Synthesis of biantennary LacNAc-linked O-glycan (core 4) and glycopeptide thioester by benzyl protection strategy: rapid zinc reduction of GlcNTCA to GlcNAc by microwave irradiation

Article abstract of DOI:10.1016/j.tet.2006.12.088

A synthetic method for the core 4 O-glycan-linked Ser and Thr was developed. Highly stereoselective 3-O- and 6-O-glycosylation was achieved by using two distinctively protected N-trichloroacetyllactosaminyl fluorides (3 and 12). Microwave-assisted Zn redu

Full text of DOI:10.1016/j.tet.2006.12.088

Tetrahedron 63 (2007) 2170–2181
Synthesis of biantennary LacNAc-linked O-glycan (core 4) and
glycopeptide thioester by benzyl protection strategy: rapid zinc
reduction of GlcNTCA to GlcNAc by microwave irradiation
*
Akiharu Ueki, Yuko Nakahara, Hironobu Hojo and Yoshiaki Nakahara
*
Department of Applied Biochemistry, Institute of Glycotechnology, Tokai University, 1117 Kitakaname,
Hiratsuka-shi, Kanagawa 259-1292, Japan
Received 1 December 2006; revised 22 December 2006; accepted 25 December 2006
Available online 22 January 2007
Abstract—A synthetic method for the core 4 O-glycan-linked Ser and Thr was developed. Highly stereoselective 3-O- and 6-O-glycosylation
was achieved by using two distinctively protected N-trichloroacetyllactosaminyl fluorides (3 and 12). Microwave-assisted Zn reduction
rapidly and efficiently converted N-trichloroacetylglucosamine (GlcNTCA) to N-acetylglucosamine (GlcNAc). In order to demonstrate
the usefulness of the protected core 4 O-glycan a segment (Gly34-Gly58) of emmprin (extracellular matrix metalloproteinase inducer), a cancer
metastasis-related glycoprotein, was synthesized by the solid-phase method, utilizing the pentasaccharyl Thr (2) to introduce an O-glycan in
place of the native N-glycan at Asn44.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
4, which has been identified in the oligosaccharides from
human bronchial mucins of cystic fibrosis patients,4 secreted
mucins of a human colonic cancer cell line,5 human meco-
nium mucins,6 and sheep gastric mucins.7 The core 4 oligo-
saccharides bearing the N-acetyllactosamine branches are of
particular interest regarding an unanswered question,
whether their physical, structural, and biological properties
are different from those of the complex-type N-glycan as
well as those of the core 2 O-glycan having an extension
of N-acetyllactosamine to the core galactose residue.6,8 To
this end our investigations were directed to the synthesis
of a glycopeptide with core 4 O-glycan. In this paper, we
describe preparation of the core 4 glycoserine and glyco-
threonine building blocks, 1 and 2, and performance of the
solid-phase glycopeptide synthesis with 2 according to the
established protocol.9
Mucins and their O-glycans are of great importance and in-
terest in a number of biological processes. Aberrant features
of neoplastic mucins, such as overexpression and altered
glycosylation, have attracted particular attention in connec-
tion with metastasis.1 However, only limited knowledge of
the biological roles of the alteration in mucins has been ob-
tained so far. By considering the inaccessibility of a homo-
geneous mucin sample from natural sources, we have studied
a synthetic approach to the glycoproteins with O-glycan, and
recently established an original protocol using the benzyl-
protected glycoamino acid building blocks in solid-phase
glycopeptide synthesis.2 In a previous study, we have syn-
thesized the core 3 and core 6 oligosaccharides by glyco-
sylating either the 3- or 6-hydroxyl group of the core
N-acetylgalactosamine precursor with an N-trichloroacetyl-
lactosaminyl glycosyl donor of high reactivity and b-selec-
tivity. Usefulness of the synthetic O-glycan building
blocks was demonstrated by the synthesis of MUC2 and
MUC6 related glycopeptides.3
2. Synthesis of the building blocks 1 and 2
We first attempted selective di-O-glycosylation of 3,4,6-
O-unmasked GalN3–Thr derivative 410 with known N-tri-
chloroacetyllactosaminyl fluoride 311 (2.2 equiv) by using
Cp2ZrCl2/AgClO4 as the promoter12 in CH2Cl2 at ꢀ15 ꢁC,
since the 4-hydroxyl group of the GalN3 residue was hardly
glycosylated in many cases (Fig. 1).3,10,11 This simple strat-
egy, however, was unsuccessful and gave a complex mixture
of a heptasaccharide and three pentasaccharides each in
5–14% yield after consuming the glycosyl fluoride for 3 h.
As the second attempt, we reacted 3 and 6-O-silylated
The N-acetylglucosaminyl substitution at both 3- and 6-
position gives another core class O-glycan, known as core
Keywords: Glycopeptide; Core 4 O-glycan; Solid-phase synthesis; Micro-
wave reaction.
* Corresponding authors. Tel./fax: +81 463 50 2075; e-mail addresses:
hojo@keyaki.cc.u-tokai.ac.jp; yonak@keyaki.cc.u-tokai.ac.jp
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.12.088

A. Ueki et al. / Tetrahedron 63 (2007) 2170–2181
2171
OBn
O
Ph
BnO
BnO
O
O
OBn
O
O
BnO
O
O
TCAHN
O
F
O
O
O
BnO
3
BnO
BnO
AcNH
HO
Ph
OR3
OBn
R2O
HO
BnO
O
BnO
BnO
O
O
O
O
BnO
O
OBn
AcNH
R
AcNH
O
N3
O
R1
FmocNH
CO2H
FmocNH
CO2All
1: R = H
2: R = CH3
4: R1 = CH3, R2, R3 = H
5: R1 = CH3, R2 = H, R3 = SiMe2But
6: R1 = H, R2,R3 = -CH(Ph)-
7: R1 = CH3, R2, R3 = -CH(Ph)-
Figure 1. Structures of the protected core 4 pentasaccharyl Ser/Thr (1 and 2) and the known intermediates (3–7).
acceptor 510 in expectation of attaining selective 3-O-glyco-
sylation. But a pentasaccharide (12%) derived by 3,4-di-
O-glycosylation was produced along with the desired
trisaccharide (13%). In this reaction, an additional complica-
tion arose from the departure of the acid-labile silyl group of
5 under the reaction conditions. Thus, we were convinced
that the side reaction on the 4-hydroxyl group was unavoid-
able, when this reactive glycosyl donor was used with the
3,4-unprotected glycosyl acceptors. In order to secure
mono 3-O-glycosylation, we decided to use 4,6-O-benzyl-
idene GalN3–Ser/Thr derivatives, 613 and 7,10 as the glycosyl
acceptors, and instead needed a benzylidene group-free gly-
cosyl donor that allowed selective deprotection of the 6-O
position of the GalN3 residue at a later stage. Thus,
a perbenzylated N-trichloroacetyllactosaminyl fluoride was
synthesized as an alternative to glycosyl donor 3. Known
lactosamine derivative 814 was heated with ethylenediamine
in n-BuOH to remove the N-phthaloyl group (Scheme 1).
The resulting amine 9 was reacted with trichloroacetyl chlo-
ride in pyridine to give 10 (81% in two steps). Desilylation of
10 with n-Bu4NF in THF in the presence of excess AcOH af-
forded hemiacetal 11, which upon treatment with Et2NSF3
gave fluoride 12 (82% in two steps) as a mixture of anomers
(a/b¼19/1). Fluoride 12 seemed more reactive than 3, and
reacted with glycosyl serine 6 within 0.5 h by activation
with Cp2Zr(ClO4)2 at ꢀ15 ꢁC to afford trisaccharide 13 as
OBn
BnO
OBn
BnO
BnO
BnO
BnO
BnO
O
O
OSiPh2But
OSiPh2But
NHR
a, b
O
O
BnO
BnO
O
O
PhthN
OBn
OBn
8
9: R = H
10: R = -COCCl3 (TCA)
OBn
OBn
O
BnO
BnO
BnO
BnO
O
BnO
BnO
d
O
c
O
BnO
O
OBn
BnO
O
OBn
TCANH
TCANH
OH
F
12
11
OR3
R2O
O
OBn
BnO
O
BnO
BnO
O
e, f
O
BnO
O
OBn
N3
O
TCANH
R1
FmocNH
CO2All
13: R1 = H, R2, R3 = -CH(Ph)-
14: R1 = CH3, R2, R3 = -CH(Ph)-
15: R1 = H, R2, R3 = H
16: R1 = CH3, R2, R3 = H
Scheme 1. Synthesis of hexabenzylated glycosyl fluoride 12 and trisaccharyl serine/threonine, 15 and 16. Reaction conditions: (a) 1,2-diaminoethane, n-BuOH,
90 ꢁC, 2 days, 96%; (b) trichloroacetyl chloride, pyridine, 0 ꢁC, 1.5 h, 84%; (c) n-Bu4NF, AcOH, THF, room temperature, overnight, 89%; (d) diethylamino-
sulfur trifluoride, THF, 0 ꢁC, 1 h, 92%; (e) 6 or 7, Cp2ZrCl2, AgClO4, CH2Cl2, ꢀ15 ꢁC, 1 h, 13 (75%), 14 (80%); (f) 80% aq TFA, CH2Cl2, 94% (15), 83% (16).

2172
A. Ueki et al. / Tetrahedron 63 (2007) 2170–2181
the sole coupling product in 75% yield. Similarly, trisac-
charyl threonine 14 was obtained from 7 and 12 in 80%
yield. Stereochemistry of the newly generated linkage was
lowered temperature or with other solvents was ineffective
for suppressing the 4-O-glycosylation. On the other hand,
we had achieved 6-O-selective glycosylation to the desired
extent by using glycosyl donor 3 in the synthesis of core 2
and core 6. Thus, condensation of 3 and 16 was next inves-
tigated. When 3 was reacted with excess 16 (3/16¼0.83) at
ꢀ20 ꢁC for 2.5 h, the desired pentasaccharide 20 was pro-
duced in a moderate yield (40%) and acceptor 16 (54%)
was recovered. In contrast, the use of excess donor (3/
16¼1.2) promoted better conversion of 16, but resulted in
formation of the byproducts, 22 and 24. Through several at-
tempts to reduce the side reactions, an improved yield (71%)
of 20 was attained under the reaction of 3 and 16 (3/16¼1.1)
at ꢀ40 to ꢀ15 ꢁC for 2 h. However, the formation of 22 (9%)
and 24 (8%) could not be suppressed. Structures of the prod-
ucts were assigned by 1H and 13C NMR. The same reaction
conditions were applied to the condensation of 3 and serine
derivative 15 to produce 19 (71%), 21 (6%), and 23 (4%).
With no further optimization, synthesis was forwarded using
1
evidenced by the JCH value (168.4 Hz for 13 and
165.9 Hz for 14). Removal of the benzylidene group of 13
and 14 with 80% TFA led to 4,6-dihydroxy compounds,
15 and 16.
Selective 6-O-glycosylation was first tested with glycosyl
donor 12 and diol 16. The reaction, however, again gave
an unsatisfactory result. Two pentasaccharides were pro-
duced, when donor 12 and excess acceptor 16 (12/
16¼0.83) were reacted in CH2Cl2 at ꢀ15 ꢁC for 1 h. 4-O-
Glycosylated product 18 was obtained in a considerable
amount (30%) along with the desired 17 (44%). The struc-
tures of 17 and 18 were determined after acetylation of the
product samples (Fig. 2). The characteristic lower field shift
of the H-4 signal (GalN3) was observed at 5.36 ppm for the
acetate derivative of 17. The altered condition with the
OBn
O
OBn
O
BnO
BnO
OBn
O
OBn
O
BnO
BnO
BnO
BnO
O
O
BnO
BnO
O
TCANH
HO
O
BnO
BnO
BnO
OH
OBn
TCANH
O
BnO
BnO
O
O
BnO
BnO
O
O
O
O
O
BnO
BnO
O
OBn
O
OBn
N
3 O
N
3 O
TCANH
TCANH
H3C
H3C
FmocNH
FmocNH
CO2All
CO2All
17
18
Ph
O
O
Ph
OBn
O
O
O
OBn
O
O
O
O
BnO
O
BnO
BnO
O
TCANH
HO
O
BnO
BnO
BnO
BnO
BnO
OH
OBn
TCANH
O
BnO
BnO
O
O
BnO
BnO
O
O
O
O
O
BnO
BnO
O
OBn
O
N
3 O
N
3 O
BnO
TCANH
TCANH
OBn
R
R
FmocNH
CO2All
FmocNH
CO2All
19: R = H
21: R = H
20: R = CH3
22: R = CH3
Ph
O
OBn
Ph
O
O
O
O
O
Ph
OBn
O
O
BnO
O
BnO
O
O
BnO
O
TCANH
O
O
BnO
O
O
BnO
O
BnO
BnO
O
BnO
AcNH
HO
BnO
BnO
BnO
BnO
OBn
O
TCANH
O
BnO
BnO
O
O
BnO
BnO
O
O
O
O
BnO
BnO
O
O
OBn
AcNH
R
N
3 O
AcNH
BnO
O
TCANH
OBn
R
FmocNH
CO2All
FmocNH
25: R = H
CO2All
23: R = H
24: R = CH3
26: R = CH3
Figure 2. Structures of glycosylation products.

A. Ueki et al. / Tetrahedron 63 (2007) 2170–2181
2173
the obtained pentasaccharides 19 and 20. In the previous
studies, we have accomplished dechlorination of the tri-
chloroacetamido group and simultaneous reduction of the
azido group with large excess of Zn and AcOH. Despite giv-
ing a fairly good yield, the tedious procedure of the reaction
was a drawback. Intermittent additions of Zn powder and
AcOH to the reactant in CH2Cl2 were necessary at 3–5 h in-
tervals during 1–2 days until completion of the dechlorina-
tion. In addition, Zn(OAc)2 resulted in a large quantity,
which gave some difficulty in the following acetylation of
the crude reduction product. In recent years, acceleration
of reactions by microwave irradiation has received consider-
able attention.15 The apparent effectiveness reported for the
reactions in a heterogeneous system prompted us to inves-
tigate the Zn reduction under microwave. We examined
several conditions by varying the amount of the reductant,
microwave power, reaction time, and solvent with a model
monosaccharide as well as 19. After optimization, the best
result was obtained with a reduced amount of Zn by micro-
wave heating gently at refluxing temperature in EtOAc for
30 min. The reduction products derived from 19 and 20
were then acetylated with Ac2O in CH2Cl2/MeOH to give
25 (81%) and 26 (80%), respectively. To fulfill the synthesis
of the building blocks suitable for the solid-phase synthesis
of glycopeptide, 25 and 26 were converted to carboxylic
acids 1 (93%) and 2 (92%) by deallylation with Pd(PPh3)4
and 5,5-dimethyl-1,3-cyclohexanedione in THF.
oligosaccharide at the Asn44 site, and the first Ig domain
(Gly34-Met94) (Fig. 3) including the N-glycan is responsible
for the inducer activity. Recently, we have established an ef-
ficient synthetic route to the first Ig domain and its extended
peptide bearing chitobiosyl or core pentasaccharyl motif of
the N-glycan by taking advantage of the thioester method
of segment condensation.16 In the present study, building
block 2 was introduced into the significant segment in place
of the N-glycan at the Asn44 position. The synthetic imita-
tion would be helpful to explore the structural requirement
of the oligosaccharide attached to the bioactive Ig domain,
since the biantennary LacNAc in the core 4 O-glycan penta-
saccharide resembles the peripheral structure of the com-
plex-type N-glycan.
Glycopentacosapeptide 27, which carries a thioester func-
tionality necessary for the segment condensation in the Ig
domain synthesis, was synthesized from commercial Fmoc-
CLEAR-amide resin (Scheme 2). Attachment of Fmoc–
Gly–SCH2CH2CO2H to the N-deprotected resin was
followed by removal of the Fmoc group using a cocktail of
1-methylpyrrolidine (25%), hexamethyleneimine (2%),
and 1-hydroxybenzotriazole (2%) in NMP/DMSO (1:1).17
According to the reported procedure,16b the tripeptide-resin
was prepared by coupling first with N6-benzyloxycarbonyl-
N2-triisopropylsilyloxycarbonyl-lysine pentafluorophenyl
ester [(Tsoc)–Lys(Z)–OPfp] and then with Fmoc–Leu–F in
the presence of tetrabutylammonium fluoride. This two-
step protocol is indispensable to get rid of the side reaction
caused by a diketopiperazine formation in the early stage
of thioester synthesis. Further elongation with 11 amino
acids was performed with an automated peptide synthesizer
using Fmoc amino acids activated with HBTU, HOBt, and
DIEA. All the N-deprotection steps were carried out with the
above cocktail under the automated program. Benzyl-
oxycarbonyl group (Z), tert-butoxycarbonyl group (Boc),
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group
(Pbf), and triphenylmethyl group (Trt) were employed for
protection of the functional side chain amino groups of
3. Solid-phase synthesis of glycopeptide
As a suitable platform that allows us to test the effectiveness
of the benzyl and benzylidene-protected core 4 building
blocks in the solid-phase synthesis, the chosen model glyco-
peptide was a glycosylated pentacosapeptide thioester that
mimics a segment (Gly34-Gly58) of the extracellular matrix
metalloproteinase inducer (emmprin). Emmprin is produced
by tumor cells and is thought to function in the early stage
of metastasis. The native emmprin carries an N-linked
CHO
34
40
50
-Gly-Ser-Lys-Ile-Leu-Leu-Thr-Cys-Ser-Leu-Asn-Asp-Ser-Ala-Thr-Glu-Val-Thr-Gly-His-Arg-
60
70
Trp-Leu-Lys-Gly-Gly-Val-Val-Leu-Lys-Glu-Asp-Ala-Leu-Pro-Gly-Gln-Lys-Thr-Glu-Phe-Lys-
80
90
94
Val-Asp-Ser-Asp-Asp-Gln-Trp-Gly-Glu-Tyr-Ser-Cys-Val-Phe-Leu-Pro-Glu-Pro-Met-
OH
HO
AcNH
HO
O
O
O
HO
O
HO
O
OH
HO
OH
O
OH
O
O
HO
HO
O
HO
H2N
HN
AcNH
NH
O
AcNH
H2N
OH
CH3
CH3
CH3
CH3
HO
HO
CH3
O
H3C
O
O
CH3
CH3
HO
HO
O
CH3
58
34
O
O
O
O
O
H
H
N
O
H
N
H
N
H
N
O
H
O
O
H
N
H
N
H
N
H
N
H
O
H
N
S
N
N
NH2
N
N
H
N
N
N
H
N
H
N
H
N
N
N
H
N
H
N
H
N
H
H2N
H
H
H
H
O
O
O
O
O
O
O
HO
O
O
O
O
O
CH3
CH3
CH3
CH3
CO2H
S
CH3
HO
CH3
N
N
NH
H2N
CO2H
CH3
CH3
NHAc
H
27
Figure 3. Structures of the emmprin Ig domain (Gly34-Met94) and synthetic glycopeptide 27.

2174
A. Ueki et al. / Tetrahedron 63 (2007) 2170–2181
Fmoc-CLEAR-amide resin
a - e
c, h
O-protected Gal 1-4GlcNAc 1-6-(Gal 1-4GlcNAc 1-3)-GalNAc
Fmoc-Leu-Lys(Z)-Gly-SCH2CH2CO-CLEAR-amide resin
c, f
Fmoc-Gly-Ser(tBu)-Lys(Boc)-Ile-Leu-Leu-Thr(tBu)-Cys(Acm)-Ser(tBu)-Leu-Thr-
Asp(OtBu)-Ser(tBu)-Ala-Thr(tBu)-Glu(OtBu)-Val-Thr(tBu)-Gly-His(Trt)-
Arg(Pbf)-Trp(Boc)-Leu-Lys(Z)-Gly-SCH2CH2CO-CLEAR-amide resin
Fmoc-Asp(OtBu)-Ser(tBu)-Ala-Thr(tBu)-Glu(OtBu)-Val-Thr(tBu)-Gly-His(Trt)-
Arg(Pbf)-Trp(Boc)-Leu-Lys(Z)-Gly-SCH2CH2CO-CLEAR-amide resin
c, i, j
Gal 1-4GlcNAc 1-6-(Gal 1-4GlcNAc 1-3)-GalNAc
c, g
H-Gly-Ser-Lys-Ile-Leu-Leu-Thr-Cys(Acm)-Ser-Leu-Thr-Asp-Ser-Ala-Thr-Glu-
Val-Thr-Gly-His-Arg-Trp-Leu-Lys-Gly-SCH2CH2CONH2
O-protected Gal 1-4GlcNAc 1-6-(Gal 1-4GlcNAc 1-3)-GalNAc
27
Fmoc-Thr-Asp(OtBu)-Ser(tBu)-Ala-Thr(tBu)-Glu(OtBu)-Val-Thr(tBu)-Gly-
His(Trt)-Arg(Pbf)-Trp(Boc)-Leu-Lys(Z)-Gly-SCH2CH2CO-CLEAR-amide resin
Scheme 2. Solid-phase synthesis of glycopeptide 27. Reaction conditions: (a) 20% piperidine/NMP; (b) Fmoc–Gly–SCH2CH2CO2H, DCC/HOBt/NMP;
(c) 1-methylpyrrolidine/hexamethyleneimine/1-hydroxybenzotriazole/NMP/DMSO; (d) Tsoc–Lys(Z)–OPfp; (e) Fmoc–Leu–F, TBAF, CH2Cl2; (f) Fmoc-
amino acid, HBTU, HOBt, DIEA, ABI 433A peptide synthesizer, FastMoc program; (g) 2, DCC/HOBt/NMP, 50 ꢁC, 3 h; (h) Fmoc amino acid, DCC/
HOBt/NMP, 50 ꢁC; (i) TFA/phenol/thioanisole/EDT/H2O, room temperature, 2 h; (j) DMS/TFA/m-cresol, ꢀ15 ꢁC, then TfOH, 2 h.
Lys, Trp, Arg, and His, respectively, whereas the tert-butyl
group (tBu) was used for masking the hydroxyl groups of
Ser and Thr, and the carboxyl groups of Glu and Asp resi-
dues. These protecting groups are readily removed under
acidic conditions. The N-deprotected tetradecapeptide-resin
(25 mmol) was coupled with 2 (2 equiv) by manual operation
using DCC and HOBt at 50 ꢁC for 3 h in a polypropylene
vessel with stirring by a vortex mixer. Among the tested con-
ditions including other condensing agents, the combination
of DCC and HOBt gave the most acceptable coupling effi-
ciency. The coupling was monitored by HPLC and MS anal-
yses of the glycopeptide detached from the resin sample. The
glycopeptide chain was further elongated manually to com-
plete the pentacosapeptide synthesis. For persistent protec-
tion of the thiol group of Cys, an acetamidomethyl group
(Acm) was employed. According to our standard protocol
for isolation of synthetic glycopeptide, a series of deprotec-
tion procedures were conducted. The resin was treated with
reagent K (aq CF3CO2H, thioanisole, 1,2-ethanedithiol, and
phenol)18 for 2 h to split the glycopeptide from the resin. The
crude product was thoroughly debenzylated under the ‘low-
acidity TfOH’ conditions19 at ꢀ15 ꢁC for 2 h and then puri-
fied by HPLC. Figure 4 shows that the desired product 27
was obtained as the major product (peak 1). The structure
of the synthesized glycopeptide thioester was evidenced by
MALDI TOF MS [m/z 3764.95 (M+H+)]. A byproduct
eluted in the less mobile fraction (peak 2) was assigned as
a glycopeptide lacking a LacNAc moiety [m/z 3399.62
(M+H+)]. Although the resulting glycan structure of the lat-
ter glycopeptide has not been elucidated, it is probable that
an acid-labile 6-O-glycosidic linkage might have been
cleaved as observed in the synthetic glycopeptide carrying
core 6 O-glycans.3 The overall yield of purified 27 was de-
duced to be 2.2% from the data of amino acid analysis of
the acid-hydrolyzed sample, and the value is comparable
to that obtained previously for the synthetic N-glycan-linked
glycopeptide.16b The LacNAc-missing glycopeptide was
also isolated in 1.3% overall yield. The isolated 27 (total
(a)
(b)
1 (27)
45
45
35
25
15
2
35
25
15
0
10
20
30
0
10
20
30
Retention time/min
Retention time/min
Figure 4. HPLC profiles of synthetic glycopeptide 27 (a) and isolated 27 (b). Conditions: column, Mightysil RP-18, 4.6ꢂ150 mm (5 mm); eluant A, distilled
water containing 0.1% TFA; eluant B, acetonitrile containing 0.1% TFA; flow rate, 1 ml/min.

A. Ueki et al. / Tetrahedron 63 (2007) 2170–2181
2175
0.6 mmol, Fig. 4) is a useful intermediate to synthesize an
O-glycan analog of the emmprin first Ig domain, through
N-tert-butoxycarbonylation, condensation with the known
segment (Gly59-Met94), and disulfide formation.
4.1.1. tert-Butyldiphenylsilyl 2,3,4,6-tetra-O-benzyl-b-
D-galactopyranosyl-(1/4)-2-amino-3,6-di-O-benzyl-2-
deoxy-b-D-glucopyranoside 9. A mixture of 8 (703 mg,
0.56 mmol) and 1,2-diaminoethane (0.75 ml, 11.2 mmol)
in n-BuOH (25 ml) was heated at 90 ꢁC with stirring for 2
days under Ar, before being concentrated in vacuo. The resi-
due 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 CHCl3 to give 9 (603 mg, 96%). [a]D ꢀ5.3 (c 1.0).
In summary, the biantennary LacNAc-linked O-glycosyl Ser
and Thr were synthesized both in a benzyl and benzylidene-
protected form by using two distinctively protected
LacNTCA glycosyl donors. The dechlorination of the tri-
chloroacetamido group by Zn/AcOH reduction was greatly
accelerated by employing microwave irradiation. By the
solid-phase synthetic procedure suited for the labile Fmoc
peptide thioester, the glycothreonine derivative was success-
fully introduced into a glycopeptide, which mimics the gly-
cosylated segment in the metastasis-related emmprin first Ig
domain. The benzyl-protecting groups of the glycan moiety
were readily removed under the low-acidity TfOH condi-
tions to complete the desired glycopeptide thioester. Synthe-
sis toward the 61 amino acid structure for the Ig domain and
bioassay with the glycan-mutated sample will be reported in
due course.
1
Rf 0.43 (1:1 hexane/EtOAc). H NMR: d 7.71–7.67, 7.49–
7.10 (m, 40H, Ar), 5.16 (d, 1H, J¼10.5 Hz, –CH2Ph), 4.97
(d, 1H, J¼11.2 Hz, –CH2Ph), 4.76–4.69 (m, 4H, 4ꢂ
–CH2Ph), 4.58–4.47 (m, 2H, 2ꢂ –CH2Ph), 4.45 (d, 1H,
J¼7.8 Hz, H-1b), 4.39 (d, 1H, J¼12.0 Hz, –CH2Ph), 4.37
(d, 1H, J¼7.1 Hz, H-1a), 4.36 (d, 1H, J¼12.2 Hz,
–CH2Ph), 4.26 (d, 1H, J¼11.7 Hz, –CH2Ph), 4.20 (d, 1H,
J¼12.2 Hz, –CH2Ph), 4.01 (t, 1H, J¼9.3 Hz, H-4a), 3.91
(d, 1H, J¼2.4 Hz, H-4b), 3.72 (t, 1H, J¼8.1 Hz, H-2b),
3.67 (dd, 1H, J¼2.9, 11.2 Hz, H-6a), 3.53 (m, 1H, H-6a),
3.43–3.37 (m, 3H, H-3b, H-5b, and H-6b), 3.26–3.21 (m,
2H, H-3a and H-6b), 2.91–2.98 (m, 2H, H-2a and H-5a),
t
1.10 (s, 9H, Bu). 13C NMR: d 102.4 (C-1b), 98.8 (C-1a).
4. Experimental
4.1. General
MALDI TOF MS calcd for C70H77NO10Si m/z: 1119.53.
Found: 1142.75 (M+Na+). Anal. Calcd for C70H77NO10Si:
C, 75.04; H, 6.93; N, 1.25. Found: C, 75.01; H, 6.83; N, 1.14.
Optical rotation values were determined with a Jasco DIP-
370 polarimeter at 20ꢃ2 ꢁC for solutions in CHCl3, unless
noted otherwise. Column chromatography was performed
on silica gel PSQ 100B (Fuji Silysia). TLC and HPTLC
4.1.2. tert-Butyldiphenylsilyl 2,3,4,6-tetra-O-benzyl-b-
D-galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-
2-trichloroacetamido-b-D-glucopyranoside 10. To an
ice-cooled solution of 9 (3.43 g, 3.06 mmol) in pyridine
(40 ml) was added CCl3COCl (513 ml, 4.06 mmol). The mix-
ture was stirred for 1.5 h at 0 ꢁC, and then concentrated
in vacuo. The residue was extracted with CHCl3, washed
successively with water and brine, dried over Na2SO4, and
concentrated invacuo. Chromatography of the crude on silica
gel with hexane/EtOAc (4:1) afforded 10 (3.23 g, 84%). [a]D
1
were performed on silica gel 60 F254 (E. Merck). H and
13C NMR spectra were recorded with a Jeol AL400 spec-
trometer [1H (400 MHz) and 13C (100 MHz)]. Chemical
shifts are expressed in parts per million downfield from the
signal for internal Me4Si for solutions in CDCl3. For assign-
ment of the signals of pentasaccharides, the reducing termi-
nal residue GalNAc is described as a. GlcNAc and Gal
residues in the 3-O-substituent of GalNAc are described as
b and c, respectively, while GlcNAc and Gal residues in
the 6(or 4)-O-substituent of GalNAc are shown as d and e.
For heptasaccharides 23 and 24, GlcNAc and Gal residues
in the 6-O-substituent of GalNAc are described as f and g,
respectively. MALDI TOF mass spectra were obtained with
a PerSeptive Voyager-DE PRO spectrometer (2,5-dihydroxy-
benzoic acid was used as a matrix). Microwave irradiation
was carried out with a CEM Discover microwave reactor.
Automated solid-phase peptide synthesis was performed
with Applied Biosystems Model 433A peptide synthesizer.
Manual solid-phase reactions were undertaken in capped
polypropylene test tubes equipped with a filter and three-
way stopcock by stirring with an EYELA CM-1000 vortex
mixer. HPLC was performed with Mightysil RP-18
(4.6ꢂ150 mm for analysis and 10ꢂ250 mm for preparation,
Kanto Chemical Co.). Amino acids were analyzed by a Hita-
chi L-8500 amino acid analyzer. Fmoc-CLEAR-amide resin
was purchased from Peptide International Inc. Due to the
thermolabile azide group, N analysis values of compounds
13, 14, and 15 showed some inconsistency. The yield of gly-
copeptide was determined by amino acid analysis after
a measured volume from the whole sample solution was
hydrolyzed in a sealed tube with 20% HCl and 0.5% phenol
at 150 ꢁC for 2 h.
1
ꢀ3.3 (c 1). Rf 0.66 (1:1 hexane/EtOAc). H NMR: d 7.69–
7.62, 7.39–7.10 (m, 40H, Ar), 6.88 (d, 1H, J¼7.8 Hz,
–NH), 4.95 (d, 1H, J¼10.5 Hz, –CH2Ph), 4.94 (d, 1H,
J¼11.5 Hz, –CH2Ph), 4.89 (d, 1H, J¼7.1 Hz, H-1a), 4.76–
4.66 (m, 4H, 4ꢂ –CH2Ph), 4.54 (d, 1H, J¼10.5 Hz,
–CH2Ph), 4.52 (d, 1H, J¼11.5 Hz, –CH2Ph), 4.44 (d, 1H,
J¼7.6 Hz, H-1b), 4.37 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.33
(d, 1H, J¼11.7 Hz, –CH2Ph), 4.23 (d, 1H, J¼11.7 Hz,
–CH2Ph), 4.22 (d, 1H, J¼12.0 Hz, –CH2Ph), 4.04 (t, 1H,
J¼8.8 Hz, H-4a), 3.89 (d, 1H, J¼2.4 Hz, H-4b), 3.83 (t,
1H, J¼7.8 Hz, H-3a), 3.77 (dd, 1H, J¼7.8, 9.8 Hz, H-2b),
3.71 (dd, 1H, J¼7.8, 9.8 Hz, H-2b), 3.66 (dd, 1H, J¼3.4,
11.2 Hz, H-6a), 3.41 (dd, 1H, J¼2.9, 9.8 Hz, H-3b), 3.40–
3.37 (m, 1H, H-5b), 3.33–3.29 (m, 2H, H-6a and H-6b),
t
3.07–3.05 (m, 1H, H-5a), 1.05 (s, 9H, Bu). 13C NMR:
d 161.2 (Cl3CCONH), 102.7 (C-1b), 94.8 (C-1a), 92.5
(–CCl3). MALDI TOF MS calcd for C72H76Cl3NO11Si m/z:
1263.43. Found: 1286.26 (M+Na+). Anal. Calcd for
C72H76Cl3NO11Si: C, 68.32; H, 6.05; N, 1.11. Found: C,
68.51; H, 5.84; N, 1.01.
4.1.3. 2,3,4,6-Tetra-O-benzyl-b-D-galactopyranosyl-(1/
4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-a-D-
glucopyranose 11. To an ice-cooled mixture of 10 (2.03 g,
1.60 mmol) and AcOH (912 ml, 16.0 mmol) in freshly dis-
tilled THF (20 ml) was added 1 M n-Bu4NF/THF (6.41 ml,

2176
A. Ueki et al. / Tetrahedron 63 (2007) 2170–2181
˚
dried molecular sieves 4 A powder (1 g) in anhydrous
6.41 mmol). Then the mixture was stirred at room tempera-
ture overnight and 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
hexane/EtOAc (4:1–2:1) to give 11 as a mixture of anomers
CH2Cl2 (3 ml) was stirred at room temperature under Ar
for 30 min and then cooled at ꢀ15 ꢁC. To the mixture was
added a mixture of 6 (415 mg, 0.65 mmol) and 12
(554 mg, 0.54 mmol) in anhydrous CH2Cl2 (7 ml). The mix-
ture was stirred at ꢀ15 ꢁC for 1 h, before the reaction was
quenched with aq NaHCO3. The mixture was diluted with
EtOAc and filtered through Celite. The filtrate was succes-
sively washed with satd NaHCO3, water, and brine, dried
over MgSO4, and concentrated in vacuo. The residue was
chromatographed on silica gel with toluene/EtOAc (88:12)
to give 13 (670 mg, 75%). [a]D +62.9 (c 1). Rf 0.51 (4:1
1
(1.46 g, 89%, a/b>10). Rf 0.50 (1:1 hexane/EtOAc). H
NMR: d 7.70–7.69, 7.44–7.11 (m, 30H, Ar), 6.88 (d, 1H,
J¼7.8 Hz, –NH), 5.33 (br t, 1H, J¼3.4 Hz, H-1a), 5.02
(d, 1H, J¼11.0 Hz, –CH2Ph), 4.94 (d, 1H, J¼11.5 Hz,
–CH2Ph), 4.83 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.78 (d, 1H,
J¼11.0 Hz, –CH2Ph), 4.71 (d, 1H, J¼12.0 Hz, –CH2Ph),
4.68 (d, 1H, J¼12.0 Hz, –CH2Ph), 4.53 (d, 1H,
J¼11.5 Hz, –CH2Ph), 4.50 (d, 1H, J¼12.0 Hz, –CH2Ph),
4.32 (d, 1H, J¼8.1 Hz, H-1b), 4.30 (d, 1H, J¼12.6 Hz,
–CH2Ph), 4.29 (d, 1H, J¼12.0 Hz, –CH2Ph), 4.19 (d, 1H,
J¼11.7 Hz, –CH2Ph), 4.14–4.08 (m, 1H, H-2a), 4.02–3.93
(m, 2H, H-4a and H-5a), 3.89 (d, 1H, J¼2.4 Hz, H-4b),
3.82–3.74 (m, 3H, H-2b, H-3a, and H-6a), 3.59 (br d, 1H,
J¼9.8 Hz, H-6a), 3.43–3.34 (m, 4H, H-3b, H-5b, H-6b,
OH), 3.32–3.28 (m, 1H, H-6b). 13C NMR: d 161.4
(Cl3CCONH), 102.9 (C-1b), 92.5 (–CCl3), 90.8 (C-1a, a-
anomer). MALDI TOF MS calcd for C56H58Cl3NO11 m/z:
1025.31. Found: 1048.27 (M+Na+). Anal. Calcd for
C56H58Cl3NO11: C, 65.46; H, 5.69; N, 1.36. Found: C,
65.73; H, 5.72; N, 1.24.
1
toluene/EtOAc). H NMR: d 7.76 (d, 2H, J¼7.6 Hz, Ar),
7.62 (d, 2H, J¼7.3 Hz, Ar), 7.61–7.06 (m, 40H, Ar and
Cl3CCONH), 5.98–5.85 (m, 2H, –CH2CH]CH2, FmocNH),
5.351 [s, 1H, PhCH(O)2], 5.35 (d, 1H, J¼17.1 Hz,
–CH]CH2), 5.28 (d, 1H, J¼10.7 Hz, –CH]CH2), 5.26
(d, 1H, J¼7.1 Hz, H-1b), 5.04 (d, 1H, J¼10.3 Hz,
–CH2Ph), 4.97 (d, 1H, J¼11.5 Hz, –CH2Ph), 4.95 (d, 1H,
J¼3.5 Hz, H-1a), 4.85 (d, 1H, J¼11.6 Hz, –CH2Ph), 4.82
(d, 1H, J¼11.6 Hz, –CH2Ph), 4.74 (d, 1H, J¼12.2 Hz,
–CH2Ph), 4.73–4.68 (m, 1H, –CH2Ph), 4.69 (d, 2H,
J¼5.9 Hz, –CH2C]CH2), 4.55 (d, 1H, J¼10.3 Hz,
–CH2Ph), 4.54 (d, 1H, J¼11.5 Hz, –CH2Ph), 3.92 (d, 1H,
J¼2.7 Hz, H-4c), 3.83 (dd, 1H, J¼3.5, 10.7 Hz, H-2a),
3.73–3.67 (m, 1H, H-5b), 3.59 (br s, 1H, H-4a), 3.45 (dd,
1H, J¼2.7, 9.8 Hz, H-3c), 3.39 (dd, 1H, J¼4.9, 7.6 Hz,
H-5c), 3.32 (dd, 1H, J¼4.9, 8.5 Hz, H-6c). 13C NMR: d
169.2 (–CO2), 161.7 (Cl3CCONH), 155.7 (OCONH), 131.1
4.1.4. 2,3,4,6-Tetra-O-benzyl-b-D-galactopyranosyl-(1/
4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-b-
D-glucopyranosyl fluoride 12. To a stirred solution of 11
(635 mg, 0.62 mmol) in freshly distilled THF (10 ml) was
added Et2NSF3 (121 ml, 0.93 mmol) at 0 ꢁC. The mixture
was stirred for 1 h before the reaction was quenched with
MeOH and concentrated in vacuo. The residue was extracted
with EtOAc, successively washed with water and brine, dried
over Na2SO4, and concentrated in vacuo. The crude product
was chromatographed on silica gel with toluene/EtOAc (9:1)
to give 12 as a mixture of anomers (582 mg, 92%, a/b¼19/1).
Rf 0.40 (9:1 toluene/EtOAc). 1H NMR: d 7.36–7.14 (m, 30H,
Ar), 6.54 (d, 1H, J¼8.1 Hz, ꢀNH), 5.75 (dd, 1H, J¼2.4,
53.7 Hz, H-1a), 5.03 (d, 1H, J¼11.0 Hz, ꢀCH2Ph), 4.96
(d, 1H, J¼11.5 Hz, ꢀCH2Ph), 4.84 (d, 1H, J¼11.2 Hz,
ꢀCH2Ph), 4.76 (d, 1H, J¼11.2 Hz, ꢀCH2Ph), 4.72–4.67
(m, 2H, –CH2Ph), 4.66 (d, 1H, J¼8.3 Hz, H-1b), 4.63 (d,
1H, J¼11.2 Hz, –CH2Ph), 4.56 (d, 1H, J¼12.0 Hz,
–CH2Ph), 4.54 (d, 1H, J¼11.5 Hz, –CH2Ph), 4.38–4.36 (m,
4H, –CH2Ph), 4.24 (d, 1H, J¼11.3 Hz, –CH2Ph), 4.15 (t,
1H, J¼9.5 Hz, H-2a), 3.92–3.89 (m, 2H, H-4b and H-5a),
3.86 (br d, 1H, J¼11.4 Hz, H-2b), 3.79–3.72 (m, 2H, H-3a
and H-6a), 3.56 (dd, 1H, J¼1.2, 11.0 Hz, H-6a), 3.52–3.43
(m, 1H, H-6b), 3.38–3.32 (m, 3H, H-4a, H-5b, and H-6b).
13C NMR: d 161.5 (Cl3CCONH), 105.3 (d, JCF¼220.1 Hz,
C-1a, a-F), 102.4 (C-1b), 91.9 (–CCl3). MALDI TOF MS
calcd for C56H57Cl3FNO10 m/z: 1027.30. Found: 1050.39
(M+Na+). Anal. Calcd for C56H57Cl3FNO10: C, 65.34; H,
5.58; N, 1.36. Found: C, 65.45; H, 5.58; N, 1.33.
(–CH]), 119.0 (]CH2), 103.1 (1JCH¼160.1 Hz, C-1c),
1
100.4 [PhCH(O)2], 99.8 (1JCH¼168.4 Hz, C-1b, JCH
¼
171.8 Hz, C-1a), 92.3 (–CCl3). MALDI TOF MS calcd for
C90H90N5O19Cl3 m/z: 1649.53. Found: 1672.56 (M+Na+),
1688.34 (M+K+). Anal. Calcd for C90H90N5O19Cl3: C,
65.43; H, 5.49; N, 4.24. Found: C, 65.61; H, 5.64; N, 3.90.
4.1.6. N-(9-Fluorenylmethoxycarbonyl)-O-[2,3,4,6-tetra-
O-benzyl-b-D-galactopyranosyl-(1/4)-3,6-di-O-benzyl-
2-deoxy-2-trichloroacetamido-b-D-glucopyranosyl-(1/
3)-2-azido-4,6-O-benzylidene-2-deoxy-a-D-galactopyr-
anosyl]-L-threonine allyl ester 14. Condensation of 7
(368 mg, 0.56 mmol) and 12 (481 mg, 0.47 mmol) was per-
formed in a similar manner as described for 13. Chromato-
graphy of the crude product on silica gel with toluene/
EtOAc (88:12) gave 14 (622 mg, 80%). [a]D +58.8 (c 1).
Rf 0.34 (7:1 toluene/EtOAc). 1H NMR: d 7.77 (d, 2H,
J¼7.6 Hz, Ar), 7.64 (d, 2H, J¼7.3 Hz, Ar), 7.52–7.06 (m,
40H, Ar and Cl3CCONH), 5.92 (ddt, 1H, J¼6.1, 10.5,
17.1 Hz, –CH2CH]CH2), 5.72 (d, 1H, J¼9.5 Hz,
FmocNH), 5.36 [s, 1H, PhCH(O)2], 5.35 (dd, 1H, J¼1.2,
17.1 Hz, –CH]CH2), 5.33 (d, 1H, J¼7.8 Hz, H-1b), 5.25
(dd, 1H, J¼1.2, 10.5 Hz, –CH]CH2), 5.05 (d, 1H,
J¼10.3 Hz, –CH2Ph), 5.01 (d, 1H, J¼3.7 Hz, H-1a),
4.97 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.85 (d, 1H, J¼
11.2 Hz, –CH2Ph), 4.82 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.74
(d, 1H, J¼12.0 Hz, –CH2Ph), 4.70 (d, 1H, J¼12.0 Hz,
–CH2Ph), 4.66 (m, 2H, –CH2C]CH2), 4.54 (d, 2H,
J¼11.2 Hz, –CH2Ph), 4.49–4.39 (m, 4H, Thr–bH, H-6a,
and –OCH2CHAr2), 4.40 (d, 1H, J¼7.8 Hz, H-1c), 4.33 (d,
1H, J¼11.5 Hz, –CH2Ph), 4.32 (d, 1H, J¼11.7 Hz,
–CH2Ph), 4.31–4.27 (m, 3H, H-3b, H-6a, and –CH2CHAr2),
4.22 (d, 1H, J¼11.7 Hz, –CH2Ph), 4.18 (d, 1H, J¼11.5 Hz,
–CH2Ph), 4.11–4.04 (m, 2H, H-6b and H-3a), 3.92 (d, 1H,
4.1.5. N-(9-Fluorenylmethoxycarbonyl)-O-[2,3,4,6-tetra-
O-benzyl-b-D-galactopyranosyl-(1/4)-3,6-di-O-benzyl-
2-deoxy-2-trichloroacetamido-b-D-glucopyranosyl-(1/
3)-2-azido-4,6-O-benzylidene-2-deoxy-a-D-galactopyra-
nosyl]-L-serine allyl ester 13. A mixture of Cp2ZrCl2
(315 mg, 1.08 mmol), AgClO4 (447 mg, 2.16 mmol), and

A. Ueki et al. / Tetrahedron 63 (2007) 2170–2181
2177
J¼2.7 Hz, H-4c), 3.93–3.78 (m, 2H, H-4b and H-5a), 3.85
(dd, 1H, J¼3.7, 10.7 Hz, H-2a), 3.80 (dd, 1H, J¼7.8,
9.3 Hz, H-2c), 3.76–3.72 (m, 2H, H-5b and Thr–aH), 3.63
(br d, 1H, J¼12.0 Hz, H-6b), 3.58 (br s, 1H, H-4a), 3.52–
3.45 (m, 3H, H-2b, H-6c, and H-3c), 3.41 (dd, 1H, J¼5.1,
7.8 Hz, H-5c), 3.32 (dd, 1H, J¼5.1, 8.8 Hz, H-6c), 1.30 (d,
3H, J¼6.3 Hz, Thr–gH). 13C NMR: d 169.7 (–CO2), 161.8
(Cl3CCONH), 156.6 (OCONH), 131.1 (–CH]), 119.2
(]CH2), 103.1 (1JCH¼160.1 Hz, C-1c), 100.5 [PhCH(O)2],
99.6 (1JCH¼165.9 Hz, C-1b), 99.3 (1JCH¼173.4 Hz, C-1a),
92.3 (–CCl3). MALDI TOF MS calcd for C91H92N5O19Cl3
m/z: 1663.55. Found: 1686.85 (M+Na+), 1702.75 (M+K+).
Anal. Calcd for C91H92N5O19Cl3: C, 65.60; H, 5.57; N,
4.20. Found: C, 65.84; H, 5.74; N, 3.87.
5.92 (ddt, 1H, J¼5.9, 10.5, 17.1 Hz, –CH2CH]CH2), 5.66
(d, 1H, J¼9.3 Hz, FmocNH), 5.34 (br d, 1H, J¼17.1 Hz,
–CH]CH2), 5.29 (d, 1H, J¼7.6 Hz, H-1b), 5.25 (d,
1H, J¼10.5 Hz, –CH]CH2), 5.02 (d, 1H, J¼10.5 Hz,
–CH2Ph), 4.97 (d, 1H, J¼11.5 Hz, –CH2Ph), 4.95 (d, 1H,
J¼5.1 Hz, H-1a), 4.85 (d, 1H, J¼11.0 Hz, –CH2Ph), 4.79
(d, 1H, J¼11.0 Hz, –CH2Ph), 4.75 (d, 1H, J¼12.0 Hz,
–CH2Ph), 4.71 (d, 1H, J¼12.0 Hz, –CH2Ph), 4.65 (d,
2H, J¼5.9 Hz, –CH2CH]CH2), 4.54 (d, 2H, J¼11.2 Hz,
–CH2Phꢂ2), 4.48–4.20 (m, 1H, H-1c), 4.15 (br s, 1H,
H-4a), 4.01 (dd, 1H, J¼2.7, 10.5 Hz, H-3a), 3.94 (d, 1H,
J¼2.4 Hz, H-4c), 3.35 (dd, 1H, J¼4.9, 8.5 Hz, H-6c), 3.25
(br s, 1H, –OH), 2.30 (br s, 1H, –OH), 1.30 (d, 3H,
J¼6.3 Hz, Thr–gH); 13C NMR: d 169.8 (–CO2), 162.1
(Cl3CCONH), 156.6 (OCONH), 131.2 (–CH]), 119.2
4.1.7. N-(9-Fluorenylmethoxycarbonyl)-O-[2,3,4,6-tetra-
O-benzyl-b-D-galactopyranosyl-(1/4)-3,6-di-O-benzyl-
2-deoxy-2-trichloroacetamido-b-D-glucopyranosyl-(1/
3)-2-azido-2-deoxy-a-D-galactopyranosyl]-L-serine allyl
ester 15. To an ice-cooled solution of 13 (266 mg,
0.16 mmol) in CH2Cl2 (8 ml) was added 80% aq CF3CO2H
(4 ml) with stirring. The mixture was stirred at 0 ꢁC for
30 min, neutralized by addition of satd NaHCO3, and ex-
tracted with EtOAc. The extract was successively washed
with satd NaHCO3, water, and brine, dried over MgSO4,
and concentrated in vacuo. The crude product was chromato-
graphed on silica gel with toluene/EtOAc (2:1) to give 15
(237 mg, 94%). [a]D +41.9 (c 1). Rf 0.18 (3:2 toluene/
(]CH2), 103.2 (1JCH¼160.1 Hz, C-1c), 98.9 (1JCH
¼
175.1 Hz, C-1a), 98.4 (1JCH¼161.8 Hz, C-1b), 92.1
(ꢀCCl3). MALDI TOF MS calcd for C84H88N5O19Cl3 m/z:
1575.51. Found: 1598.69 (M+Na+). Anal. Calcd for
C84H88N5O19Cl3: C, 63.94; H, 5.62; N, 4.44. Found: C,
64.11; H, 5.46; N, 4.15.
4.1.9. N-(9-Fluorenylmethoxycarbonyl)-O-{2,3,4,6-tetra-
O-benzyl-b-D-galactopyranosyl-(1/4)-3,6-di-O-benzyl-
2-deoxy-2-trichloroacetamido-b-D-glucopyranosyl-(1/
3)-[2,3,4,6-tetra-O-benzyl-b-D-galactopyranosyl-(1/4)-
3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-b-D-glu-
copyranosyl-(1/6)]-2-azido-2-deoxy-a-D-galactopyr-
anosyl}-L-threonine allyl ester 17 and N-(9-fluorenyl-
methoxycarbonyl)-O-{2,3,4,6-tetra-O-benzyl-b-D-galacto-
pyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-trichloro-
acetamido-b-D-glucopyranosyl-(1/3)-[2,3,4,6-tetra-O-
benzyl-b-D-galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-
deoxy-2-trichloroacetamido-b-D-glucopyranosyl-(1/4)]-
2-azido-2-deoxy-a-D-galactopyranosyl}-L-threonine allyl
ester 18. Condensation of 12 (29 mg, 28 mmol) and 16
(54 mg, 34 mmol) was performed at ꢀ15 ꢁC for 1 h in a sim-
ilar manner as described for 13. Chromatography of the
crude product on silica gel with toluene/EtOAc (2:1) gave
17 (32 mg, 44%) and isomer 18 (22 mg, 30%). Unconsumed
16 (11 mg, 21%) was also recovered.
1
EtOAc). H NMR: d 7.74 (d, 2H, J¼7.6 Hz, Ar), 7.62 (d,
2H, J¼7.3 Hz, Ar), 7.57–7.06 (m, 35H, Ar and Cl3CCONH),
6.09 (d, 1H, J¼8.3 Hz, FmocNH), 5.90 (ddt, 1H, J¼5.9,
10.5, 16.8 Hz, –CH2CH]CH2), 5.33 (d, 1H, J¼16.8 Hz,
–CH]CH2), 5.25 (1H, d, J¼10.5 Hz, –CH]CH2), 5.21
(1H, d, J¼7.8 Hz, H-1b), 5.02 (1H, d, J¼10.5 Hz,
–CH2Ph), 4.97 (d, 1H, J¼11.5 Hz, –CH2Ph), 4.85 (d, 1H,
J¼11.0 Hz, –CH2Ph), 4.84 (m, 1H, H-1a), 4.79 (d, 1H,
J¼11.0 Hz, –CH2Ph), 4.74 (d, 1H, J¼12.0 Hz, –CH2Ph),
4.71 (d, 1H, J¼12.0 Hz, –CH2Ph), 4.69–4.63 (m, 2H,
–CH2CH]CH2), 4.55 (d, 1H, J¼10.5 Hz, –CH2Ph), 4.54
(d, 1H, J¼11.5 Hz, –CH2Ph), 4.54 (m, 1H, Ser–aH),
4.44–4.28 (m, 6H, H-1c, –NCO2CH2CH, –CH2Phꢂ3),
4.23 (d, 1H, J¼11.5 Hz, –CH2Ph), 4.10 (br s, 1H, H-4a),
3.34 (dd, 1H, J¼4.9, 8.5 Hz, H-6c), 3.24 (br s, 1H, –OH),
2.41 (br s, 1H, –OH). 13C NMR: d 169.3 (–CO2), 161.9
(Cl3CCONH), 155.6 (OCONH), 131.1 (–CH]), 118.9
Compound 17: [a]D +20.1 (c 1.7). Rf 0.39 (3:1 toluene/
1
EtOAc). H NMR: d 7.75 (d, 2H, J¼7.6 Hz, Ar), 7.62 (d,
1H, J¼6.8 Hz, Ar), 7.60 (d, 1H, J¼6.8 Hz, Ar), 7.45–7.06
(m, 65H, Ar and Cl3CCONH), 7.01 (d, 1H, J¼7.8 Hz,
Cl3CCONH), 5.91 (ddd, 1H, J¼17.1, 10.7, 5.9 Hz,
–CH]CH2), 5.61 (d, 1H, J¼9.3 Hz, FmocNH), 5.34 (dd,
1H, J¼17.1, 1.2 Hz, –CH]CH2), 5.24 (d, 1H, J¼10.7 Hz,
–CH]CH2), 5.23 (d, 1H, J¼6.8 Hz, H-1b), 5.01 (d, 1H,
J¼10.5 Hz, –CH2Ph), 4.97 (d, 1H, J¼11.4 Hz, –CH2Ph),
4.95 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.88 (d, 1H, J¼7.1 Hz,
H-1d), 4.87 (d, 1H, J¼3.2 Hz, H-1a), 4.81 (d, 1H, J¼
11.2 Hz, –CH2Ph), 4.80 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.78
(d, 1H, J¼11.2 Hz, –CH2Ph), 3.02 (br s, 1H, –OH), 1.30
(d, 3H, J¼6.3 Hz, Thr–gH). 13C NMR: d 169.9 (–CO2),
162.1 (Cl3CCONH), 161.4 (Cl3CCONH), 156.6 (OCONH),
119.3 (]CH2), 103.02 (1JCH¼165.9 Hz, C-1c or C-1e),
(]CH2), 103.1 (1JCH¼160.9 Hz, C-1c), 99.3 (1JCH
¼
170.9 Hz, C-1a), 98.8 (1JCH¼167.6 Hz, C-1b), 92.1
(–CCl3). MALDI TOF MS calcd for C83H86N5O19Cl3 m/z:
1561.50. Found: 1584.27 (M+Na+), 1600.24 (M+K+).
Anal. Calcd for C83H86N5O19Cl3: C, 63.74; H, 5.54; N,
4.48. Found: C, 63.42; H, 5.72; N, 4.09.
4.1.8. N-(9-Fluorenylmethoxycarbonyl)-O-[2,3,4,6-tetra-
O-benzyl-b-D-galactopyranosyl-(1/4)-3,6-di-O-benzyl-
2-deoxy-2-trichloroacetamido-b-D-glucopyranosyl-(1/
3)-2-azido-2-deoxy-a-D-galactopyranosyl]-L-threonine
allyl ester 16. Compound 14 (403 mg, 0.24 mmol) was
debenzylidenated in a similar manner as described for 15.
The crude product was purified by column chromatography
on silica gel with toluene/EtOAc (2:1–3:2) to give 16
(319 mg, 83%). [a]D +34.5 (c 1). Rf 0.50 (1:1 toluene/
102.96 (1JCH¼165.9 Hz, C-1c or C-1e), 99.9 (1JCH
¼
167.6 Hz, C-1b or C-1d), 99.1 (1JCH¼174.3 Hz, C-1a),
98.6 (1JCH¼160.9 Hz, C-1b or C-1d), 92.5 (–CCl3), 92.2
(–CCl3), 18.8 (Thr–gC). MALDI TOF MS calcd for
C140H144N6O29Cl6 m/z: 2582.81. Found: 2606.11 (M+Na+).
1
EtOAc). H NMR: d 7.76 (d, 2H, J¼7.3 Hz, Ar), 7.63 (d,
2H, J¼7.3 Hz, Ar), 7.41–7.12 (m, 35H, Ar and Cl3CCONH),

2178
A. Ueki et al. / Tetrahedron 63 (2007) 2170–2181
Anal. Calcd for C140H144N6O29Cl6$H2O: C, 64.54; H, 5.65;
N, 3.23. Found: C, 64.61; H, 5.67; N, 2.93.
C-1e), 101.2 [PhCH(O)2], 99.3 (1JCH¼163.4 Hz, C-1d),
99.0 (1JCH¼169.3 Hz, C-1b), 97.5 (1JCH¼173.4 Hz, C-1a),
92.4 (–CCl3), 92.1 (–CCl3). MALDI TOF MS calcd
for C132H134N6O29Cl6 m/z: 2476.73. Found: 2499.82
(M+Na+). Anal. Calcd for C132H134N6O29Cl6: C, 63.90; H,
5.44; N, 3.39. Found: C, 63.68; H, 5.47; N, 3.20.
Compound 18: [a]D +35.5 (c 1.4). Rf 0.35 (3:1 toluene/
1
EtOAc). H NMR: d 7.75 (d, 2H, J¼7.3 Hz, Ar), 7.64 (d,
2H, J¼7.3 Hz, Ar), 7.48–7.01 (m, 65H, Ar and Cl3CCONH),
6.96 (d, 1H, J¼8.5 Hz, Cl3CCONH), 5.90 (ddd, 1H, J¼
17.1, 10.5, 5.9 Hz, –CH]CH2), 5.64 (d, 1H, J¼9.5 Hz,
FmocNH), 5.33 (d, 1H, J¼17.1 Hz, –CH]CH2), 5.32
(d, 1H, J¼8.1 Hz, H-1b or H-1d), 5.24 (d, 1H, J¼10.5 Hz,
–CH]CH2), 5.14 (d, 1H, J¼8.3 Hz, H-1b or H-1d), 5.05
(d, 1H, J¼10.0 Hz, –CH2Ph), 4.93 (d, 2H, J¼11.2 Hz,
–CH2Phꢂ2), 4.85 (d, 1H, J¼3.7 Hz, H-1a), 4.81 (d, 1H,
J¼10.5 Hz, –CH2Ph), 4.80 (d, 1H, J¼11.2 Hz, –CH2Ph),
1.29 (d, 3H, J¼6.3 Hz, Thr–gH). 13C NMR: d 169.8
(–CO2), 162.0 (Cl3CCONH), 161.5 (Cl3CCONH), 156.7
(OCONH), 119.3 (]CH2), 103.3 (1JCH¼159.2 Hz, C-1c
or C-1e), 102.7 (1JCH¼159.2 Hz, C-1c or C-1e), 100.4
(1JCH¼162.5 Hz, C-1b or C-1d), 100.3 (1JCH¼162.5 Hz,
C-1b or C-1d), 98.9 (1JCH¼172.5 Hz, C-1a), 92.6 (–CCl3),
92.2 (–CCl3), 19.1 (Thr–gC). MALDI TOF MS calcd for
C140H144N6O29Cl6 m/z: 2582.81. Found: 2606.11
(M+Na+), 2622.05 (M+K+).
Compound 21: [a]D +43.7 (c 1.4). Rf 0.36 (3:2 toluene/
1
EtOAc). H NMR: d 7.75 (d, 2H, J¼7.3 Hz, Ar), 7.63 (d,
1H, J¼7.8 Hz, Ar), 7.61 (d, 1H, J¼7.8 Hz, Ar), 7.56–7.01
(m, 61H, Ar and Cl3CCONHꢂ2), 5.97–5.81 (m, 2H,
–CH]CH2 and FmocNH), 5.43 [s, 1H, PhCH(O)2], 5.32
(d, 1H, J¼17.1 Hz, –CH]CH2), 5.25 (br d, 2H, J¼9.3 Hz,
–CH]CH2 and H-1b or H-1d), 5.12 (d, 1H, J¼8.3 Hz,
H-1b or H-1d), 5.05 (d, 1H, J¼10.2 Hz, –CH2Ph), 5.02
(d, 1H, J¼10.7 Hz, –CH2Ph), 4.94 (d, 1H, J¼11.7 Hz,
–CH2Ph), 4.36 (d, 1H, J¼8.3 Hz, H-1c or H-1e), 2.99 (br s,
1H, H-4e). 13C NMR: d 169.5 (–CO2), 162.0 (Cl3CCONH),
161.6 (Cl3CCONH), 156.0 (OCONH), 119.2 (]CH2),
103.4 (1JCH¼162.2 Hz, C-1c or C-1e), 102.8 (1JCH
¼
159.7 Hz, C-1c or C-1e), 101.1 [PhCH(O)2], 100.7 (1JCH
¼
166.3 Hz, C-1b or C-1d), 100.3 (1JCH¼168.8 Hz, C-1b or
C-1d), 99.2 (1JCH¼170.5 Hz, C-1a), 92.6 (–CCl3), 92.3
(–CCl3). MALDI TOF MS calcd for C132H134N6O29Cl6
m/z: 2476.73. Found: 2499.74 (M+Na+). Anal. Calcd for
C132H134N6O29Cl6$H2O: C, 63.44; H, 5.48; N, 3.36. Found:
C, 63.68; H, 5.54; N, 3.07.
4.1.10. N-(9-Fluorenylmethoxycarbonyl)-O-{2,3-di-O-
benzyl-4,6-O-benzylidene-b-D-galactopyranosyl-(1/4)-
3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-b-D-glu-
copyranosyl-(1/3)-[2,3,4,6-tetra-O-benzyl-b-D-galacto-
pyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-trichloro-
acetamido-b-D-glucopyranosyl-(1/6)]-2-azido-2-de-
oxy-a-D-galactopyranosyl}-L-serine allyl ester 19. A
mixture of Cp2ZrCl2 (89 mg, 0.30 mmol), AgClO4
Compound 23: [a]D +26.5 (c 0.7). Rf 0.56 (2:1 toluene/
1
EtOAc). H NMR: d 7.71 (d, 2H, J¼7.3 Hz, Ar), 7.57 (d,
1H, J¼7.8 Hz, Ar), 7.54 (d, 1H, J¼7.3 Hz, Ar), 7.51–6.97
(m, 88H, Ar and Cl3CCONHꢂ3), 5.95–5.76 (m, 2H,
–CH]CH2 and FmocNH), 5.43 [s, 2H, PhCH(O)2ꢂ2],
5.30 (d, 1H, J¼17.1 Hz, –CH]CH2), 5.25–5.09 (m, 5H,
–CH]CH2, GlcNTCA H-1ꢂ2, –CH2Phꢂ2), 5.04 (d, 1H,
J¼10.2 Hz, –CH2Ph), 4.92 (d, 1H, J¼11.2 Hz, –CH2Ph),
4.87 (d, 1H, J¼2.9 Hz, H-1a), 2.94 (br s, 1H, H-4e or
H-4g), 2.84 (br s, 1H, H-4e or H-4g). 13C NMR: d 169.6
(–CO2), 162.0 (Cl3CCONH), 161.8 (Cl3CCONH), 161.4
(Cl3CCONH), 156.0 (OCONH), 119.2 (]CH2), 103.1
(1JCH¼159.7 Hz), 102.8 (1JCH¼163.9 Hz), and 102.5
(1JCH¼159.7 Hz) (C-1c, C-1e, and C-1g), 101.3 [PhCH(O)2],
˚
(127 mg, 0.61 mmol), and dried molecular sieves 4 A pow-
der (700 mg) in anhydrous CH2Cl2 (5 ml) was stirred at
room temperature under Ar for 30 min and then cooled at
ꢀ40 ꢁC. To the stirred mixture was added a mixture of 3
(143 mg, 0.15 mmol) and 15 (218 mg, 0.14 mmol) in an-
hydrous CH2Cl2 (15 ml). Then the temperature was raised
to ꢀ15 ꢁC for 30 min and stirring was continued for further
1.5 h, before the reaction was quenched with aq NaHCO3.
The mixture was diluted with EtOAc and filtered through
Celite. The filtrate was successively washed with satd
NaHCO3, water, and brine, dried over MgSO4, and concen-
trated in vacuo. The residue was chromatographed on silica
gel with toluene/EtOAc (4:1 to 3:1) to give 19 (233 mg,
68%). Heptasaccharide 23 (20 mg, 4%) and isomer 21
(20 mg, 6%) were obtained in the less polar and the more po-
lar fractions, respectively. Unconsumed 15 (13 mg, 6%) was
also recovered. Compound 19: [a]D +28.5 (c 1). Rf 0.71 (3:2
toluene/EtOAc). 1H NMR (CDCl3): d 7.71 (d, 1H, J¼6.6 Hz,
Ar), 7.70 (d, 1H, J¼6.8 Hz, Ar), 7.61 (d, 1H, J¼7.6 Hz, Ar),
7.55 (d, 1H, J¼7.3 Hz, Ar), 7.54–7.09 (m, 61H, Ar and
Cl3CCONHꢂ2), 5.90 (m, 1H, –CH2CH]CH2), 5.80 (d,
1H, J¼7.3 Hz, FmocNH), 5.46 [s, 1H, PhCH(O)2], 5.31 (d,
1H, J¼16.6 Hz, –CH]CH2), 5.22 (br d, 2H, J¼10.3 Hz,
–CH]CH2, –CH2Ph), 5.13 (d, 1H, J¼7.6 Hz, H-1b), 5.01
(d, 1H, J¼10.5 Hz, –CH2Ph), 4.97 (d, 1H, J¼11.5 Hz,
–CH2Ph), 4.95 (d, 1H, J¼7.8 Hz, H-1d), 4.80 (d, 1H,
J¼5.1 Hz, H-1a), 4.84 (d, 1H, J¼11.5 Hz, –CH2Ph), 2.98
(br s, 1H, –OH), 2.95 (s, 1H, H-4e). 13C NMR: d 169.0
(–CO2), 161.8 (Cl3CCONH), 161.4 (Cl3CCONH), 155.5
101.26 [PhCH(O)2], 100.9 (1JCH¼158.9 Hz), 99.8 (1JCH
¼
162.2 Hz), and 99.4 (1JCH¼164.7 Hz) (C-1b, C-d, and
C-1f), 98.0 (1JCH¼170.5 Hz, C-1a), 92.7 (–CCl3), 92.6
(–CCl3), 92.3 (–CCl3). MALDI TOF MS calcd for
C182H184N7O39Cl9 m/z: 3391.97. Found: 3431.09 (M+K+).
Anal. Calcd for C181H182N7O39Cl9$2H2O: C, 63.30; H,
5.46; N, 2.85. Found: C, 63.21; H, 5.40; N, 2.62.
4.1.11. N-(9-Fluorenylmethoxycarbonyl)-O-{2,3-di-O-
benzyl-4,6-O-benzylidene-b-D-galactopyranosyl-(1/4)-
3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-b-D-glu-
copyranosyl-(1/3)-[2,3,4,6-tetra-O-benzyl-b-D-galacto-
pyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-trichloro-
acetamido-b-D-glucopyranosyl-(1/6)]-2-azido-2-deoxy-
a-D-galactopyranosyl}-L-threonine allyl ester 20. In a sim-
ilar manner as described for 19, condensation of 3 (204 mg,
0.22 mmol) and 16 (312 mg, 0.20 mmol) was performed
with Cp2ZrCl2 (127 mg, 0.43 mmol), AgClO4 (181 mg,
˚
0.87 mmol), and dried molecular sieves 4 A powder (1 g)
(OCONH), 131.1 (–CH]), 118.9 (]CH2), 102.9 (1JCH
¼
in anhydrous CH2Cl2 (30 ml) at ꢀ40 to ꢀ15 ꢁC for 2 h.
160.1 Hz, C-1c or C-1e), 102.6 (1JCH¼160.9 Hz, C-1c or
Chromatography of the crude product on silica gel with

A. Ueki et al. / Tetrahedron 63 (2007) 2170–2181
2179
toluene/EtOAc (88:12 to 80:20) afforded 20 (351 mg,
71%), 22 (43 mg, 9%), 24 (55 mg, 8%), and unreacted 16
(13 mg, 4%).
1.27 (d, 3H, J¼7.3 Hz, Thr–gH). 13C NMR: d 169.9
(–CO2), 162.1 (Cl3CCONH), 161.6 (Cl3CCONH), 161.4
(Cl3CCONH), 156.7 (OCONH), 119.6 (]CH2), 103.0
(1JCH¼158.1 Hz), 102.8 (1JCH¼162.2 Hz), and 102.5
(1JCH¼160.6 Hz) (C-1c, C-1e, and C-1g), 101.3
[PhCH(O)2], 101.2 [PhCH(O)2], 100.8 (1JCH¼163.9 Hz),
100.2 (1JCH¼164.7 Hz), and 99.4 (1JCH¼165.5 Hz) (C-1b,
C-1d, and C-1f), 99.0 (1JCH¼172.1 Hz, C-1a), 92.6
(–CCl3), 92.6 (–CCl3), 92.3 (–CCl3), 18.6 (Thr–gC). MALDI
TOF MS calcd for C182H184N7O39Cl9 m/z: 3405.98. Found:
3444.23 (M+K+). Anal. Calcd for C182H184N7O39Cl9: C,
64.06; H, 5.44; N, 2.87. Found: C, 63.97; H, 5.40; N, 2.70.
Compound 20: [a]D +21.7 (c 1). Rf 0.54 (2:1 toluene/
1
EtOAc). H NMR: d 7.74 (d, 2H, J¼7.3 Hz, Ar), 7.60 (t,
2H, J¼7.3 Hz, Ar), 7.60–7.05 (m, 60H, Ar and Cl3CCONH),
6.95 (d, 1H, J¼7.3 Hz, Cl3CCONH), 5.90 (m, 1H,
–CH2CH]CH2), 5.59 (d, 1H, J¼9.5 Hz, FmocNH), 5.45
[s, 1H, PhCH(O)2], 5.33 (d, 1H, J¼17.1 Hz, –CH]CH2),
5.24 (d, 1H, J¼10.3 Hz, –CH]CH2), 5.23 (d, 1H, J¼
7.6 Hz, H-1b), 5.19 (d, 1H, J¼10.5 Hz, –CH2Ph), 5.00 (d,
1H, J¼10.5 Hz, –CH2Ph), 4.96 (d, 1H, J¼11.5 Hz,
–CH2Ph), 4.88 (d, 1H, J¼8.8 Hz, H-1d), 4.87 (d, 1H,
J¼4.1 Hz, H-1a), 4.84 (d, 1H, J¼11.5 Hz, –CH2Ph), 4.49–
4.28 (m, 10H, H-1c, H-1e, Thr–aH, ꢀCH2CHAr2, Thr–
bH, and –CH2Phꢂ4), 2.99 (br s, 1H, –OH), 2.90 (s, 1H,
H-4e), 1.29 (d, 3H, J¼6.3 Hz, Thr–gH). 13C NMR: d
169.6 (–CO2), 161.9 (Cl3CCONH), 161.3 (Cl3CCONH),
156.5 (OCONH), 131.1 (–CH¼), 119.1 (]CH2), 102.9
(1JCH¼160.1 Hz, C-1c or C-1e), 102.5 (1JCH¼160.1 Hz,
C-1c or C-1e), 101.2 [PhCH(O)2], 99.9 (1JCH¼165.9 Hz,
C-1d), 99.0 (1JCH¼172.5 Hz, C-1a), 98.6 (1JCH¼169.2 Hz,
C-1b), 92.4 (–CCl3), 92.1 (–CCl3). MALDI TOF MS calcd
for C133H136N6O29Cl6 m/z: 2490.75. Found: 2514.13
(M+Na+). Anal. Calcd for C133H136N6O29Cl6: C, 64.02; H,
5.49; N, 3.37. Found: C, 64.02; H, 5.32; N, 3.22.
4.1.12. N-(9-Fluorenylmethoxycarbonyl)-O-{2,3-di-O-
benzyl-4,6-O-benzylidene-b-D-galactopyranosyl-(1/4)-
2-acetamido-3,6-di-O-benzyl-2-deoxy-b-D-glucopyrano-
syl-(1/3)-[2,3,4,6-tetra-O-benzyl-b-D-galactopyrano-
syl-(1/4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-b-D-
glucopyranosyl-(1/6)]-2-acetamido-2-deoxy-a-D-galac-
topyranosyl}-L-serine allyl ester 25. A mixture of 19
(254 mg, 102.4 mmol), powdered Zn (1 g, 15.3 mmol), and
AcOH (1 ml, 17.5 mmol) in EtOAc (10 ml) was placed in
a round-bottom flask equipped with a reflux condenser.
The atmosphere was replaced with a balloon of Ar. The re-
action mixture was stirred under microwave irradiation at
150 W for 30 min. The microwave machine was controlled
so as to continuously irradiate the flask during this period.
The mixture was diluted with CHCl3 and filtered through
Celite. The filtrate was successively washed with satd
NaHCO3, water, and brine, dried over Na2SO4, and concen-
trated in vacuo. The residue was dissolved in a mixture of
CH2Cl2 (8 ml) and MeOH (2 ml), and stirred with Ac2O
(1 ml) at room temperature for 1 h. The mixture was concen-
trated in vacuo to the residue, which was chromatographed
on silica gel with toluene/EtOAc (1:4) and then with
CHCl3/MeOH (95:5) to afford 25 (200 mg, 85%). [a]D
+26.7 (c 1.1). Rf 0.59 (9:1 CHCl3/MeOH). 1H NMR
(DMSO-d6): d 7.85 (d, 3H, J¼7.8 Hz, Ar), 7.75 (d, 2H,
J¼8.3 Hz, Ar), 7.70–7.67 (m, 2H, Ar), 7.56–7.02 (m, 60H,
Ar and NHꢂ4), 5.87 (m, 1H, –CH2CH]CH2), 5.64 [s,
1H, PhCH(O)2], 5.29 (d, 1H, J¼17.3 Hz, –CH]CH2),
5.18 (d, 1H, J¼10.5 Hz, –CH]CH2), 5.09 (d, 1H,
J¼11.0 Hz, –CH2Ph), 4.88 (d, 1H, J¼10.7 Hz, –CH2Ph),
4.84 (d, 1H, J¼11.7 Hz, –CH2Ph), 1.83 (s, 3H, Ac), 1.77
(s, 6H, Acꢂ2). 13C NMR (CDCl3): d 170.5, 170.1, 170.0,
169.7 (CH3CONHꢂ3, –CO2 All), 155.8 (OCONH), 131.1
(–CH¼), 118.8 (]CH2), 102.8 (1JCH¼160.9 Hz, C-1c or
C-1e), 102.5 (1JCH¼165.1 Hz, C-1c or C-1e), 101.0
[PhCH(O)2], 100.3 (1JCH¼165.9 Hz, C-1b and C-1d), 97.8
(1JCH¼172.6 Hz, C-1a), 23.41, 23.36, 23.2 (CH3COꢂ3).
MALDI TOF MS calcd for C134H144N4O30 m/z: 2288.99.
Found: 2312.28 (M+Na+), 2328.31 (M+K+). Anal. Calcd
for C134H144N4O30$H2O: C, 69.71; H, 6.37; N, 2.43. Found:
C, 69.78; H, 6.41; N, 2.37.
Compound 22: [a]D +38.0 (c 1.8, CHCl3). Rf 0.21 (2:1 tolu-
1
ene/EtOAc). H NMR: d 7.75 (d, 2H, J¼7.3 Hz, Ar), 7.63
(d, 2H, J¼7.3 Hz, Ar), 7.57–7.00 (m, 61H, Ar and
Cl3CCONHꢂ2), 5.90 (ddd, 1H, J¼16.6, 10.7, 5.9 Hz,
–CH]CH2), 5.67 (d, 1H, J¼9.8 Hz, FmocNH), 5.43 [s,
1H, PhCH(O)2], 5.33 (d, 1H, J¼16.6 Hz, –CH]CH2),
5.30 (d, 1H, J¼7.8 Hz, H-1b or H-1d), 5.24 (d, 1H,
J¼10.2 Hz, –CH2Ph), 5.13 (d, 1H, J¼8.3 Hz, H-1b or
H-1d), 5.06 (d, 1H, J¼10.2 Hz, –CH2Ph), 5.00 (d, 1H,
J¼11.2 Hz, –CH2Ph), 4.93 (d, 1H, J¼11.7 Hz, –CH2Ph),
4.85 (d, 1H, J¼3.4 Hz, H-1a), 4.81 (d, 1H, J¼11.2 Hz,
–CH2Ph), 4.80 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.78 (d, 1H,
J¼11.2 Hz, –CH2Ph), 2.98 (br s, 1H, H-4e), 1.29 (d, 3H,
J¼6.3 Hz, Thr–gH). 13C NMR: d 170.0 (–CO2), 162.1
(Cl3CCONH), 161.6 (Cl3CCONH), 156.8 (OCONH),
119.4 (]CH2), 103.4 (1JCH¼158.9 Hz, C-1c or C-1e),
102.8 (1JCH¼158.9 Hz, C-1c or C-1e), 101.1 [PhCH(O)2],
100.5 (1JCH¼169.6 Hz, C-1b or C-1d), 100.4 (1JCH
¼
169.6 Hz, C-1b or C-1d), 99.1 (1JCH¼173.0 Hz, C-1a),
92.6 (–CCl3), 92.2 (–CCl3), 18.9 (Thr–gC). MALDI TOF
MS calcd for C133H136N6O29Cl6 m/z: 2490.75. Found:
2513.67 (M+Na+). Anal. Calcd for C133H136N6O29Cl6: C,
64.02; H, 5.49; N, 3.37. Found: C, 63.89; H, 5.58; N, 3.18.
Compound 24: [a]D +25.9 (c 1.0). Rf 0.64 (2:1 toluene/
1
EtOAc). H NMR: d 7.73 (d, 2H, J¼7.3 Hz, Ar), 7.61 (d,
1H, J¼7.3 Hz, Ar), 7.57 (d, 1H, J¼9.3 Hz, Ar), 7.55–6.99
(m, 87H, Ar and Cl3CCONHꢂ2), 6.97 (d, 1H, J¼8.3 Hz,
Cl3CCONH), 5.95–5.81 (m, 1H, –CH]CH2), 5.59 (d, 1H,
J¼9.8 Hz, FmocNH), 5.43 [s, 1H, PhCH(O)2], 5.42 [s, 1H,
PhCH(O)2], 5.31 (d, 1H, J¼16.6 Hz, –CH]CH2), 5.27–
5.08 (m, 5H, –CH]CH2, GlcNTCA H-1ꢂ2, –CH2Phꢂ2),
5.04 (d, 1H, J¼10.2 Hz, –CH2Ph), 4.92 (d, 1H, J¼11.2 Hz,
–CH2Ph), 4.87 (d, 1H, J¼2.9 Hz, H-1a), 2.96 (br s, 1H,
H-4c, H-4e, or H-4g), 2.80 (br s, 1H, H-4c, H-4e, or H-4g),
4.1.13. N-(9-Fluorenylmethoxycarbonyl)-O-{2,3-di-O-
benzyl-4,6-O-benzylidene-b-D-galactopyranosyl-(1/4)-
2-acetamido-3,6-di-O-benzyl-2-deoxy-b-D-glucopyrano-
syl-(1/3)-[2,3,4,6-tetra-O-benzyl-b-D-galactopyrano-
syl-(1/4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-b-D-
glucopyranosyl-(1/6)]-2-acetamido-2-deoxy-a-D-galac-
topyranosyl}-L-threonine allyl ester 26. Reduction of 20
(46 mg, 18.4 mmol) was performed with Zn (180 mg,

2180
A. Ueki et al. / Tetrahedron 63 (2007) 2170–2181
2.75 mmol) and AcOH (0.2 ml, 3.49 mmol) in EtOAc (2 ml)
under microwave irradiation for 30 min, according to the
procedure described above. The crude product was acety-
lated and purified by column chromatography. Toluene/
EtOAc (1:4) followed by CHCl3/MeOH (95:5) eluted 26
(33 mg, 80%). [a]D +29.5 (c 1). Rf 0.61 (9:1 CHCl3/
44.3 mmol) was deallylated according to the procedure de-
scribed for the synthesis of 1. Chromatography of the crude
product gave 2 (92 mg, 92%). [a]D +32.2 (c 1). Rf 0.48 (9:1
1
CHCl3/MeOH, 2% AcOH). H NMR (DMSO-d6): d 7.93–
7.77, 7.77–7.55, 7.48–7.03 (m, 67H, Ar and NHꢂ4), 5.64
[s, 1H, PhCH(O)2], 5.08 (d, 1H, J¼10.7 Hz, –CH2Ph), 4.88
(d, 1H, J¼10.7 Hz, –CH2Ph), 4.84 (d, 1H, J¼11.7 Hz,
–CH2Ph), 4.82–4.73 (m, 5H, H-1b, and –CH2Phꢂ4), 4.70
(d, 1H, J¼11.5 Hz, –CH2Ph), 4.69 (d, 3H, J¼12.7 Hz,
–CH2Phꢂ3), 4.63 (d, 1H, J¼12.5 Hz, –CH2Ph), 4.61 (d,
2H, J¼12.0 Hz, –CH2Phꢂ2), 1.91 (s, 3H, Ac), 1.794 and
1.790 (2s, 3Hꢂ2, Acꢂ2), 1.11 (d, 3H, J¼6.1 Hz, Thr–gH).
13C NMR (DMSO-d6): d 171.4 (–CO2H), 169.0, 168.8, and
1
MeOH). H NMR (DMSO-d6): d 7.91–7.05 (m, 67H, Ar
and NHꢂ4), 5.87 (m, 1H, –CH2CH]CH2), 5.64 [s, 1H,
PhCH(O)2], 5.33 (d, 1H, J¼16.8 Hz, –CH]CH2), 5.22 (d,
1H, J¼10.5 Hz, –CH]CH2), 4.89 (d, 1H, J¼12.9 Hz,
–CH2Ph), 4.85 (d, 1H, J¼11.7 Hz, –CH2Ph), 4.84 (d, 1H,
J¼11.0 Hz, –CH2Ph), 4.81–4.73 (m, 4H, H-1b and
–CH2Phꢂ3), 4.70 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.69 (d,
1H, J¼11.2 Hz, –CH2Phꢂ2), 1.89 (s, 3H, Ac), 1.81 (s,
3H, Ac), 1.80 (s, 3H, Ac), 1.13 (d, 3H, J¼4.9 Hz, Thr–
gH). 13C NMR (CDCl3): d 170.6, 170.34, 170.26, 169.8
(CH3CONHꢂ3, –CO2 All), 156.3 (OCONH), 130.9
(–CH]), 119.3 (]CH2), 103.0 (1JCH¼160.9 Hz, C-1c or
C-1e), 102.7 (1JCH¼165.1 Hz, C-1c or C-1e), 101.1
[PhCH(O)2], 100.5 (1JCH¼163.4 Hz, C-1b or C-1d), 100.3
(1JCH¼167.6 Hz, C-1b or C-1d), 99.0 (1JCH¼170.1 Hz,
C-1a), 23.6, 23.4 (CH3COꢂ3), 18.5 (Thr C-4). MALDI
TOF MS calcd for C135H146N4O30 m/z: 2303.00. Found:
2325.96 (M+Na+), 2341.92 (M+K+). Anal. Calcd for
C135H146N4O30$H2O: C, 69.81; H, 6.42; N, 2.43. Found:
C, 69.84; H, 6.16; N, 2.30.
168.4 (CH3CONHꢂ3), 156.3 (OCONH), 102.0 (1JCH
¼
161.8 Hz, C-1c and C-1e), 101.3 (1JCH¼157.6 Hz, C-1b
and C-1d), 99.7 [PhCH(O)2], 98.5 (1JCH¼168.4 Hz, C-1a),
23.03, and 22.97 (CH3COꢂ3), 18.8 (Thr C-4). MALDI
TOF MS calcd for C132H142N4O30 m/z: 2262.97. Found:
2285.75 (M+Na+), 2301.72 (M+K+). Anal. Calcd for
C131H140N4O30$H2O: C, 69.46; H, 6.36; N, 2.45. Found:
C, 69.22; H, 6.22; N, 2.35.
4.2. Synthesis of glycopeptide 27
Commercial Fmoc-CLEAR-amide resin (435 mg, 0.2 mmol)
was stirred with 20% piperidine/NMP (5 ml) in a polypropyl-
ene tube by a vortex mixer for 5 min. After filtration the resin
was againstirredwith20%piperidine/NMP(5 ml)for15 min
to complete N-deprotection. Then the resin was repeatedly
washed with NMP to remove piperidine and reacted for 1 h
with Fmoc–Gly–SCH2CH2COOBt prepared from the car-
boxylic acid (154 mg, 0.4 mmol) by activation with 1 M
DCC in NMP (0.4 ml, 0.4 mmol) and 1 M HOBt in NMP
(0.4 ml, 0.4 mmol). The Fmoc group was removed by stirring
with a mixture of 1-methylpyrrolidine (25%), hexamethyl-
eneimine (2%), and 1-hydroxybenzotriazole (4.8%) in NMP/
DMSO (1:1, 3 ml) for 2 min and again with the same amount
of the mixture for 15 min. After washing the resin with NMP,
Tsoc–Lys(Z)–OPfp (259 mg, 0.4 mmol) in THF (2.5 ml) was
reactedwithitfor30 min. Thecouplingreactionwasrepeated
with the same amount of Tsoc–Lys(Z)–OPfp. After washing
with THF and dichloromethane, Fmoc–Leu–F (142 mg,
0.4 mmol) dissolved in CH2Cl2 (2.5 ml) was added to the
resin. Then 1 M Bu4NF in THF (20 ml, 0.02 mmol)was added
and the resin was stirred for 1 h before washing with THF and
NMP. The resulting tripeptide-resin was subjected to the au-
tomated synthesis by ABI 433A peptide synthesizer using
FastMoc protocol. The Fmoc deprotection protocol was mod-
ified so that the premixed reagent was introduced to the reac-
tion vessel without dilution. The deprotection time was 5 and
20 min. After the synthesis of tetradecapeptide (Asp45-Gly58)
was completed, an eighth part of the resin placed in a propyl-
ene tube was stirred with the Fmoc deblocking reagent (1 ml)
for 5 min and then for 20 min as already mentioned. The resin
was washed withNMPandreactedwith2 (113 mg, 50 mmol),
1 M DCC in NMP (100 ml, 100 mmol), and 1 M HOBt in
NMP (100 ml, 100 mmol) in NMP (75 ml) at 50 ꢁC for 3 h
by using a vortex mixer. The remaining sequence was also
manually introduced using Fmoc amino acid (100 mmol),
DCC (150 mmol), and HOBt (150 mmol) in NMP to give gly-
copeptide (Gly34-Gly58)-resin (108 mg). The experiment for
deprotection was performed with the resin (27 mg), which
was treated with the Fmoc deblocking reagent as mentioned
4.1.14. N-(9-Fluorenylmethoxycarbonyl)-O-{2,3-di-O-
benzyl-4,6-O-benzylidene-b-D-galactopyranosyl-(1/4)-
2-acetamido-3,6-di-O-benzyl-2-deoxy-b-D-glucopyrano-
syl-(1/3)-[2,3,4,6-tetra-O-benzyl-b-D-galactopyrano-
syl-(1/4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-b-
D-glucopyranosyl-(1/6)]-2-acetamido-2-deoxy-a-D-
galactopyranosyl}-L-serine 1. A mixture of 25 (49 mg,
0.02 mmol), 5,5-dimethyl-1,3-cyclohexanedione (60 mg,
0.43 mmol), and Pd(PPh3)4 (3 mg, 2.6 mmol) in freshly dis-
tilled THF (5 ml) was stirred under Ar at room temperature
for 30 min, before concentrated in vacuo. The residue was
chromatographed on silica gel with CHCl3/MeOH (95:5)
to elute less polar 5,5-dimethyl-1,3-cyclohexanedione and
the byproducts. Then addition of AcOH (2%) to the eluant
afforded 1 (44 mg, 93%). [a]D +31.1 (c 1). Rf 0.46 (9:1
1
CHCl3/MeOH, 2% AcOH). H NMR (DMSO-d6): d 7.85
(d, 2H, J¼7.6 Hz, Ar), 7.80–7.03 (m, 65H, Ar and
NHꢂ4), 5.63 [s, 1H, PhCH(O)2], 5.09 (d, 1H, J¼10.7 Hz,
–CH2Ph), 4.87 (d, 1H, J¼11.0 Hz, –CH2Ph), 4.84 (d, 1H,
J¼11.7 Hz, –CH2Ph), 1.85 (s, 3H, Ac), 1.79 (s, 3H, Ac),
1.77 (s, 3H, Ac). 13C NMR (DMSO-d6): d 171.5 (–CO2H),
169.0, 168.8, 168.7 (CH3CONHꢂ3), 155.8 (OCONH),
102.0 (C-1c and C-1e), 101.8, 101.4 (C-1b and C-1d), 99.7
[PhCH(O)2], 97.7 (C-1a), 23.0 (CH3COꢂ3). MALDI TOF
MS calcd for C131H140N4O30 m/z: 2248.96. Found:
2271.87 (M+Na+), 2287.85 (M+K+). Anal. Calcd for
C131H140N4O30$H2O: C, 69.36; H, 6.31; N, 2.47. Found:
C, 69.34; H, 6.33; N, 2.47.
4.1.15. N-(9-Fluorenylmethoxycarbonyl)-O-{2,3-di-O-
benzyl-4,6-O-benzylidene-b-D-galactopyranosyl-(1/4)-
2-acetamido-3,6-di-O-benzyl-2-deoxy-b-D-glucopyrano-
syl-(1/3)-[2,3,4,6-tetra-O-benzyl-b-D-galactopyrano-
syl-(1/4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-b-D-
glucopyranosyl-(1/6)]-2-acetamido-2-deoxy-a-D-galac-
topyranosyl}-L-threonine 2. Compound 26 (102 mg,

A. Ueki et al. / Tetrahedron 63 (2007) 2170–2181
2181
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above. The resulting N-deprotected glycopeptide-resin was
stirred with reagent K (TFA/phenol/water/thioanisole/
ethanedithiol, 33:2:2:2:1, 500 ml) by the vortex mixer at
room temperature for 2 h. The volatile materials in the mix-
ture were evaporated in a stream of N2. Ether was added to
the residue to precipitate the product, which was separated
by centrifugation. The precipitate was washed several times
by suspending in ether and then centrifuging to give a crude
product, which was dissolved in a mixture of TFA/DMS/
m-cresol (5:3:1, 180 ml) and cooled at ꢀ15 ꢁC. TfOH
(20 ml) was added to the mixture and the plastic vessel was
shaken in the cooling bath. The reaction mixture was left at
ꢀ15 ꢁC for 2 h, before the reaction was terminated by the ad-
dition of ether. The mixture was centrifuged to precipitate the
debenzylated product, which was washed three times with
ether as mentioned above. The crude product was purified
by HPLC to give 27 (0.14 mmol, 2.2% overall yield based
on the amino group on the initial resin). MALDI TOF MS
calcd for C158H262N38O63S2 m/z: 3763.79. Found: 3764.95
(M+H+). Amino acid analysis: Asp1.00Thr3.26Ser2.42Glu0.94
-
Gly3Ala1.14Val1.36Ile0.94Leu3.74Lys2.01His0.97Arg1.25
.
Acknowledgements
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This work was supported by grant-in-aid for creative scien-
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tion of Science and in part by NEDO (New Energy and
Industrial Technology Development Organization, Japan).
We thank Dr. T. Chihara and his staff at Riken for the com-
bustion analyses. We also thank Tokai University for support
by grant-in-aid for high-technology research.
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