Solid-phase synthesis of core 8 O-glycan-linked MUC5AC glycopeptide

Article abstract of DOI:10.1271/bbb.69.1575

The benzyl-protected disaccharide building blocks of core 8 O-glycan (15a/15b) for glycopeptide were stereoselectively synthesized by two glycosidation reactions with the glycosyl fluoride method. The building blocks were utilized in the solid-phase synth

Full text of DOI:10.1271/bbb.69.1575

Biosci. Biotechnol. Biochem., 69 (8), 1575–1583, 2005
Solid-Phase Synthesis of Core 8 O-Glycan-Linked MUC5AC Glycopeptide
y
Makoto MAEMURA, Atsushi OHGAKI, Yuko NAKAHARA, Hironobu HOJO,y and Yoshiaki NAKAHARA
Department of Applied Biochemistry, Institute of Glycotechnology, Tokai University,
Kitakaname 1117, Hiratsuka, Kanagawa 259-1292, Japan
Received April 1, 2005; Accepted May 16, 2005
The benzyl-protected disaccharide building blocks of
core 8 O-glycan (15a/15b) for glycopeptide were stereo-
selectively synthesized by two glycosidation reactions
with the glycosyl fluoride method. The building blocks
were utilized in the solid-phase synthesis of a glycopep-
tide carrying two O-glycans with the consensus sequence
of the tandem-repeat domain of MUC5AC. The syn-
thetic glycopeptide was detached from the resin with
reagent K, and subsequent debenzylation under con-
ditions of low-acidity TfOH afforded glycopeptide 2.
The synthetic sample will be used as a suitable standard
in studies of the physicochemical or immunochemical
characterization of mucin glycoforms.
will also be used as a standard in developing the
technique for higher-ordered MS analyses of the mucin
fragments.
To this end, we have been studying the solid-phase
synthesis of glycopeptides especially by taking advant-
age of the benzyl group for consistent protection of the
carbohydrate moiety. The discovery of appropriate
acidic conditions for cleavage of the benzyl groups
have made our syntheses of several glycopeptides
carrying core 14–6) and core 2 O-glycans7–9) successful.
Our synthetic studies were then directed toward the
other core class glycan displayed on the mucin peptide
backbone. The rare core 8 glycan [ꢀ-D-Gal-(1!3)-D-
GalNAc-] was the latest characterized in a rather simple
trisaccharide form (1) amongst the complex oligosac-
charides isolated from human respiratory mucin collect-
ed from a patient suffering from chronic bronchitis.10)
There has been a report on the synthesis of the related ꢀ-
D-Gal-(1!3)-D-GalNAc analog with a lipophilic tag,11)
but no extensive study on the synthesis of the core 8 O-
glycan-linked glycopeptide has previously appeared.
From the easy access to ꢀ glycoside with the fully
benzylated glycosyl donor and its compatibility with our
strategy in solid-phase synthesis, we decided to synthe-
size model glycopeptides 2 carrying duplicate O-glycans
that will be a useful standard in elaborating a facile
physicochemical or immunological method for identi-
fication of this rare glycan structure. Since the backbone
amino acid sequence has not been identified for the
natural core 8 glycan, we designated an octapeptide
(TTSTTSAP) corresponding to the consensus sequence
of MUC5AC, a tracheobronchial mucin, as a putative
scaffold.12)
Key words: core 8 O-glycan; glycopeptide; solid-phase
synthesis; MUC5AC
O-Glycans are important types of post-translationally
modified proteins. Most abundant O-glycans with the
common structure of ꢀ-D-GalNAc-(1!3)-L-Ser/Thr are
grouped into eight core classes on the basis of glycosyl
substitution at the GalNAc residue.1) Mucin glycopro-
teins are densely O-glycosylated by a variety of
heterogeneous oligosaccharides with or without nega-
tively charged sialic acid or sulfate, and are assumed to
form a barrier to protect the epithelial cell surfaces from
desiccation and invading pathogenic microbes. On the
other hand, altered glycoforms that express mucins have
been found as the result of malignant transformation,
thereby being potential immunotherapeutic targets.2,3)
However, studies on the analyses of intact glycoproteins
as well as on the functions of O-glycan have been
obstructed due to difficulties in the purification of
natural mucin molecules of huge molecular mass. The
whole molecule of such a glycoprotein, although
difficult to synthesize, can be partly mimicked by
chemically synthesized pure glycopeptides which will
act as useful probes in studies on the immunological and
viscoelastic properties of native mucins. These samples
The target octapeptide contains a C-terminal proline
residue which reduces the total yield of the synthetic
peptide by generating an undesired diketopiperazine in
the early stage of Fmoc solid-phase synthesis. Sakamoto
et al. have recently reported an effective procedure to
prevent this diketopiperazine formation by a procedure
y
To whom correspondence should be addressed. Tel: +81-463-50-2075; Fax: +81-463-50-2012; E-mail: yonak@keyaki.cc.u-tokai.ac.jp;
hojo@keyaki.cc.u-tokai.ac.jp
Abbreviations: DAST, diethylaminosulfur trifluoride; DIEA, N,N-diisopropylethylamine; Fmoc, 9-fluorenylmethoxycarbonyl; HBTU, O-
benzotriazol-1-yl-N,N,N0,N0-tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; MS, mass spectrometry; NMP, 1-methyl-2-
pyrolidinone; Pfp, pentafluorophenyl; TBAF, tetra-n-butylammonium fluoride; TFFH, fluoro-N,N,N0,N0-tetramethylformamidinium hexafluorophos-
phate; TfOH, trifluoromethanesulfonic acid; Tsoc, triisopropylsilyloxycarbonyl

1576
M. MAEMURA et al.
that involved fluoride ion-catalyzed cleavage of N-
triisopropylsilyloxycarbonyl group on the resin-bound
dipeptide in the presence of the third Fmoc amino acid
activated as its acid fluoride.13) In order to enhance this
procedure that can be applied to glycopeptide synthesis,
we designed our solid-phase synthesis so as to use a
glycoamino acid fluoride.
Fig. 1. Structures of Naturally Occurring Core 8 O-Glycan and MUC5AC Glycopeptide.
Fig. 2. Core 8 O-Glycan Building Blocks and Synthetic Intermediates.

Synthesis of Glycopeptide with Core 8 O-Glycan
1577
Scheme 1. Solid-Phase Synthesis of the Glycopeptide.
Results and Discussion
Solid-phase synthesis of the glycopeptide and depro-
tection
Synthesis of building blocks 15a and 15b
The synthesis of glycopeptide 2 was commenced with
commercial Fmoc-Pro-CLEAR acid resin 18 (9 mmol) as
shown in Scheme 1. Solid-phase reactions were man-
ually operated in a polypropylene tube with vigorous
stirring by a vortex mixer. The Fmoc group was
removed with 20% piperidine/NMP. The second amino
acid residue was condensed to the N-deprotected proline
on the resin in THF twice with Tsoc-Ala-OPfp 17 (5
equiv.). The unreacted proline was blocked by acetyla-
tion. A solution of glycoamino acid fluoride 16 (2
equiv.), prepared from 15a and TFFH in CH2Cl2, was
then added to the resin, and the mixture was stirred for
12 h after adding a catalytic amount of TBAF. This
coupling procedure was repeated with another 2 equiv.
of 16. After capping with Ac2O, obtained resin 19 was
N-deprotected and reacted with Fmoc-Thr(But)-OH (4
equiv.) by activation with HBTU, HOBt and DIEA in
NMP at 50 ꢀC for 1 h. Analogouly, the peptide was
consecutively elongated with Fmoc-Thr(But)-OH and
Fmoc-Ser(But)-OH to give 20. The second O-glycan
was introduced by N-deprotection of the resin, this being
followed by coupling with glycothreonine 15b (2 equiv.)
under similar conditions. The condensation procedure
was duplicated with another two equivalents of 15b.
Finally, the N-terminal threonine was attached to
complete the solid-phase synthesis. The synthesized
glycopeptide was released from resin 21 by a treatment
with reagent K.26) The acid-labile But groups, benzyli-
dene groups and part of the benzyl groups were removed
by this treatment. The crude product, which was
precipitated from ether, was subsequently debenzylated
Synthesis of the disaccharide building blocks was
carried out via two complementary routes, using the
glycosyl fluoride method with known compounds 3,14)
4,15,16) 5a,17,18) 5b,17,18) 6a,19,20) and 6b.21,22) Glycosyla-
tion of 4 with fluoride 3 (ꢀ=ꢁ ¼ 1=2) was promoted by
Cp2ZrCl2–AgClO423) in CH2Cl2 to form an ꢀ=ꢁ mixture
of disaccharides (ꢀ=ꢁ ¼ 3:7=1) in a 93% yield. Minor ꢁ-
isomer 8 was identical with the known disaccharide
reported in the synthesis of core 1 O-glycan.24) Sepa-
rated ꢀ-isomer 7 was desilylated with TBAF-AcOH in
THF, and resulting hemiacetal 9 was treated with DAST
in THF to afford ꢀ- and ꢁ-fluoride 10 in an 81% yield in
two steps. The reaction between serine derivative 5a and
10 proceeded under the above mentioned glycosidation
conditions to furnish coupling product 11a (62%) and its
ꢁ-isomer 12a (16%). Analogously, threonine 5b was
glycosylated with 10 to afford 11b (52%) and isomer
12b (17%). Disaccharyl amino acids 11a and 11b were
alternatively obtained by glycosylation of 6a and 6b
with 3 in 62% and 63% yields, respectively. Corre-
sponding ꢁ-isomers 13a and 13b, the known core 1
derivatives,24,25) were also generated as minor products
in 10% and 8% yields, respectively. The azide com-
pounds were susceptible to reduction with Zn–AcOH
and subsequent N-acetylation with Ac2O to give
acetamides 14a (88%) and 14b (92%). Cleavage of the
allyl esters with Pd(Ph3P)4-dimedone completed the
preparation of building blocks 15a and 15b, both in a
quantitative yield.

1578
M. MAEMURA et al.
CHCl3, unless noted otherwise. Column chromatogra-
phy was performed on silica gel PSQ 100B (Fuji
Silysia). TLC and HPTLC were performed on silica gel
60 F254 (E. Merck). 1H and 13C NMR spectra were
recorded with
a
Jeol AL400 spectrometer [1H
(400 MHz) and 13C (100 MHz)]. Chemical shifts are
expressed in ppm downfield from the signal for internal
Me4Si for solutions in CDCl3. MALDI TOF mass
spectra were obtained with a PerSeptive Voyager-DE
PRO spectrometer (2,5-dihydroxybenzoic acid was used
as a matrix). High-resolution Fab mass spectra were
measured with Jeol JMS HX-110 spectrometer (2,5-
dihydroxybenzoic acid was used as a matrix). All solid-
phase reactions were performed at room temperature in
capped polypropylene test tubes equipped with a filter
and three-way stopcock by stirring on an EYELA CM-
1000 vortex mixer. HPLC was performed with Mightysil
RP-8 and RP-18 (150 ꢂ 4:6 mm for analysis and
250 ꢂ 10 mm for preparation, Kanto Chemical Co.).
Amino acids were analyzed by a Hitachi L-8500 amino
acid analyzer. Fmoc-Pro-CLEAR-acid resin was pur-
chased from Peptide International Inc.
Fig. 3. HPLC Profile of the Synthesized Glycopeptide.
Column: Mightysil RP-18 (150 ꢂ 4:6 mm). eluent A: distilled
water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA;
flow rate: 1 ml/min.
under the low-acidity TfOH conditions reported pre-
viously.7) The resulting mixture was analyzed by
reversed-phase HPLC with a C18 column. Figure 3
presents a chromatogram of the product, in which the
major peak corresponds to glycopeptide 2. The structure
was identified by MALDI TOF MS of the separated
sample (m=z 1739.73). MS analysis of the less-mobile
fraction containing minor peaks indicated the presence
of a by-product missing one galactose residue that had
probably been generated during the debenzylation
process. The overall yield of 2 was estimated to be
11% by the amino acid analysis, after an acid hydrolysis
of part of the separated sample. The N-terminal Fmoc
group, which could be removed with piperidine, was
retained in synthetic sample 2 in anticipation of ready
characterization by HPLC in further studies such as
enzymatic glycosyltransfer.
In conclusion, core 8 O-linked disaccharide building
blocks 15a and 15b were stereoselectively synthesized
via two complementary synthetic pathways. The benzyl-
protected disaccharides were successfully introduced
into the MUC5AC octapeptide, a putative scaffold of
core 8 glycans, by the Fmoc solid-phase synthesis with
an application of Sakamoto’s procedure. The benzyl-
protecting groups in the synthetic glycopeptide were
finally removed with minimum loss of glycosidic
linkages by a combination of hard TfOH and soft
nucleophiles to furnish title compound 2. Synthesis of
the glycopeptide carrying native sialoglycan 1 is
currently being investigated.
tert-Butyldiphenylsilyl
2,3,4,6-tetra-O-benzyl-ꢀ-D-
galactopyranosyl-(1!3)-2-azido-4,6-O-benzylidene-2-
deoxy-ꢀ-D-galactopyranoside (7). A mixture of 4
(505 mg, 0.95 mmol), Cp2ZrCl2 (416 mg, 1.42 mmol),
AgClO4 (295 mg, 1.42 mmol), and dried 4A molecular
sieves (3 g) in anhydrous CH2Cl2 (20 ml) was stirred at
ꢃ15 ꢀC under Ar for 1 h. A solution of 3 (759 mg,
1.43 mmol) in anhydrous CH2Cl2 (20 ml) was then
added to the stirred mixture. The mixture was stirred at
ꢃ15 ꢀC-room temperature for 14 h, before the reaction
was quenched with aq. NaHCO3. The mixture was
diluted with CHCl3 and filtered through Celite. The
filtrate was successively washed with sat. NaHCO3,
water and brine, dried over Na2SO4, and concentrated in
vacuo. The product was chromatographed on silica gel
with toluene–EtOAc (9:1) to afford 7 (730 mg, 73%) and
8 (196 mg, 20%). Compound 7: ½ꢀꢄD ¼ þ35:9ꢀ (c 1). Rf
1
0.41 (9:1 toluene–EtOAc). H-NMR ꢂ: 7.79–7.77 (m,
2H, Ar), 7.73–7.71 (m, 2H, Ar), 7.52–7.49 (m, 2H, Ar),
7.42–7.10 (m, 27H, Ar), 7.09–7.02 (m, 2H, Ar), 5.41 [s,
1H, PhCH(O–)2], 5.17 (d, 1H, J ¼ 2:2 Hz, H-1b), 4.93
(d, 1H, J ¼ 11:5 Hz, –CH2Ph), 4.85 (d, 1H, J ¼ 11:8 Hz,
–CH2Ph), 4.71 (d, 1H, J ¼ 11:7 Hz, –CH2Ph), 4.56 (d,
1H, J ¼ 11:5 Hz, –CH2Ph), 4.54 (d, 1H, J ¼ 12:0 Hz,
–CH2Ph), 4.50 (d, 1H, J ¼ 11:7 Hz, –CH2Ph), 4.43 (brs,
2H, –CH2Ph), 4.40 (d, 1H, J ¼ 7:8 Hz, H-1a), 4.12–4.06
(m, 4H, H-4a, H-2b, H-3b, H-5b), 4.02 (dd, 1H, J ¼ 7:8,
10.5 Hz, H-2a), 3.92 (brs, 1H, H-4b), 3.87 (dd, 1H,
J ¼ 1:2, 12.2 Hz, H-6a), 3.77 (dd, 1H, J ¼ 1:7, 12.2 Hz,
H-6a), 3.54 (dd, 1H, J ¼ 6:9, 10.0 Hz, H-6b), 3.50 (dd,
1H, J ¼ 3:4, 10.5 Hz, H-3a), 3.44 (dd, 1H, J ¼ 5:8,
10.0 Hz, H-6b), 2.75 (brs, 1H, H-5a), 1.13 (s, 9H, t-Bu).
Anal. Found: C, 71.74; H, 6.51; N, 3.87%. Calcd. for
C63H67N3O10Si: C, 71.77; H, 6.41; N, 3.99%.
Experimental
Optical rotation values were determined with a Jasco
DIP-370 polarimeter at 20 ꢁ 2 ꢀC for solutions in

Synthesis of Glycopeptide with Core 8 O-Glycan
1579
2,3,4,6-Tetra-O-benzyl-ꢀ-D-galactopyranosyl-(1!3)-
2-azido-4,6-O-benzylidene-2-deoxy-D-galactopyranose
(9). To a mixture of 7 (497 mg, 0.46 mmol) and AcOH
(0.28 ml, 4.60 mmol) in freshly distilled THF (20 ml)
was added 1 M n-Bu4NF/THF (2.76 ml, 2.76 mmol). The
mixture was stirred overnight at room temperature and
then concentrated in vacuo. The residue was extracted
with CHCl3, successively washed with water and brine,
dried over Na2SO4, and concentrated in vacuo. The
crude product was chromatographed on silica gel with
toluene–EtOAc (3:2) to give 9 (351 mg, 91%). Rf 0.21
(4:1 toluene–EtOAc). 1H-NMR ꢂ: 7.49–7.46 (m, 3H,
Ar), 7.48–7.20 (m, 14H, Ar), 7.19–7.05 (m, 8H, Ar),
5.49 and 5.46 [2s, 1H, PhCH(O–)2], 5.42 and 5.28 (2d,
1H, J ¼ 3:2 and 2.2 Hz, H-1b), 5.20 (brs, 0.5H, H-1aꢀ).
Anal. Found: C, 69.16; H, 6.07; N, 4.96%. Calcd. for
C47H49N3O10: C, 69.19; H, 6.05; N, 5.15%.
(brs, 1H, H-5a). Anal. Found: C, 68.42; H, 5.91; N,
.
5.00%. Calcd. for C47H48FN3O9 0.3H2O: C, 68.57; H,
5.95; N, 5.10%.
N-(9-Fluorenylmethoxycarbonyl)-O-[2,3,4,6-tetra-O-
benzyl-ꢀ-D-galactopyranosyl-(1!3)-2-azido-4,6-benzyl-
idene-2-deoxy-ꢀ-D-galactopyranosyl]-L-serine allyl es-
ter (11a).
Procedure A (reaction of 5a and 10): A mixture of 5a
(45 mg, 0.12 mmol), Cp2ZrCl2 (36 mg, 0.12 mmol),
AgClO4 (26 mg, 0.12 mmol), and dried 4A molecular
sieves (0.2 g) in anhydrous CH2Cl2 (3 ml) was stirred at
ꢃ15 ꢀC under Ar for 1 h. A solution of 10 (68 mg,
0.08 mmol) in anhydrous CH2Cl2 (1 ml) was then added
to the stirred mixture. The mixture was stirred at
ꢃ15 ꢀC-room temperature for 16 h, before the reaction
was quenched with aq. NaHCO3. The mixture was
diluted with CHCl3 and filtered through Celite. The
filtrate was successively washed with sat. NaHCO3,
water and brine, dried over Na2SO4, and concentrated in
vacuo. The product was chromatographed on Bio-beads
S ꢂ 3 with toluene and then on silica gel with toluene–
EtOAc (4:1) to afford 11a (60 mg, 62%) and 12a (15 mg,
16%). Compound 11a: ½ꢀꢄD ¼ þ101:9ꢀ (c 1). Rf 0.40
(4:1 toluene–EtOAc). 1H-NMR ꢂ: 7.76–7.72 (m, 2H,
Ar), 7.58–7.54 (m, 2H, Ar), 7.49–7.46 (m, 2H, Ar),
7.40–7.22 (m, 21H, Ar), 7.18–7.08 (m, 6H, Ar), 5.91
(d, 1H, J ¼ 7:3 Hz, –NH), 5.88 (m, 1H, –CH=CH2),
5.38 [s, 1H, PhCH(O–)2], 5.32 (brd, J ¼ 17:1 Hz,
–CH=CH2), 5.25–5.23 (m, 2H, –CH=CH2, H-1b),
4.99 (d, 1H, J ¼ 3:2 Hz, H-1a), 4.93 (d, 1H, J ¼ 11:2
Hz, –CH2Ph), 4.82 (d, 1H, J ¼ 12:0 Hz, –CH2Ph), 4.70
(d, 1H, J ¼ 11:7 Hz, –CH2Ph), 4.66 (m, 2H, –CH2CH=
CH2), 4.59 (brs, 2H, –CH2Ph), 4.55–4.45 (m, 3H,
{CH2Ph ꢂ 2, Ser-ꢀH), 4.42 (d, 1H, J ¼ 12:0 Hz,
–CH2Ph), 4.38–4.29 (m, 3H, –OCH2CHAr2), 4.22–
4.05 (m, 6H, Ser-ꢁH, H-3a, H-4a, H-6a, H-2b, H-5b),
3.98–3.94 (m, 3H, H-2a, H-4b, Ser-ꢁH), 3.86 (brd, 1H,
J ¼ 12:2 Hz, H-6a), 3.64–3.52 (m, 3H, H-5a, H-6b ꢂ 2).
Anal. Found: C, 70.17; H, 5.95; N, 4.23%. Calcd. for
C68H68N4O14: C, 70.09; H, 5.88; N, 4.81%.
Compound 12a: ½ꢀꢄD ¼ þ38:4ꢀ (c 1). Rf 0.24 (4:1
toluene–EtOAc). 1H-NMR ꢂ; 7.74 (brd, 2H, J ¼ 7:6 Hz,
Ar), 7.62–7.58 (m, 2H, Ar), 7.50–7.48 (m, 2H, Ar),
7.38–7.04 (m, 27H, Ar), 5.97 (d, 1H, J ¼ 8:3 Hz, NH),
5.90 (m, 1H, –CH=CH2), 5.49 [s, 1H, PhCH(O–)2],
5.34 (brd, 1H, J ¼ 17:3 Hz, –CH=CH2), 5.20–5.17
(m, 2H, H-1b, –CH=CH2), 4.94 (d, 1H, J ¼ 11:7 Hz,
–CH2Ph), 4.86 (d, 1H, J ¼ 11:7 Hz, –CH2Ph), 4.73
(d, 1H, J ¼ 11:7 Hz, –CH2Ph), 4.71–4.68 (m, 2H,
–CH2CH=CH2), 4.65–4.52 (m, 4H, –CH2Ph ꢂ 3,
Ser-ꢀH), 4.45–4.20 (m, 8H, Ser-ꢁH, {CH2Ph ꢂ 2,
–OCH2CHAr2, H-4a, H-6a), 4.18 (d, 1H, J ¼ 8:0 Hz,
H-1a), 4.12–4.04 (m, 3H, H-2b, H-3b, H-5b), 4.00–3.92
(m, 3H, H-2a, H-6a, H-4b), 3.87 (dd, 1H, J ¼ 3:0,
10.0 Hz, Ser-ꢁH), 3.61 (dd, 1H, J ¼ 3:2, 10.5 Hz, H-3a),
3.58 (dd, 1H, J ¼ 6:8, 9.7 Hz, H-6b), 3.39 (dd, 1H,
J ¼ 5:3, 9.7 Hz, H-6b). Anal. Found: C, 69.94; H, 5.99;
2,3,4,6-Tetra-O-benzyl-ꢀ-D-galactopyranosyl-(1!3)-
2-azido-4,6-O-benzylidene-2-deoxy-ꢀ and ꢁ-D-galacto-
pyranosyl fluoride (10). To a stirred solution of 9
(273 mg, 0.35 mmol) in freshly distilled THF (10 ml)
was added Et2NSF3 (0.19 ml, 1.41 mmol) at 0 ꢀC. The
mixture was stirred for 40 min before the reaction was
quenched with MeOH, and the mixture concentrated in
vacuo. The residue was extracted with EtOAc, succes-
sively washed with water and brine, dried over Na2SO4,
and concentrated in vacuo. The crude product was
chromatographed on silica gel with toluene–EtOAc (4:1)
to give 10ꢀ (195 mg, 66%) and 10ꢁ (65 mg 22%).
Compound 10ꢀ: ½ꢀꢄD ¼ þ68:6ꢀ (c 1). Rf 0.65 (4:1
toluene–EtOAc). 1H-NMR ꢂ: 7.49–7.46 (m, 2H, Ar),
7.36–7.25 (m, 19H, Ar), 7.19–7.07 (m, 4H, Ar), 5.77
(dd, 1H, J ¼ 2:4, 52.7 Hz, H-1a), 5.47 [s, 1H,
PhCH(O–)2], 5.28 (d, 1H, J ¼ 2:3 Hz, H-1b), 4.95 (d,
1H, J ¼ 11:4 Hz, –CH2Ph), 4.82 (d, 1H, J ¼ 11:3 Hz,
–CH2Ph), 4.69 (d, 1H, J ¼ 11:3 Hz, –CH2Ph), 4.59 (brs,
2H, –CH2Ph), 4.57 (d, 1H, J ¼ 11:5 Hz, –CH2Ph), 4.50–
4.43 (m, 3H, –CH2Ph, H-4a), 4.26 (dd, 1H, J ¼ 1:4,
12.7 Hz, H-6a), 4.19 (dd, 1H, J ¼ 2:7, 9.5 Hz, H-3a),
4.17–4.06 (m, 4H, H-2a, H-2b, H-3b, H-5b), 4.00–3.97
(m, 2H, H-4b, H-6a), 3.75 (brs, 1H, H-5a), 3.60 (dd, 1H,
J ¼ 6:4, 9.5 Hz, H-6b), 3.52 (dd, 1H, J ¼ 6:3, 9.5 Hz,
H-6b). Anal. Found: C, 69.02; H, 6.01; N, 4.60%. Calcd.
for C47H48FN3O9: C, 69.02; H, 5.92; N, 5.14%.
Compound 10ꢁ: ½ꢀꢄD ¼ þ58:9ꢀ (c 1). Rf 0.45 (4:1
toluene–EtOAc). 1H-NMR ꢂ: 7.52–7.49 (m, 2H, Ar),
7.37–7.23 (m, 19H, Ar), 7.18–7.05 (m, 4H, Ar), 5.50 [s,
1H, PhCH(O–)2], 5.21 (d, 1H, J ¼ 3:4 Hz, H-1b), 4.95
(d, 1H, J ¼ 11:4 Hz, –CH2Ph), 4.91 (dd, 1H, J ¼ 7:6,
52.5 Hz, H-1a), 4.84 (d, 1H, J ¼ 11:5 Hz, –CH2Ph), 4.71
(d, 1H, J ¼ 11:7 Hz, –CH2Ph), 4.58 (d, 1H, J ¼ 10:5
Hz, –CH2Ph), 4.57 (brs, 2H, –CH2Ph), 4.46 (brs, 2H,
–CH2Ph), 4.32 (dd, 1H, J ¼ 1:4, 12.6 Hz, H-6a), 4.26
(br, 1H, H-4a), 4.11–3.99 (m, 5H, H-2a, H-6a, H-2b,
H-3b, H-5b), 3.94 (brd, 1H, J ¼ 1:5 Hz, H-4b), 3.67 (dd,
1H, J ¼ 2:9, 10.5 Hz, H-3a), 3.60 (dd, 1H, J ¼ 7:1,
9.7 Hz, H-6b), 3.44 (dd, 1H, J ¼ 5:4, 9.7 Hz, H-6b), 3.33

1580
M. MAEMURA et al.
N, 4.53%. Calcd. for C68H68N4O14: C, 70.09; H, 5.88;
N, 4.81%.
–CH=CH2), 5.19 (d, 1H, J ¼ 2:7 Hz, H-1b), 5.11 (brd,
1H, J ¼ 10:5 Hz, –CH=CH2), 4.95 (d, 1H, J ¼ 11:5 Hz,
–CH2Ph), 4.86 (d, 1H, J ¼ 11:7 Hz, –CH2Ph), 4.73
(d, 1H, J ¼ 11:5 Hz, –CH2Ph), 4.65–4.63 (m, 2H,
–CH2CH=CH2), 4.60–4.55 (m, 4H, {CH2Ph ꢂ 3, Thr-
ꢁH), 4.50–4.43 (m, 3H, {CH2Ph ꢂ 2, Thr-ꢀH), 4.41–
4.22 (m, 6H, –OCH2CHAr2, –OCH2CHAr2, H-1a, H-4a,
H-6a), 4.15 (brt, 1H, J ¼ 6:4 Hz, H-5b), 4.12–4.05 (m,
2H, H-2b, H-3b), 3.99–3.96 (m, 2H, H-6a, H-4b), 3.62–
3.56 (m, 2H, H-3a, H-6b), 3.46 (dd, 1H, J ¼ 5:6, 9.8 Hz,
H-6b), 3.16 (brs, 1H, H-5a), 1.34 (d, 1H, J ¼ 6:4 Hz,
Thr-ꢃH). Anal. Found: C, 69.90; H, 6.03; N, 4.52%.
Procedure B (reaction of 3 and 6a): A mixture of
Cp2ZrCl2 (67 mg, 0.23 mmol), AgClO4 (94 mg, 0.45
mmol), and dried 4A molecular sieves (0.5 g) in
anhydrous CH2Cl2 (2 ml) was stirred at ꢃ15 ꢀC under
Ar for 1 h. A mixture of 3 (82 mg, 0.15 mmol) and 6a
(152 mg, 0.24 mmol) in anhydrous CH2Cl2 (1 ml) was
then added to this mixture. The mixture was stirred at
ꢃ15 ꢀC-room temperature for 16 h, before the reaction
was quenched with aq. NaHCO3. The mixture was
diluted with CHCl3 and filtered through Celite. The
filtrate was successively washed with sat. NaHCO3,
water and brine, dried over Na2SO4, and concentrated in
vacuo. The product was chromatographed on Bio-beads
S ꢂ 3 with toluene and then on silica gel with toluene–
EtOAc (4:1) to afford 11a (108 mg, 62%) and 13a
(17 mg, 10%). The latter was identical in all respects
with the sample previously prepared in this laboratory.
.
Calcd. for C69H70N4O14 0.5H2O: C, 69.74; H, 6.02; N,
4.71%.
Procedure B (reaction of 3 and 6b): As described for
11a, disaccharyl threonine 11b was prepared by glyco-
sidation of 3 (60 mg, 0.11 mmol) and 6b (90 mg,
0.14 mmol) with Cp2ZrCl2 (48 mg, 0.16 mmol), AgClO4
(69 mg, 0.33 mmol), and dried 4A molecular sieves
(0.5 g) in CH2Cl2 (5 ml). The product was chromato-
graphed on Bio-beads S ꢂ 3 with toluene and then on
silica gel with toluene–EtOAc (4:1) to afford 11b
(82 mg, 63%) and the 13b (10 mg, 8%). The latter was
identical in all respects with the sample previously
prepared in this laboratory.
N-(9-Fluorenylmethoxycarbonyl)-O-[2,3,4,6-tetra-O-
benzyl-ꢀ-D-galactopyranosyl-(1!3)-2-azido-4,6-benzyl-
idene-2-deoxy-ꢀ-D-galactopyranosyl]-L-threonine allyl
ester (11b).
Procedure A (reaction of 5b and 10): Glycosylation
of 5b (116 mg, 0.36 mmol) with 10 (200 mg, 0.24 mmol)
was conducted with Cp2ZrCl2 (105 mg, 0.36 mmol),
AgClO4 (74 mg, 0.36 mmol), and dried 4A molecular
sieves (0.4 g) in CH2Cl2 (12 ml) as described for 11a.
The product was chromatographed on Bio-beads S ꢂ 3
with toluene and then on silica gel with toluene–EtOAc
(4:1) to afford 11b (149 mg, 52%) and 12b (50 mg,
17%). Compound 11b: ½ꢀꢄD ¼ þ91:4ꢀ (c 1). Rf 0.47
(4:1 toluene–EtOAc). 1H-NMR ꢂ: 7.76–7.74 (m, 2H,
Ar), 7.60–7.59 (m, 2H, Ar), 7.49–7.47 (m, 2H, Ar),
7.38–7.20 (m, 21H, Ar), 7.19–6.99 (m, 6H, Ar), 5.91 (m,
1H, –CH=CH2), 5.82 (d, 1H, J ¼ 7:3 Hz, –NH), 5.43
[s, 1H, PhCH(O–)2], 5.33 (dd, 1H, J ¼ 1:2, 17.1 Hz,
–CH=CH2), 5.28 (d, 1H, J ¼ 2:4 Hz, H-1b), 5.24 (brd,
1H, J ¼ 10:2 Hz, –CH=CH2), 5.05 (d, 1H, J ¼ 3:4 Hz,
H-1a), 4.92 (d, 1H, J ¼ 11:5 Hz, –CH2Ph), 4.82 (d, 1H,
J ¼ 11:7 Hz, –CH2Ph), 4.74–4.62 (m, 3H, –CH2Ph,
–CH2CH=CH2), 4.59 (brs, 2H, –CH2Ph), 4.54 (d, 1H,
J ¼ 11:4 Hz, –CH2Ph), 4.47–4.36 (m, 7H, H-4a,
–CH2Ph, –OCH2CHAr2, Thr-ꢀH, Thr-ꢁH), 4.28 (dd,
1H, J ¼ 7:4, 10.3 Hz, –OCH2CHAr2), 4.23–4.06 (m,
5H, H-3a, H-6a, H-2b, H-3b, H-5b), 4.02–3.96 (m, 3H,
H-2a, H-6a, H-4b), 3.63 (brs, 1H, H-5a), 3.59 (dd, 1H,
J ¼ 6:6, 9.6 Hz, H-6b), 3.50 (dd, 1H, J ¼ 6:6, 9.3 Hz,
H-6b), 1.25 (d, 3H, J ¼ 6:6 Hz, Thr-ꢃH). Anal. Found:
C, 70.08; H, 6.05; N, 4.47%. Calcd. for C69H70N4O14:
C, 70.27; H, 5.98; N, 4.75%.
N-(9-Fluorenylmethoxycarbonyl)-O-[2,3,4,6-tetra-O-
benzyl-ꢀ-D-galactopyranosyl-(1!3)-2-acetamido-4,6-
benzylidene-2-deoxy-ꢀ-D-galactopyranosyl]-L-serine allyl
ester (14a). A mixture of 11a (56 mg, 48 mmol), AcOH
(39 ml, 0.68 mmol) and powdered Zn (195 mg, 2.98
mmol) in CH2Cl2 (1 ml) was stirred for 3 h. The mixture
was filtered through Celite, and the filtrate was con-
centrated with toluene in vacuo. The residue dissolved in
a mixture of MeOH (3 ml) and CH2Cl2 (2 ml) was
stirred with Ac2O (69 ml, 0.68 mmol) for 1 h. The
mixture was concentrated in vacuo. The product was
extracted with EtOAc, successively washed with sat.
NaHCO3, water and brine, dried over Na2SO4, and
concentrated in vacuo. Chromatography of the crude
product on silica gel with toluene–EtOAc (1:1) afforded
14a (50 mg, 88%). ½ꢀꢄD ¼ þ92:3ꢀ (c 1). Rf 0.43 (1:1
toluene–EtOAc). 1H-NMR ꢂ: 7.76 (brd, 2H, J ¼ 7:3 Hz,
Ar), 7.57 (m, 2H, Ar), 7.47 (m, 2H, Ar), 7.39 (brt, 2H,
J ¼ 7:3 Hz, Ar), 7.31–7.11 (m, 25H, Ar), 5.89–5.85
(m, 2H, –CH=CH2, –NH), 5.33 (brd, 1H, J ¼ 17:6 Hz,
–CH=CH2), 5.28 (brd, 1H, J ¼ 11:2 Hz, –CH=CH2),
5.24 [s, 1H, PhCH(O–)2], 5.04 (d, 1H, J ¼ 2:9 Hz, H-
1b), 5.02 (d, 1H, J ¼ 3:0 Hz, H-1a), 3.59 (brs, 1H, H-
5a), 3.52 (brt, J ¼ 7:5 Hz, H-6b), 3.30 (dd, 1H, J ¼ 4:1,
9.8 Hz, H-6b), 1.85 (s, 3H, Ac). Anal. Found: C, 70.53;
.
H, 6.09; N, 2.31%. Calcd. for C70H72N2O15 0.5H2O: C,
70.63; H, 6.18; N, 2.35%.
Compound 12b: ½ꢀꢄD ¼ þ26:1ꢀ (c 1). Rf 0.29 (4:1
toluene–EtOAc). 1H-NMR ꢂ: 7.74 (brd, 2H, J ¼ 7:6 Hz,
Ar), 7.62 (m, 2H, Ar), 7.49 (brd, 2H, J ¼ 6:6 Hz, Ar),
7.47–7.23 (m, 21H, Ar), 7.18–7.04 (m, 6H, Ar), 5.87 (m,
1H, –CH=CH2), 5.79 (d, 1H, J ¼ 9:6 Hz, NH), 5.49
[s, 1H, PhCH(O–)2], 5.26 (dd, 1H, J ¼ 1:5, 17.1 Hz,
N-(9-Fluorenylmethoxycarbonyl)-O-[2,3,4,6-tetra-O-
benzyl-ꢀ-D-galactopyranosyl-(1!3)-2-acetamido-4,6-
benzylidene-2-deoxy-ꢀ-D-galactopyranosyl]-L-threonine
allyl ester (14b). Azide 11b (137 mg, 0.12 mmol) was

Synthesis of Glycopeptide with Core 8 O-Glycan
1581
reduced with Zn and AcOH, and then treated with Ac2O
as described for 14a. The crude product was purified by
chromatography on silica gel with toluene–EtOAc (1:1)
to give 14b (128 mg, 92%). ½ꢀꢄD ¼ þ87:9ꢀ (c 1.2). Rf
(m, 2H, Ar), 7.68–7.67 (m, 1H, Ar), 7.54–7.16 (m,
28H, Ar), 5.20 (d, 1H, J ¼ 2:0 Hz, H-1b), 5.15 [s, 1H,
PhCH(O–)2], 4.78 (d, 1H, J ¼ 3:7 Hz, H-1a), 1.77
(s, 3H, Ac), 1.14 (d, 3H, J ¼ 6:3 Hz, Thr-ꢃH). Anal.
Found: C, 70.13; H, 6.07; N, 2.32%. Calcd. for
1
0.47 (1:1 toluene–EtOAc). H-NMR ꢂ: 7.75–7.73 (m,
.
C68H70N2O15 0.5H2O: C, 70.15; H, 6.15; N, 2.41%.
2H, Ar), 7.59–7.54 (m, 2H, Ar), 7.47–7.45 (m, 3H, Ar),
7.38–7.23 (m, 24H, Ar), 7.20–7.11 (m, 2H, Ar), 5.94 (d,
1H, J ¼ 8:8 Hz, –NH), 5.87 (m, 1H, –CH=CH2), 5.59
(d, 1H, J ¼ 9:8 Hz, –NH), 5.33 (brd, 1H, J ¼ 17:3 Hz,
–CH=CH2), 5.28 (brd, 1H, J ¼ 10:5 Hz, –CH=CH2),
5.22 [s, 1H, PhCH(O–)2], 5.06 (d, 1H, J ¼ 3:2 Hz, H-
1b), 5.01 (d, 1H, J ¼ 3:2 Hz, H-1a), 3.80 (brs, 1H, H-
4b), 3.59 (brs, 1H, H-5a), 3.50 (brt, 1H, J ¼ 7:6 Hz, H-
6b), 3.30 (dd, 1H, J ¼ 4:1, 9.6 Hz, H-6b), 1.99 (s, 3H,
Ac), 1.25 (d, 3H, J ¼ 4:8 Hz, Thr-ꢃH). Anal. Found: C,
N-(9-Fluorenylmethoxycarbonyl)-O-[2,3,4,6-tetra-O-
benzyl-ꢀ-D-galactopyranosyl-(1!3)-2-acetamido-4,6-
benzylidene-2-deoxy-ꢀ-D-galactopyranosyl]-L-seryl fluo-
ride (16). A mixture of compound 15a (23 mg, 20 mmol),
TFFH (5 mg, 19 mmol), and DIEA (4.7 ml, 36 mmol) in
anhydrous CH2Cl2 (2.5 ml) was stirred at room temper-
ature for 2 h. This mixture was used for the coupling
reaction without isolating 16.
.
70.67; H, 6.29; N, 2.18%. Calcd. for C71H74N2O15
0.5H2O: C, 70.81; H, 6.28; N, 2.33%.
L-Threonyl-O-[ꢀ-D-galactopyranosyl-(1!3)-2-acet-
amido-2-deoxy-ꢀ-D-galactopyranosyl]-L-threonyl-L-seryl-
L-threonyl-L-threonyl-O-[ꢀ-D-galactopyranosyl-(1!3)-
2-acetamido-2-deoxy-ꢀ-D-galactopyranosyl]-L-seryl-L-
alanyl-L-proline (2). Commercial Fmoc-Pro-CLEAR-
acid resin (31 mg, 9 mmol) was stirred with 20%
piperidine/NMP (2 ml) by a vortex mixer for 2 min,
and then filtered. The resin was again stirred with 20%
piperidine/NMP (2.5 ml) for 3 min to complete N-
deprotection. After filtration, the resin was washed with
NMP (2.5 ml). This washing was repeated five more
times with the same amount of NMP. The resin was then
stirred with Tsoc-Ala-OPfp (21 mg, 45 mmol) in freshly
distilled THF (2.5 ml) for 15 min and filtered. Tsoc-Ala-
OPfp (21 mg, 45 mmol) in THF (2.5 ml) was again added
to the resin, and the mixture was stirred for a further
15 min. The resin was washed with NMP (2.5 ml) and
treated with 10% Ac2O–5% DIEA/NMP (2.5 ml) for
5 min to acetylate the unreacted amino moiety. The resin
was successively washed with NMP (2.5 ml) and
CH2Cl2 (2.5 ml). To the resin were added a mixture of
16 prepared as already described, 1 M TBAF/THF (5 ml,
0.1 equiv.) and DIEA (5 ml). The mixture was stirred for
12 h, before successively washing with CH2Cl2 and
NMP. The unreacted amino terminal was masked by
acetylation, and the resin was washed with NMP. The
resulting glycopeptide-loaded resin was N-deprotected
with 20% piperidine/NMP (2.5 ml) for 5 min and again
with the same amount of piperidine for 15 min. After
washing six times with NMP (2.5 ml each) for 1 min, a
mixture of Fmoc-Thr(But)-OH (40 mg, 0.1 mmol),
0.45 M TBTU/NMP (200 ml, 0.09 mmol), and DIEA
(24 ml, 0.18 mmol) was added to the resin, which was
then stirred at 50 ꢀC for 1 h. The resin was washed and
N-deprotected before being subjected to further con-
densation. After two amino-acid-elongation with Fmoc-
Thr(But)-OH and Fmoc-Ser(But)-OH, glycothreonine
15b (23 mg, 0.02 mmol) was analogously condensed in
the presence of 0.45 M TBTU/NMP (40 ml, 18 mmol) and
DIEA (5 ml, 36 mmol) in NMP (2.5 ml). The latter
condensation procedure was repeated with the same
amount of 15b. Finally, Fmoc-Thr(But)-OH was con-
N-(9-Fluorenylmethoxycarbonyl)-O-[2,3,4,6-tetra-O-
benzyl-ꢀ-D-galactopyranosyl-(1!3)-2-acetamido-4,6-
benzylidene-2-deoxy-ꢀ-D-galactopyranosyl]-L-serine
(15a). A mixture of 14a (62 mg, 52 mmol), Pd(Ph3P)4
(6 mg, 5 mmol), and 5,5-dimethyl-1,3-cyclohexanedione
(146 mg, 1.04 mmol) in freshly distilled THF (1 ml) was
stirred at room temperature for 1.5 h under Ar. The
mixture was concentrated in vacuo, and the resulting
residue was chromatographed on silica gel with CHCl3–
MeOH–AcOH (38:2:1). The product was further puri-
fied in a column of Biobeads S ꢂ 3 with toluene–EtOAc
(1:1) to afford 15a (60 mg, quant.). ½ꢀꢄD ¼ þ127:7ꢀ (c
1.1). Rf 0.29 (9:1 CHCl3–MeOH). 1H-NMR (DMSO-d6)
ꢂ: 7.99 (brd, 2H, J ¼ 7:6 Hz, Ar), 7.72–7.69 (m, 3H, Ar,
AcNH), 7.43–7.20 (m, 29H, Ar), 7.12 (d, 1H, J ¼
9:5 Hz, Ser-NH), 5.36 [s, 1H, PhCH(O–)2], 5.22 (d, 1H,
J ¼ 2:9 Hz, H-1b), 4.81 (d, 1H, J ¼ 3:5 Hz, H-1a), 4.78
(d, 1H, J ¼ 11:2 Hz, –CH2Ph), 4.73 (d, 1H, J ¼ 11:9
Hz, –CH2Ph), 4.69 (d, 1H, J ¼ 11:9 Hz, –CH2Ph), 4.61
(d, 1H, J ¼ 11:7 Hz, –CH2Ph), 4.57 (d, 1H, J ¼ 11:7
Hz, –CH2Ph), 4.51 (d, 1H, J ¼ 12:2 Hz, –CH2Ph), 4.45
(d, 1H, J ¼ 11:2 Hz, –CH2Ph), 4.43 (d, 1H, J ¼ 12:0 Hz,
–CH2Ph), 4.39–4.34 (m, 2H, Ser-ꢁH, –OCH2CHAr2),
4.30–4.22 (m, 4H, –OCH2CHAr2, –OCH2CHAr2, Ser-
ꢀH, H-2a), 4.05–3.78 (m, 9H, Ser-ꢁH, H-3a, H-4a,
H-6a ꢂ 2, H-2b, H-3b, H-4b, H-5b), 3.66 (brs, 1H, H-
5a), 3.51 (m, 2H, H-6b ꢂ 2), 1.80 (s, 3H, Ac). Anal.
Found: C, 69.76; H, 5.98; N, 2.31%. Calcd. for
.
C67H68N2O15 0.5H2O: C, 69.96; H, 6.04; N, 2.44%.
N-(9-Fluorenylmethoxycarbonyl)-O-[2,3,4,6-tetra-O-
benzyl-ꢀ-D-galactopyranosyl-(1!3)-2-acetamido-4,6-
benzylidene-2-deoxy-ꢀ-D-galactopyranosyl]-L-threonine
(15b). Compound 14b (262 mg, 0.22 mmol) was de-
allylated with Pd(Ph3P)4 (26 mg, 22 mmol) and 5,5-
dimethyl-1,3-cyclohexanedione (617 mg, 4.40 mmol) in
THF (10 ml) as described for 15a. Chromatography on
silica gel and then on Biobeads afforded 15b (252 mg,
quant.). ½ꢀꢄD ¼ þ106:5ꢀ (c 1.1). Rf 0.36 (9:1 CHCl3–
1
MeOH). H-NMR ꢂ: 7.90–7.88 (m, 2H, Ar), 7.75–7.73

1582
M. MAEMURA et al.
sialyl-T antigen. Carbohydr. Res., 329, 773–780 (2000).
7) Takano, Y., Habiro, M., Someya, M., Hojo, H., and
Nakahara, Y., Preparation of core 2 type tetrasaccharide
carrying decapeptide by benzyl protection-based solid-
phase synthesis strategy. Tetrahedron Lett., 43, 8395–
8399 (2002).
8) Takano, Y., Kojima, N., Nakahara, Y., Hojo, H., and
Nakahara, Y., Solid-phase synthesis of core 2 O-linked
glycopeptide and its enzymatic sialylation. Tetrahedron,
59, 8415–8427 (2003).
9) Takano, Y., Hojo, H., Kojima, N., and Nakahara, Y.,
Synthesis of a mimic for the heterogeneous surface of
core 2 sialoglycan-linked glycoprotein. Org. Lett., 6,
3135–3138 (2004).
10) Van Halbeek, H., Stang, A.-M., Lhermitte, M.,
Rahmoune, H., Lamblin, G., and Roussel, P., Structure
of monosialyl oligosaccharides isolated from the respi-
ratory mucins of a non-secretor (O, Leaþbꢃ) patient
suffering from chronic bronchitis. Characterization of a
novel type of mucin carbohydrate core structure.
Glycobiology, 4, 203–219 (1994).
11) Paulsen, H., and Adermann, K., Synthese von 8-me-
thoxycarbonyloctyl-2-acetamido-2-deoxy-3-O-ꢀ-D-gal-
actopyranosyl-ꢀ-D-galactopyranosid. Carbohydr. Res.,
175, 163–172 (1988).
12) Guyonnet Duperat, V., Audie, J.-P., Debailleul, V.,
Laine, A., Buisine, M.-P., Galiegue-Aouitina, S., Pigny,
P., Aubert, J.-P., and Porchet, N., Characterization of the
human mucin gene MUC5AC: a consensus cysytein-rich
domain for 11p15 mucin genes? Biochem. J., 305, 211–
219 (1995).
densed to complete the desired sequence. The resin
(44 mg) was successively washed with NMP and
CH2Cl2, before being stirred with reagent K [TFA-
phenol-deionized
water-thioanisole-1,2-ethanedithiol
(82.5:5:5:5:2.5), 440 ml] at room temperature for 1 h to
cleave the synthetic glycopeptide from the resin. Blow-
ing in a nitrogen stream evaporated the volatile materials
in the mixture. Ether was added to the resulting mixture
to precipitate the crude product, which was separated by
centrifugation. The precipitate was further washed with
ether before being dissolved in a mixture of dimethyl
sulfide (132 ml), m-cresole (44 ml), and TFA (220 ml),
and the magnetically stirred mixture was cooled at
ꢃ15 ꢀC. TfOH (44 ml) was added to the mixture, which
was stirred for 2 h at ꢃ15 ꢀC. The reaction was
terminated by precipitating with cold (ꢃ80 ꢀC) ether.
The ethereal layer was removed by decantation after
centrifuging the mixture. The resulting precipitate was
again washed with cold ether before being purified by
reversed-phase HPLC on C-18 silica gel with gradient
elution by CH3CN/H2O containing 0.1% TFA as
depicted in Fig. 3. Glycopeptide 2 was collected, and
part of the sample was subjected to an amino acid
analysis in order to estimate the total efficiency (11%).
MALDI TOF MS m=z (Mþ þ Na): calcd. for
.
C73H108N10O37 Na, 1739.68; found, 1739.73.
Acknowledgments
13) Sakamoto, K., Nakahara, Y., and Ito, Y., Combination of
silyl carbamate and amino acid fluoride for solid phase
peptide synthesis. Tetrahedron Lett., 43, 1515–1518
(2002).
14) Nicolaou, K. C., Caulfield, T. J., and Kataoka, H., Total
synthesis of globotriaosylceramide (Gb3) and lysoglobo-
triaosylceramide (lysoGb3). Carbohydr. Res., 202, 177–
191 (1990).
15) Nakahara, Y., Iijima, H., Shibayama, S., and Ogawa, T.,
A highly stereoselective synthesis of di- and trimeric
sialosyl Tn epitope: a partial structure of glycophorin A.
Tetrahedron Lett., 31, 6897–6900 (1990).
We thank Dr. T. Chihara and his staff at Riken for the
combustion analyses. This work was supported by
NEDO (New Energy and Industrial Technology Devel-
opment Organization, Japan) and in part by grant-in-aid
for scientific research from the Ministry of Education,
Culture, Sports, Science and Technology of Japan. We
also thank Tokai University for support by grant-aid for
high-technology research.
References
16) Nakahara, Y., Iijima, H., Shibayama, S., and Ogawa, T.,
Stereoselective total synthesis of glycopeptides bearing
the dimeric and trimeric sialosyl-Tn epitope. Carbohydr.
Res., 216, 211–225 (1991).
1) Kim, Y. S., Gum Jr., J., and Brockhausen, I., Mucin
glycoproteins in neoplasia. Glycoconj. J., 13, 693–707
(1996).
´
2) Hanisch, F.-G., and Muller, S., MUC1: the polymorphic
¨
17) De la Torre, B. G., Torrea, J. L., Bardaji, E., Clapes, P.,
appearance of a human mucin. Glycobiology, 10, 439–
449 (2000).
Xaus, N., Jorba, X., Calvet, S., Albericio, F., and
Valentia, G., Improved method for the synthesis of O-
glycosylated Fmoc amino acids to be used in solid-phase
glycopeptide synthesis (Fmoc = fluoren-9-ylmethoxy-
carbonyl). J. Chem. Soc., Chem. Commun., 965–967
(1990).
3) Brockhausen, I., Yang, J. M., Burcell, J., Whitehouse,
C., and Taylor-Papadimitriou, J., Mechanisms under-
lying aberrant glycosylation of MUC1 mucin in breast
cancer cells. Eur. J. Biochem., 233, 607–617 (1995).
4) Nakahara, Y., Nakahara, Y., Ito, Y., and Ogawa, T.,
Total synthesis of B-chain of human ꢀ2HS glycoprotein.
Tetrahedron Lett., 38, 7211–7214 (1997).
5) Nakahara, Y., Nakahara, Y., Ito, Y., and Ogawa, T.,
Solid-phase synthesis of B-chain of human ꢀ2HS
glycoprotein. Carbohydr. Res., 309, 287–296 (1998).
6) Ando, S., Nakahara, Y., Ito, Y., Ogawa, T., and
Nakahara, Y., Solid-phase synthesis of the glycopeptide
of human glycophorin AM, bearing the consecutive
18) Ciommer, M., and Kunz, H., Synthesis of glycopeptides
with partial structure of human glycophorin using the
fluorenylmethoxycarbonyl/allyl ester protecting group
combination. Synlett, 593–595 (1991).
19) Macindoe, W. M., Iijima, H., Nakahara, Y., and Ogawa,
T., Synthesis of a glycotetraosyl serine, a partial
structure of an ovarian cyst mucin glycoprotein of blood
group A activity. Tetrahedron Lett., 35, 1735–1738
(1994).

Synthesis of Glycopeptide with Core 8 O-Glycan
1583
20) Macindoe, W. M., Iijima, H., Nakahara, Y., and Ogawa,
T., Stereoselective synthesis of a blood group A type
glycopeptide present in human blood mucin. Carbohydr.
Res., 269, 227–257 (1995).
and application to highly ꢁ-selective glycosidation of D-
micinose. Tetrahedron Lett., 29, 3567–3570 (1988).
24) Nakahara, Y., Nakahara, Y., and Ogawa, T., Solid-phase
synthesis of an O-linked glycopeptide based on a benzyl-
protected glycan approach. Carbohydr. Res., 292, 71–81
(1996).
25) Nakamura, K., Ishii, A., Ito, Y., and Nakahara, Y., A
novel silyl linker: Motif for side chain tethered approach
to solid-phase glycopeptide synthesis. Tetrahedron, 55,
11253–11266 (1999).
21) Singh, L., Nakahara, Y., Ito, Y., and Nakahara, Y., The
first total synthesis of the core class II disialylated
hexasaccharide as a building block for glycopeptide
synthesis. Tetrahedron Lett., 40, 3769–3772 (1999).
22) Singh, L., Nakahara, Y., Ito, Y., and Nakahara, Y., An
efficient access to protected disialylated glycohexaosyl
threonine present on the leukosialin of activated T-
lymphocytes. Carbohydr. Res., 325, 132–142 (2000).
23) Suzuki, K., Maeta, T., Matsumoto, T., and Tsuchihashi,
G., New glycosidation reaction 1. Combinational use of
Cp2ZrCl2–AgClO4 for activation of glycosyl fluorides
26) King, D. S., Fields, C. G., and Fields, G. B., A cleavage
method which minimizes side reactions following Fmoc
solid phase peptide synthesis. Int. J. Peptide Protein
Res., 36, 255–266 (1990).