817.3; found:2840.0 [ M+Na]+; calcd
ter, grad:CH 3CN/H2O
+
0.1% TFA (5:95)!(30:70), 80 min, Rt
=
27.3 min) to yield the title compound as a colorless, amorphous solid
M+K]+; calcd 2855.4; found:2879.0
(8.6 mg, 56% over three steps). [a]2D6 = ꢀ40.7 (c = 0.86, H2O), Rt
=
2839.3; found:2856.1
[
[MꢀH+Na+K]+ calcd 2877.4.
14.5 min (Phenomenex JupiterC18, grad.:CH 3CN/H2O
+ 0.1% TFA
1
Ac-Gly-Val-Thr(a-Ac4NeuNAcCOOBn-(2 3)-b-6-O-Bn-Ac2Gal-(1 3)-a-
Ac2GalNAc)-Ser-Ala-Pro-Asp-Thr-Arg-Pro-Ala-Pro-OH (21):A solu-
tion of 20 (48.5 mg, 0.017 mmol) in a mixture of TFA (15 mL), triisopro-
pylsilane (0.9 mL), and water (0.9 mL) was stirred at room temperature
for 2.5 h. The reaction mixture was then concentrated in vacuo and co-
evaporated with toluene (4î20 mL). The product 21 was precipitated
into cold (08C) diethyl ether (20 mL) to furnish a colorless solid, which
was washed with diethyl ether (3î20 mL). The obtained glycopeptide 21
(41 mg) was sufficiently pure to be characterized and subsequently de-
protected without further purification. Rt = 20.2 min (Phenomenex Jupi-
ter C18, grad.:CH 3CN/H2O + 0.1% TFA (10:90)!(60:40), 40 min); 1H
NMR (400 MHz, CD3OD, COSY, HMQC): d = 7.53 7.45 (m, 10H; Har-
Bn), 5.61 5.52 (m, 1H; H8’’), 5.50 5.35 (m, 3H; H7’’ {5.44}, H4 {5.45},
CH2a-Bn1 {5.39}), 5.23 (d, JH3,H4 = JH4,H5 = 3.5 Hz, 1H; H4’), 5.19 5.11
(m, 1H; CH2b-Bn1), 5.07 4.97 (m, 2H; H1 {5.02}, H4’’ {4.98}), 4.83 4.56
(m, 9H; H1’ {4.80}, H2’ {4.79}, Ra {4.72}, TSaT {4.68}, Da {4.66}, H3’ {4.65},
CH2a-Bn2 {4.62}, 2îAa {4.60}), 4.55 4.44 (m, 5H; CH2b-Bn2 {4.52}, Sa
{4.48}, 3îPa {4.45}), 4.43 4.24 (m, 7H; T1a {4.37}, H2 {4.34}, TSbT {4.33},
H9a’’ {4.32}, Va {4.31}, H5 {4.29}, Tb1 {4.29}), 4.19 3.59 (m, 18H; H6a
{4.13}, H3 {4.12}, H9b’’ {4.08}, H6b {4.04}, H5’’ {3.97}, Gaa {3.95}, Gba
{3.91}, H5’ {3.88}, Sb {3.82}, 3îPad {3.81}, H6’’ {3.77}, 3îPdb {3.68}, H6a’
{3.62}), 3.45 (dd, JH6b’,H6a’ = 7.44 Hz, JH6b’,H5’ = 9.6 Hz, 1H; H6b’), 3.30
3.16 (m, 2H; Rd), 3.00 3.16 (d, JDb,Da = 5.9 Hz, 2H; Db), 2.64 (dd, JH3eq’’,H3ax’’
= 12.3 Hz, JH3eq’’,H4’’ = 4.7 Hz, 1H; H3eq’’), 2.39 1.97 (m, 13H; 3îPab
{2.29}, 3îPg {2.07}, Vb {2.12}, 3îPbb {2.04}), 2.31 (s, 3H; CH3-NHAc),
2.18, 2.14, 2.09 (3îs, 9H; 3îCH3-OAc), 2.08 (s, 3H; CH3-NHAc), 2.07,
2.05, 1.99 (3îs, 15H; 5îCH3-OAc), 1.95 1.86 (m, 1H; Rba), 1,84 (s, 3H;
(5:95)!(30:70), 40 min); H NMR (400 MHz, D2O, COSY, HMQC): d =
4.92 (d, JH1,H2 = 3.7 Hz, 1H; H1), 4.69 (t, JDa,Db = 6.4 Hz, 1H; Da), 4.65
4.58 (m, 2H; TaST {4.63}, Ra {4.62}), 4.57 4.49 (m, 1H; A1a), 4.49 4.41 (m,
3H; H1’ {4.47}, Sa {4.45}, A2a {4.44}), 4.40 4.34 (m, 3H; 3îPa), 4.33 4.26
(m, 3H; TbST {4.31}, Va {4.29}, Ta1 {4.28}), 4.22 4.14 (m, 3H; H4 {4.21}, H2
{4.19}, Tb1 {4.18}), 4.08 3.95 (m, 3H; H3’ {4.04}, H7’’ {4.03}, H3 {4.00}),
3.94 3.87 (m, 3H; Ga {3.91}, H4’ {3.90}), 3.86 3.52 (m, 20H; H5’, H8’’
{3.83}, H6a {3.80}, Sb {3.76}, 3îPad {3.73}, H5’’ {3.71}, H9a’’, H9b’’, H4’’
{3.67}, 3îPaa {3.63}, H6b {3.62}, H6’’, H5, H6a’, H6b’), 3.48 (dd, JH1’,H2’
= 7.9 Hz, JH2’,H3’ = 1.6 Hz, 1H; H2’), 3.21 3.14 (m, 2H; Rd), 2.99 2.83
(m, 2H; Db), 2.71 (dd, JH3eq’’,H3ax’’ = 12.2 Hz, JH3eq’’,H4’’
= 4.4 Hz, 1H;
H3eq’’), 2.35 2.19 (m, 3H; 3îPab), 2.10 1.94 (m, 8H; Vb {2.03}, 3îPg
{2.01}, Rba), 2.02, 1.99, 1.97 (3îs, 9H; 3îCH3-NHAc), 1.93 1.77 (m, 4H;
3îPbb {1.88}, H3ax’’ {1.80}), 1.77 1.59 (m, 3H; Rbb{1.70}, Rg {1.63}), 1.38
1.31 (m, 6H; 2îAb), 1.24 (d, JTg,Tb = 6.1 Hz, 3H; TSgT), 1.15 (d, JTg,Tb
=
6.4 Hz, 3H; Tg1), 0.94 (dd, J = 6.6 Hz, J = 2.0 Hz, 6H; Vg); MALDI-
TOF-MS (dhb, positive ion mode):found:1868.8 [ M+H]+; C76H124N17O37
calcd 1867.9; found:1890.9
[
M+Na]+; calcd 1889.9; found:1907.1
[M+K]+; calcd 1905.9; found:1912.8 [ MꢀH+2îNa]+; calcd 1911.9; ESI-
MS
(positive-ion
mode):found:977.87
[
Mꢀ2H+4Na]2+
;
[C76H121N17O37Na4]2+ calcd 977.88,
Ac-Thr-Ser-Ser-Ala-Ser-Thr-Gly-His-Ala-Thr(a-Ac4NeuNAcCOOBn-(2
3)-b-Ac2BnGal-(1 3)-a-Ac2GalNAc)-Pro-Leu-Pro-Val-Thr-Asp-OH (24):
The automated solid-phase synthesis of the MUC4 glycopeptide 24 was
performed starting from the preloaded Fmoc-Asp(OtBu)-PHB Tentagel S
resin[56] 23 (238 mg, 0.05 mmol, loading:0.21 mmolg ꢀ1). The first five
Fmoc-amino acids were coupled with excesses of 20 equivalents
(1.00 mmol), by the standard procedure. For the coupling of the glycosy-
lated threonine 18, which was incorporated in the MUC4 sequence at the
threonine-10 position, only 1.3 equivalents (100 mg, 0.066 mmol) were
used. The coupling of the a-(2,3)-sialyl-T-threonine building block 18 was
activated with a mixture of HATU (35 mg, 0.092 mmol), HOAt (13 mg,
0.092 mmol), and N-methylmorpholine (20 mL, 0.185 mmol) in N-methyl-
pyrrolidone (2 mL) and carried out over 3 h. The nine subsequent Fmoc-
amino acids were coupled to the glycopeptide by the standard procedure.
After completion of the 16-amino acid MUC4 sequence, the terminal
Fmoc group was removed with piperidine (20%) in N-methylpyrrolidone,
and the N-terminus of the glycopeptide was acetylated with capping re-
agent on the resin. In order to detach the glycopeptide from the polymer
and simultaneously to cleave the amino acid side chain protection
CH3-NHAc), 1,82 1,69 (m, 2H; Rbb {1.78}, Rg {1.73}), 1.55 (t, JH3eq’’,H3ax’’
JH3ax’’,H4’’ = 12.5 Hz, 1H; H3ax’’), 1.49 1.39 (m, 6H; 2îAb), 1.33 1.27 (d,
JTg,Tb = 6.3 Hz, 3H; TgST), 1.26 1.18 (m, 3H; Tg1), 1.02 (t, JVag,Vbg = JVg,Vb
=
=
7.4 Hz, 6H; Vg) ppm; 13C NMR (100.6 MHz, CDCl3, BB, HMQC): d =
175.30, 174.47, 174.30, 174.03, 173.87, 173.80, 173.57, 173.18, 173.06,
172.83, 172.52, 172.34, 172.08, 171.93, 171.72, 171.51, 170.95, 168.72 (C=
O), 139.40, 136.14 (Cq-Bn), 129.80, 129.64, 129.41, 128.94, 128.80 (Car-Bn),
102.54 (C1’), 101.11 (C1), 98.15 (C2’’), 79.03 (TbST), 75.50 (C3), 74.40
(CH2-Bn2), 72.91 (C6’’, C3’, C5’), 71.64 (C2’, C4), 70.90 (C4’’), 69.76
(CH2-Bn1), 69.19 (C4’), 69.03 (C8’’, Tb1, 68.79 (C6’, C5), 68.55 (C7’’), 64.35
(C6), 63.23 (C9’’, Sb), 60.28 (T1a, Pa, Va), 57.95 (TSaT), 56.54 (Sa), 51.99
(Ra), 51.82 (Da), 50.09 (C5’’), 49.99 (C2), 48.90 (Aa), 48.11 (Pd), 43.60
(Ga), 42.13 (Rd), 38.76 (C3’’), 35.80 (Db), 31.82 (Vb), 30.41 (Pb), 29.40
4160
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 4150 4162

Tumor-Associated (2,3)-Sialyl-T Antigen
4150 4162
groups, the resin was transferred into a Merrifield glass reactor and treat-
ed with a mixture of trifluoroacetic acid (7.50 mL), dist. water (0.45 mL),
and triisopropylsilane (0.45 mL) for 2 h. After filtration, the resin was
washed with trifluoroacetic acid (4î5 mL), and the combined filtrates
were concentrated in vacuo to a volume of 5 mL and added dropwise to
dry diethyl ether (20 mL) at 08C. The formed precipitate was separated
from the mother liquor by centrifugation and washed with diethyl ether
(10 mL). The residue was dissolved in dist. water and lyophilized. The
crude product was purified by semipreparative RP-HPLC (column:Phe-
nomenex Luna, gradient:CH 3CN/H2O (+ 0.1% TFA) 5:95!CH3CN/
H2O (+ 0.1% TFA) 50:50 in 60 min, Rt = 55.2 min), and the MUC4 gly-
copeptide 24 (64 mg, 46% yield over 33 steps, 98% yield per coupled
amino acid) was obtained as a colorless lyophilizate.
Rt = 16.6 min (analytical RP-HPLC, column:Phenomenex Luna, gradi-
ent:CH 3CN/H2O (+ 0.1% TFA) 5:95!CH3CN/H2O (+ 0.1% TFA)
100:0 in 60 min); MS (MALDI-TOF, dhb, positive-ion mode): m/z:
found:2576.1
[
M+H]+; C107H163N20O53 calcd 2576.1; found:2597.8
[M+Na]+; C107H162N20NaO53 calcd 2598.1; found:2613.5
[
M+K]+;
C107H162KN20O53 calcd 2614.0; found:2619.7
C107H161N20Na2O53 calcd 2620.0.
[
M+2NaꢀH]+;
The debenzylated MUC4 glycopeptide (29 mg, 0.011 mmol) was dis-
solved in anhydrous methanol (15 mL) and treated with a solution of
NaOMe (5%) in methanol. The reaction mixture was adjusted to pH 10
and stirred for 48 h at ambient temperature. The solution was neutralized
by addition of acetic acid, and the solvent was evaporated in vacuo. Ana-
lytical RP-HPLC analyses and mass spectra confirmed that the residue
contained the monoacetylated MUC4 glycopeptide as the main product.
The residue was therefore again dissolved in an aqueous solution of
NaOH (5 mm, 10 mL) and stirred at pH 11.5. The final deacetylation was
monitored carefully by analytical RP-HPLC. The reaction mixture was
neutralized with acetic acid after 36 h, and the water was removed by lyo-
philization. The residue was purified by semipreparative RP-HPLC
(column:Phenomenex Luna, gradient:CH 3CN/H2O (+ 0.1% TFA)
5:95!CH3CN/H2O (+ 0.1% TFA) 25:75 in 60 min, Rt = 40.0 min) to
give the (2,3)-sialyl-T-MUC4-glycopeptide 25 (15 mg, 64%) as a color-
less, amorphous solid. In addition, the corresponding monoacetylated
MUC4-glycopeptide was isolated as a by-product (3 mg, 12%).
Rt = 27.1 min (analytical RP-HPLC, column:Phenomenex Luna, gradi-
ent:CH 3CN/H2O (+ 0.1% TFA) 5:95!CH3CN/H2O (+ 0.1% TFA)
100:0 in 60 min); [a]D23 = ꢀ47.31 (c = 1, H2O); 1H NMR (400 MHz,
COSY, HMQC, D2O): d = 8.61 (d, JHe,NH = 1.6 Hz, 1H; He), 7.46 7.30
(m, 10H; Har-Bn (2î)), 7.26 (s, 1H; Hd), 5.44 5.38 (m, 4H; H4 {5.41},
H7’’ {5.41}, H8’’ {5.40}, CH2a-Bn {5.40}), 5.12 5.05 (m, 2H; H1 {5.09},
CH2b-Bn {5.09}), 5.00 4.47 (m, 1H; H4’), 4.92 4.73 (m, 5H; H4’’ {4.87},
H1’ {4.79}, H2’ {4.79}, Da {4.78}, Ta* {4.74}), 4.70 (dd, JHa,Hb = 5.5 Hz,
JHa,Hb’ = 8.6 Hz, 1H; Ha), 4.60 (d, Ja,b = 11.7 Hz, 1H; CH2a-Bn), 4.54
4.40 (m, 9H; Sa {4.52, 4.45 (3î)}, Pa {4.50, 4.42}, CH2b-Bn {4.49}, H3’
{4.49}, Aa {4.48}, La {4.45}), 4.38 4.28 (m, 9H; Ta {4.36, 4.33 (3î)}, H5’
{4.35}, Aa {4.34}, H9a’’ {4.33}, 2-H {4.31}, H3 {4.31}, Tb* {4.29}), 4.26 4.14
(m, 5H; Tb {4.22 (3î)}, H6a’ {4.16}, Va {4.16}), 4.12 4.02 (m, 2H; H9b’’
{4.09}, H6b’ {4.05}), 3.97 3.81 (m, 10H; Pd {3.95}, Sb {3.87 (3î)}, H5’’
{3.86}, Ga {3.85}), 3.79 3.54 (m, 6H; H5 {3.75}, H6’’ {3.75}, Pd {3.73, 3.67,
3.61}, H6a {3.56}), 3.46 3.39 (m, 1H; H6b), 3.26 (dd, JHa,Hb = 5.1 Hz,
JHb,Hb’ = 15.7 Hz, 1H; Hb), 3.10 (dd, JHa,Hb’ = 8.6 Hz, JHb,Hb’ = 15.7 Hz,
1H; Hb’), 2.96 (d, JDa,b = 5.9 Hz, 2H; Db), 2.64 2.58 (m, 1H; H3eq’’),
2.30 2.23 (m, 2H; Pb), 2.29 (s, 3H; CH3-Ac), 2.19 (s, 3H; CH3-Ac), 2.15
(s, 3H; CH3-Ac), 2.10 2.08 (m, 1H; Vb), 2.09 (s, 3H; CH3-Ac), 2.08 (s,
3H; CH3-Ac), 2.07 (s, 3H; CH3-Ac), 2.06 (s, 3H; CH3-Ac), 2.05 1.96 (m,
4H; Pg {2.03, 1.98}), 2.02 (s, 3H; CH3-Ac), 1.98 (s, 3H; CH3-Ac), 1.92
1.86 (m, 2H; Pb), 1.85 (s, 3H; CH3-Ac), 1.72 1.48 (m, 4H; Lg {1.68}, Lb
{1.58, 1.53}, H3ax’’ {1.56}), 1.39 (d, JAa,b = 7.1 Hz, 6H; Ab (2î)), 1.33 (d,
JTb*,g = 6.3 Hz, 3H; Tg*), 1.23 1.17 (m, 9H; Tg (3î)), 1.00 0.93 (m, 12H;
Ld {0.99, 0.96}, Vg {0.96, 0.94}) ppm; 13C NMR (100.6 MHz, BB, HMQC,
D2O): d 175.0, 174.9, 174.6, 174.0, 173.9, 173.5, 173.4, 173.3, 173.1, 172.7,
172.6, 172.3, 172.2, 172.1, 171.9, 171.7, 171.5, 171.1, 170.6, 169.5, 167.1
(C=O), 137.0, 134.1 (Cq-Bn (2î)), 133.3 (He), 129.0, 128.7, 128.6, 127.9
(Car-Bn (2î)), 128.2 (Hg), 117.2 (Hd), 100.5 (C1’), 99.1 (C1), 97.0 (C2’’),
75.5 (Tb*), 72.9 (C3, CH2-Bn), 71.6 (C5, C3’, C6’’), 70.5 (C8’’), 70.1 (C2’),
69.4 (C4’’), 68.8 (CH2-Bn), 68.3 (C4’), 67.5 (C4, C6, C5’, C7’’), 66.9, 66.7
(Tb (3î)), 63.1 (C6’), 61.9 (C9’’), 60.9, 60.8 (Sb (3î)), 60.2, 59.6 (Pa), 59.4
(Va), 59.1, 58.6 (Ta (3î)), 55.6, 55.3 (Sa (3î)), 55.3 (Ta*), 51.8 (Ha), 50.3
(La), 49.9, 49.3 (Aa), 48.9 (Da), 48.5 (C5’’, C2), 48.1, 47.7 (Pd), 42.3 (Ga),
39.7 (Lb), 36.7 (C3’’), 35.3 (Db), 30.0 (Vb), 29.3, 29.2 (Pb), 26.5 (Hb), 24.6,
24.3 (Pg), 24.2 (Lg), 22.4 (Ld), 22.3, 21.7 (CH3-NHAc (3î)), 21.6 (Ld),
20.8, 20.7, 20.2, 20.0, 19.9, 19.7 (CH3-OAc (8î)), 18.6 (Tg (3î), Tg*), 18.3,
17.5 (Vg), 16.6, 16.2 (Ab) ppm; MS (MALDI-TOF, dhb, positive-ion
mode):m/z:found:2755.7 [ M+H]+; C121H175N20O53 calcd 2756.2; found:
2777.6 [M+Na]+; C121H174N20NaO53 calcd 2778.1; found:2793.1 [ M+K]+;
Rt = 15.6 min (analytical RP-HPLC, column:Phenomenex Luna, gradi-
ent:CH 3CN/H2O (+ 0.1% TFA) 5:95!CH3CN/H2O (+ 0.1% TFA)
50:50 in 60 min); [a]D23 = ꢀ28.73 (c = 1, H2O); 1H NMR (600 MHz,
DQF-COSY, HMQC, D2O): d = 8.62 (s, 1H; He), 7.28 (d, JHd,NH
=
3.1 Hz, 1H; Hd), 5.06 (d, JH1,H2 = 3.5 Hz, 1H; H1), 4.78 4.65 (m, 3H; Da
{4.73}, Ha {4.73}, Ta* {4.69}), 4.55 4.14 (m, 19H; Aa {4.50, 4.35}, H1’
{4.49}, Sa {4.49, 4.43 (3î)}, Pa {4.46, 4.42}, La {4.41}, Ta {4.35, 4.34 (3î)},
Tb* {4.29}, Tb {4.26, 4.20 (3î)}, H2 {4.23}, H8’’ {4.19}, Va {4.15}), 4.10 4.01
(m, 3H; H7’’ {4.07}, H3’ {4.04}, H3 {4.03}), 3.94 3.55 (m, 26H; Ga {3.91},
H4 {3.90}, Pd {3.89, 3.74, 3.68, 3.60}, Sb {3.88 (3î)}, H4’ {3.86}, H6a, H6b
{3.82, 3.62}, H5’’ {3.82}, H9a’’, H9b’’{3.81, 3.60}, 6Ha’, 6Hb’ {3.73}, H6’’
{4.73}, H4’’ {3.67}, H5’ {3.61}, H5 {3.57}), 3.51 (dd, JH1’,H2’ = 7.8 Hz JH2’,H3’
= 9.4 Hz, 1H; H2’), 3.29 (dd, JHa,b = 5.1 Hz, JHb,Hb’ = 15.3 Hz, 1H; Hb),
3.17 3.10 (m, 1H; Hb’), 2.96 (d, JDa,b = 5.9 Hz, 2H; Db), 2.74 (dd, J3’’ax,eq
= 12.5 Hz, JH3’’eq,H4’’ = 4.3 Hz, 1H; H3eq’’), 2.32 2.22 (m, 2H; Pb), 2.10
1.96 (m, 14H; CH3-Ac {2.09, 2.02, 1.97}, Vb {2.08}, Pg {2.02, 1.99}), 1.93
1.84 (m, 2H; Pb), 1.80 (t, JH3’’ax,H3’’eq = JH3’’ax,H4 = 12.1 Hz, 1H; H3ax’’),
1.74 1.66 (m, 1H; Lg), 1.63 1.51 (m, 2H; Lb), 1.40 (d, JAa,b = 7.4 Hz,
6H; Ab (2î)), 1.33 (d, JTb*,g* = 6.3 Hz, 3H; Tg*), 1.26 1.17 (m, 9H; Tg
(3î)), 1.00 0.92 (m, 12H; Ld {0.96, 0.94}, Vg {0.95, 0.94}) ppm; 13C NMR
(150.9 MHz, BB, HMQC, D2O): d = 175.0, 174.8, 174.6, 174.3, 173.9,
173.6, 173.5, 173.4, 173.3, 172.8, 172.3, 171.5, 171.0, 170.9, 169.5 (C=O),
133.3 (He), 128.2 (Hg), 117.2 (Hd), 104.4 (C-1’), 99.4 (C2’’), 98.6 (C1), 77.9
(C3), 75.5 (C3’), 74.6 (Tb*), 72.6 (C5’), 71.6 (C4’), 70.9 (C7’’), 70.2 (C6’’),
70.0 (C2’), 68.9 (C8’’), 68.2 (C4’’), 67.9 (C5), 67.2 (C4), 66.9, 66.8 (Tb (3î
)), 63.1 (C9’’), 62.4 (C6), 61.2 (C6’), 60.9, 60.7 (Sb (3î)), 60.1, 59.6 (Pa),
59.3 (Va), 59.1, 59.0, 58.6 (Ta), 55.6 (Ta*), 55.5, 55.4 (Sa (3î)), 51.9 (Ha),
51.5 (C5’’), 50.3 (La), 49.9, 49.4 (Aa), 49.2 (Da), 48.2 (C-2), 48.0, 47.8 (Pd),
42.4 (Ga), 39.3 (C3’’), 38.9 (Lb), 35.5 (Db), 30.0 (Vb), 29.3, 29.2 (Pb), 26.4
(Hb), 24.6, 24.2 (Pg), 24.3 (Lg), 22.3 (Ld), 22.2, 21.9, 21.6 (CH3-Ac), 21.4
(Ld), 18.6 (Tg (3î), Tg*), 18.3, 17.6 (Vg), 16.6, 16.3 (Ab) ppm; MS
(MALDI-TOF, dhb, positive-ion mode): m/z:found:2242.1 [ M+H]+;
C91H147N20O45 calcd 2241.3; found:2263.9 [ M+Na]+; C91H146N20NaO45
calcd 2263.2; found:2280.1 [ M+K}+; C91H146KN20O45 calcd 2279.2.
C
121H174KN20O53
calcd
2794.1;
found:2799.6
[
M+2NaꢀH]+;
C121H173N20Na2O53 calcd 2800.1.
Ac-Thr-Ser-Ser-Ala-Ser-Thr-Gly-His-Ala-Thr(a-NeuNAc-(2 3)-b-Gal-(1
3)-a-GalNAc)-Pro-Leu-Pro-Val-Thr-Asp-OH (25):A catalytic amount of
palladium (10%) on activated charcoal was added under argon atmos-
phere to a solution of Ac-Thr-Ser-Ser-Ala-Ser-Thr-Gly-His-Ala-Thr(a-
Ac4NeuNAcCOOBn-(2 3)-b-Ac2BnGal-(1 3)-a-Ac2GalNAc)-Pro-Leu-
Pro-Val-Thr-Asp-OH 24 (36 mg, 0.013 mmol) in dry methanol (15 mL).
The reaction flask was then flooded with hydrogen, and the suspension
was stirred for 48 h under hydrogen atmosphere. After completion of the
reaction (monitoring by analytical RP-HPLC), the mixture was filtered
through Hyflo Super Cel and washed with methanol, and the solvent was
removed from the filtrate at reduced pressure. The residue was dissolved
in dist. water (20 mL) and lyophilized. The crude product of the debenzy-
lation reaction was obtained as a colorless lyophilizate (31 mg, 92%) and
subjected to the subsequent deacetylation procedure without further pu-
rification.
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft and
by the Stiftung Rheinland-Pfalz f¸r Innovation. S. D. is grateful for a doc-
toral scholarship provided by the Fonds der Chemischen Industrie and
the Bundesministerium f¸r Bildung und Forschung (BMBF).
[1] R. A. Dwek, Chem. Rev. 1996, 96, 683.
[2] K. Landsteiner, Wien. Klin. Wochenschr. 1901, 14, 1132.
Chem. Eur. J. 2004, 10, 4150 4162
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4161

H. Kunz et al.
FULL PAPER
[3] I. Brockhausen, Biochem. Soc. Trans. 2003, 31, 318.
[4] I. Brockhausen, Biochim. Biophys. Acta 1999, 67.
[5] G. F. Springer, Science 1984, 224, 1198.
[6] S. M¸ller, F.-G. Hanisch, J. Biol. Chem. 2002, 277, 26103.
[7] N. Marcos, T. , A. Cruz, F. Silva, R. Almeida, L. David, U. Mandel,
H. Clausen, S. v. Mensdorff-Pouilly, C. A. Reis, J. Histochem. Cyto-
chem. 2003, 51, 761.
[8] J. R. Gum Jr. , Biochem. Soc. Trans. 1995, 23, 795.
[9] I. Carlstedt, J. R. Davies, Biochem. Soc. Trans. 1997, 25, 214.
[10] S. Patton, S. J. Gendler, A. P. Spicer, Biochim. Biophys. Acta 1995,
407.
[30] J. B. Schwarz, S. D. Kuduk, X.-T. Chen, D. Sames, P. W. Glunz, S. J.
Danishefsky, J. Am. Chem. Soc. 1999, 121, 2662.
[31] B. Liebe, H. Kunz, Angew. Chem. 1997, 109, 629; Angew. Chem. Int.
Ed. Engl. 1997, 36, 618.
[32] B. Liebe, H. Kunz, Helv. Chim. Acta 1997, 80, 1473.
[33] G. Zemplÿn, A. Kunz, Chem. Ber. 1923, 56, 1705.
[34] S. K. George, B. Holm, C. A. Reis, T. Schwientek, H. Clausen, J.
Kihlberg, J. Chem. Soc. Perkin Trans. 1 2001, 880.
[35] X. Pannecoucke, G. Schmitt, B. Luu, Tetrahedron 1994, 50, 6569.
[36] W. Stahl, U. Sprengard, G. Kretzschmar, D. W. Schmidt, H. Kunz, J.
Prakt. Chem. 1995, 337, 441.
[11] J. Taylor-Papadimitriou, J. M. Burchell, D. W. Miles, M. Dalziel, Bio-
chim. Biophys. Acta 1999, 301.
[37] U. Sprengard, G. Kretzschmar, E. Bartnik, C. H¸ls, H. Kunz,
Angew. Chem. 1995, 107, 1104; Angew. Chem. Int. Ed. Engl. 1995,
34, 990.
[38] E. Eichler, H. J. Jennings, M. Gilbert, D. M. Whitfield, Carbohydr.
Res. 1999, 319, 1.
[39] R. R. Schmidt, J. Michel, Angew. Chem. 1980, 92, 763; Angew.
Chem. Int. Ed. Engl. 1980, 19, 731.
[40] R. R. Schmidt, Angew. Chem. 1986, 98, 213;, Angew. Chem. Int. Ed.
Engl. 1986, 25, 212.
[41] M. Lergenm¸ller, Y. Ito, T. Ogawa, Tetrahedron 1998, 54, 1381.
[42] W. Koenigs, E. Knorr, Chem. Ber. 1901, 957.
[43] B. Helferich, F. Wedemeyer, Liebigs Ann. Chem. 1949, 563, 139.
[44] G.-J. Boons, A. V. Demchenko, Chem. Rev. 2000, 100, 4539.
[12] F.-G. Hanisch, S. M¸ller, Glycobiology 2000, 10, 439.
[13] S. von Mensdorff-Pouilly, F. G. M. Snijdewint, A. A. Verstraeten,
R. H. M. Verheijen, P. Kenemans, Int. J. Biol. Markers 2000, 15, 343.
[14] J. Taylor-Papadimitriou, J. M. Burchell, T. Plunkett, R. Graham, I.
Correra, D. Miles, M. Smith, J. Mammary Gland Biol. Neoplesia
2002, 7, 209.
[15] N. Porchet, N. Van Gong, J. Dufosse, J. P. Audie, V. Guyonnet-Du-
perat, M. S. Gross, C. Denis, P. Degand, A. Bernheim, J.-P. Aubert,
Biochem. Biophys. Res. Commun. 1991, 175, 414.
[16] J. Dekker, J. W. A. Rossen, H. A. B¸ller, A. W. C. Einerhand,
Trends Biochem. Sci. 2002, 27, 126.
[17] N. Moniaux, F. Escande, S. K. Batra, N. Porchet, A. Laine, J.-P.
Aubert, Eur. J. Biochem. 2000, 267, 4536.
[18] P. L. Nguyen, G. A. Niehans, D. L. Cherwitz, Y. S. Kim, S. B. Ho,
Tumor Biol. 1992, 17, 176.
[19] S. Ogata, H. Uehara, A. Chen, S. H. Itzkowitz, Cancer Res. 1992, 52,
5971.
[20] M. A. Hollingsworth, J. M. Strawhecker, T. C. Caffrey, D. R. Mack,
Int. J. Cancer 1994, 57, 198.
[21] C. Balaguÿ, J.-P. Audiÿ, N. Porchet, F. X. Real, Gastroenterology
1995, 109, 953.
[22] A. Girling, J. Bartkova, J. M. Burchell, S. J. Gendler, C. Gillett, J.
Taylor-Papadimitriou, Int. J. Cancer 1989, 43, 1072.
[23] M. D. Walsh, M. A. McGuckin, P. L. Devine, B. G. Hohn, R. G.
Wright, J. Clin. Pathol. 1993, 46, 922.
»
[45] A. Marra, P. Sinay, Carbohydr. Res. 1989, 187, 35.
[46] S. Keil, C. Claus, W. Dippold, H. Kunz, Angew. Chem. 2001, 113,
379; Angew. Chem. Int. Ed. 2001, 40, 366.
[47] V. Martichonok, G. M. Whitesides, J. Org. Chem. 1996, 61, 1702.
[48] S. Dziadek, H. Kunz, Synlett 2003, 1623.
[49] F. Dasgupta, P. J. Garegg, Carbohydr. Res. 1988, 177, c13.
[50] C. Brocke, H. Kunz, Synlett 2003, 2052.
[51] M. Wagner, H. Kunz, Angew. Chem. 2002, 114, 315; Angew. Chem.
Int. Ed. 2002, 41, 317.
[52] M. Wagner, S. Dziadek, H. Kunz, Chem. Eur. J. 2003, 9, 6018.
[53] L. A. Carpino, H. Imazumi, A. El-Faham, F. J. Ferrer, C. Zhang, Y.
Lee, B. M. Foxmann, P. Henklein, C. Hanay, C. M¸gge, H. Wen-
schuh, J. Klose, M. Beyermann, M. Bienert, Angew. Chem. 2002,
114, 457; Angew. Chem. Int. Ed. 2002, 41, 441.
[24] C. Brocke, H. Kunz, Synthesis 2004, 4, 525.
[54] W. Kˆnig, R. Geiger, Chem. Ber. 1970, 103, 788.
[55] L. A. Carpino, J. Am. Chem. Soc. 1993, 115, 4397.
[56] E. Bayer, W. Rapp, Chem. Pept. Proteins 1986, 3, 3.
[57] S. S. Wang, J. Am. Chem. Soc. 1973, 95, 1328.
[25] S. Dziadek, C. G. EspÌnola, H. Kunz, Aust. J. Chem. 2003, 56, 519.
[26] C. Brocke, H. Kunz, Bioorg. Med. Chem. 2002, 10, 3085.
[27] H. Herzner, T. Reipen, M. Schultz, H. Kunz, Chem. Rev. 2000, 100,
4495.
[28] Y. Nakahara, Y. Nakahara, Y. Ito, T. Ogawa, Tetrahedron Lett. 1997,
38, 7211.
[29] S. Komba, M. Meldal, O. Werdelin, T. Jensen, K. Bock, J. Chem.
Soc. Perkin Trans. 1 1999, 415.
Received:March 10, 2004
Published online:July 9, 2004
4162
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 4150 4162