Solid-phase synthesis of O-sulfated glycopeptide by the benzyl-protected glycan strategy

Article abstract of DOI:10.1016/j.tet.2009.07.080

To expand the repertoire of our benzyl-protection strategy for solid-phase glycopeptide synthesis, an O-sulfated glycopeptide was chosen as the synthetic target. Trisaccharyl serine derivatives (Galβ1-4-GlcNAcβ1-2-Manα1-3-Ser) carrying (4-methoxyphenyl)me

Full text of DOI:10.1016/j.tet.2009.07.080

Tetrahedron 65 (2009) 8143–8153
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Solid-phase synthesis of O-sulfated glycopeptide by the benzyl-protected
glycan strategy
*
*
Keita Kawahira, Hiromasa Tanaka, Akiharu Ueki, Yuko Nakahara, Hironobu Hojo , Yoshiaki Nakahara
Department of Applied Biochemistry, Institute of Glycoscience, Tokai University, Kitakaname 1117, Hiratsuka, Kanagawa 259-1292, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 July 2009
Received in revised form 27 July 2009
Accepted 28 July 2009
Available online 6 August 2009
To expand the repertoire of our benzyl-protection strategy for solid-phase glycopeptide synthesis, an
O-sulfated glycopeptide was chosen as the synthetic target. Trisaccharyl serine derivatives (Gal 1–4-
GlcNAc 1–2-Man 1–3-Ser) carrying (4-methoxyphenyl)methyl (MPM) groups at either 3-O or 6-O of the
Gal residue were prepared through three stereoselective glycosylations. Cleavage of MPM followed by
reaction with Me3N$SO3 efficiently afforded 3-O- and 6-O-sulfo-glycoserines, respectively. A preliminary
debenzylation study using the sulfated glycoserines revealed that the sulfate groups persisted under
‘low-acidity TfOH’ conditions, when using a limited amount of TfOH and extending the reaction period.
b
b
a
The 3-O-sulfo-glycoserine was then introduced into an icosapeptide modeled after an a-dystroglycan
fragment by a combination of automated and manual solid-phase peptide synthesis procedures. The
synthesized glycopeptide was successfully debenzylated by the low-acidity TfOH cocktail with slight
damage to the sulfate functionality.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
the glycopeptides. In 1997, we first found that benzyl groups in the
oligosaccharide of glycopeptides were efficiently removed after
Recent advances in methodology and technology have enabled
the synthesis of complex glycopeptides up to the size of glycopro-
teins. Synthetic samples obtained in a single glycoform are neces-
sary tools to investigate the oligosaccharide-mediated biological
functions of glycoprotein. Widely used approaches to synthetic
glycopeptides employ preformed oligosaccharyl amino acids as
a building block in Fmoc-based solid-phase peptide synthesis
(SPPS). Although on-resin peptide assembly can be performed
without masking the hydroxyl groups in the oligosaccharyl moi-
ety,1 the hydroxyl groups are usually protected to prevent un-
desired O-aminoacylation. O-Acetylated oligosaccharides have
often been used in glycopeptides synthesis. The acetyl groups are
removed at the final stage of synthesis with a base, which some-
times gives rise to the concern about epimerization at the peptide
SPPS under acidic conditions with minimum scission of glycosidic
linkages.2 Since then, we have established syntheses of several
complex glycopeptides carrying N- and O-glycans by a combination
of the benzyl-protection strategy and Fmoc SPPS method.3 Re-
markable advantage of the strategy has been demonstrated by the
syntheses of the labile glycopeptide thioesters, a key intermediate
for peptide chemical ligation.
O-Sulfation is found in the outer chain of the complex-type
N-glycans, exemplified by sialyl-6-sulfoLex[Sia
a
2-3 Gal 1–4(6-sulfo)-
b
(Fuca1–3)GlcNAc] and HNK-1 [(3-sulfo)-GlcA
b1–3Galb1–4GlcNAc]
epitopes. On the other hand, sulfate groups are often functionally
backbone and b-elimination of the O-linked oligosaccharide at Ser
and Thr residues. Therefore, careful pH control of the basic condi-
tions is necessary. On the other hand, O-benzyl protection provides
reactive intermediates in oligosaccharide synthesis, and the benzyl
groups can be removed by hydrogenolysis under essentially neutral
conditions. However, the catalytic hydrogenolysis cannot be ap-
plied, when sulfur-containing amino acid residues are involved in
* Corresponding authors. Tel.: þ81 463 50 2075.
E-mail addresses: hojo@keyaki.cc.u-tokai.ac.jp (H. Hojo), yonak@keyaki.
cc.u-tokai.ac.jp (Y. Nakahara).
Figure 1. Structure of sulfated glycopeptide models.
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.07.080

8144
K. Kawahira et al. / Tetrahedron 65 (2009) 8143–8153
equivalent to the sialic acid residue in oligosaccharide, as reported
for 3-sulfoLex.4
procedure.9 Propionitrile-mediated glycosylation of 6 with 3 gave
-glycoside 7 in 71% yield. The stereoisomer 8 was produced as
b
In continuation of our research on the solid-phase synthesis of
complex glycopeptides, we have been particularly interested in
observing whether our benzyl-protection strategy is compatible
with preparation of sulfated glycopeptides. In addition, little re-
search has been conducted so far on solid-phase synthesis using
sulfated glycoamino acid.5
a minor product (13%). The structure of the disaccharide was
readily assignable by the 1H NMR spectrum. The reaction of 5 and 6
also predominated in the formation of
b-glycoside 9 (71%,
b
/
a
¼83:17). The
b
-disaccharides were desilylated to hemiacetals 11
(97%) and 14 (quant), respectively, by treatment with tetra-
n-butylammonium fluoride in the presence of AcOH, and then
converted to glycosyl donors to condense with phenyl 3,4,6-tri-O-
In this paper, we describe our approach to a synthetic sulfo-gly-
copeptide, which consists of (1) stereoselective synthesis of the
benzyl-1-thio-a-D
-mannnopyranoside 16.10 Glycosylation of 16
benzyl-protected 3-sulfo-Gal
b
1–4GlcNAc
b
1–2Man-Ser and 6-sulfo-
with glycosyl fluoride 12 derived from 11 was performed by acti-
vation with Cp2ZrCl2–AgClO4 in the standard condition11 to pro-
duce the desired trisaccharide 17 in a moderate yield (44%). In
contrast, the corresponding N-phenyltrifluoroacetimidate 13, pre-
pared by reaction with N-phenyltrifluoroacetimidoyl chloride and
K2CO3,12,13 reacted with 16 by a catalytic amount of acidic promoter,
TMSOTf, to give 17 in a 91% yield. The stereochemistry of the newly
Gal 1–4GlcNAc 1–2Man-Ser, as a sulfated model of O-mannosyl
b
b
glycans, and (2) solid-phase synthesis and debenzylation of the
sulfated glycopeptide. The corresponding sialyl oligosaccharide
[Sia
a
2–3Gal
b
1–4GlcNAc
b1–2Mana1–3-Ser/Thr] is known as a major
oligosaccharide of
a
-dystroglycan,6 whereas the trisaccharide struc-
ture is shared by the outer chain of complex-type N-glycans (Fig. 1).
formed glycosidic linkage was determined to be
at
5.15 ppm (J¼8.3 Hz). Similarly, imidate 15 derived from 14
produced trisaccharide 18 (81%,
¼97:3).
b
by the 1H signal
d
2. Synthesis of sulfated glycoserine building blocks
b/a
Before condensing the amino acid moiety, the tri-
chloroacetamido group was dechlorinated under microwave-
assisted conditions of Zn–AcOH in EtOAc3i to trisaccharides 19
(84%) and 20 (95%). Preparation of the trisaccharyl amino acid was
first attempted by glycosylation of N-Fmoc serine phenacyl ester
2514 with thioglycoside 19 in the presence of NIS–TfOH in CH2Cl2
at ꢀ20 to 0 ꢁC for 4.5 h. However, the product was obtained as
Our study began with preparation of the disaccharide pre-
cursors for an N-acetyllactosamine moiety based on the b-selective
galactosylation method recently reported by this laboratory.7
Phenyl 2,4,6-tri-O-benzyl-3-O-(4-methoxyphenyl)methyl-1-thio-
b-D
-galactopyranoside 38 and phenyl 2,3,4-tri-O-benzyl-6-O-
(4-methoxyphenyl)methyl-1-thio-
chosen as the glycosyl donors and reacted with tert-butyldiphe-
b-D-galactopyranoside 5 were
a mixture of
a
/
b
-isomers (70%,
a
/
b
¼3:2). Although other thio-
nylsilyl 3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-b-D-gluco-
pyranoside 6.7 The 2-trichloroacetamido sugar was preferred
because of its ready conversion to GlcNAc by Zn reduction and high
stereo-controlling ability in glycosylation.3e Compound 5 was
obtained by (4-methoxyphenyl)methylation of phenyl 2,3,4-tri-O-
philic promoters were not tested, thioglycoside 19 was trans-
formed to other glycosyl donors such as trichloroacetimidate,
N-phenyltrifluoroacetimidate, and fluoride via hemiacetal 21.
Among the glycosyl donors, fluoride 22 demonstrated the highest
a
-stereoselectivity (
a
/
b
¼9:1) affording the desired 26 in 80% yield,
benzyl-1-thio-b-D-galactopyranoside 4 prepared by the reported
Scheme 1. Synthesis of trisaccharides.

K. Kawahira et al. / Tetrahedron 65 (2009) 8143–8153
8145
Scheme 2. Synthesis of sulfated glycoserine building blocks 32 and 33. Structure of model debenzylation products 34 and 35.
Figure 2. HPLC profiles of debenzylation products: (a) product 34 and the byproducts, (b) product 35 and the byproducts.

8146
K. Kawahira et al. / Tetrahedron 65 (2009) 8143–8153
when the reaction was performed in the presence of a large
amount of molecular sieves, which might serve as a proton
scavenger retaining the acid-labile (4-methoxyphenyl)methyl
group. Reaction with either trichloroacetimidate or N-phenyl-
trifluoroacetimidate gave a low yield of 26. Thus, 6-MPM isomer
20 was converted to fluoride 24, which was condensed with 25 to
acidity TfOH-promoted debenzylation reaction. The crucial reaction
wasperformedbytheuseofTfOH(TfOH/benzylgroup¼1.7) at ꢀ15 ꢁC
for 7 h, whereas a large excess of TfOH resulted in considerable
scission of the sulfate group as examined with model compound 28.
An HPLC profile of the crude product is shown in Figure 3. The
major product, negative mass spectrum of which showed the desired
deprotonatedmolecularion(m/z2642.0)for40[MꢀH]ꢀ, wasisolated
by preparative HPLC, and the yield (17%) was determined by amino
acid analysis as reported earlier. From the two minor peaks, 2 and 3,
elutedafter40, massspectracorrespondingtotheanalogslackingSO3
and LacNAc groups, respectively, were obtained. Since such LacNAc-
missingproducts were not detected inthe debenzylationof34 and 35
practiced under similar conditions, the acidic and higher-tempera-
ture conditions used in the reagent K process might also be re-
sponsible for the undesired cleavage of the GlcNAc–Man linkage.
Further studies will be required to suppress this side reaction.
give 29 (75%) and the stereoisomer (
a
/b
¼15:1).
Having prepared the necessary frameworks, sulfation was per-
formed after selective removal of the MPM groups from 26 and 29
with 90% CF3CO2H aq–CH2Cl2. Compound 27 was heated with ex-
cess Me3N$SO3 in dimethylformamide at 60 ꢁC for 1 h to produce
sulfated product 28 in 97% yield. Successful 3-O-sulfation was
evidenced by the lower-field shift of Gal H-3 and C-3 signals in the
NMR spectra (for Gal H-3 at around
d 4.4 ppm and for C-3 at
81.7 ppm) and by the mass spectral data. 6-O-Sulfo-trisaccharyl
serine 31 was also obtained through cleavage of MPM (30: 85%) and
sulfation (76%). Then, both phenacyl esters were converted to car-
boxylic acids 32 (90%) and 33 (82%) by Zn–AcOH reduction. Before
shifting to the SPPS study, we tested debenzylation of sulfated
oligosaccharides 28 and 31. When compound 28 was exposed to
our standard debenzylation conditions using an excess of the low-
acidity TfOH cocktail (TFA/Me2S/m-cresol/TfOH, 5:3:1:1) at ꢀ15 ꢁC
for 2 h, a substantial quantity of sulfate-group-lacking product was
generated, along with the desired debenzylated glycoserine.
Therefore, we needed to tune the conditions appropriately. In
general, an elevated temperature accelerated scission of GlcNAc–
Man linkages in addition to departure of the sulfate group, whereas
a lowered temperature gave rise to incomplete debenzylation. The
optimum result was obtained by using a limited amount of TfOH
(TfOH/benzyl group¼1.5) in the TFA/Me2S/m-cresol cocktail and by
extending the reaction time (7 h)at ꢀ15 ꢁC (Schemes 1 and 2).
HPLC profiles of the products obtained from 28 and 31 under
these conditions are shown in Figure 2a and b, respectively. The
major products (peak 1) represent the desired 34 and 35,
respectively, while the following fractions (peak 2) show the mass
spectra of the sulfate-missing products (Fig. 2a: m/z 995.6, Fig. 2b:
m/z 995.3). In a less-mobile fraction of the crude mixture of 34
(Fig. 2a), an incompletely debenzylated product (m/z 1188.2) is
present (peak 3). Having confirmed the considerable stability of the
O-sulfate group in the conditions with TfOH, we next consi-
dered whether the synthesized sulfo-glycoserine acts as a suita-
ble building block in solid-phase synthesis of glycopeptide (Fig. 2).
Figure 3. HPLC profile of crude glycopeptide 40.
In summary, we have synthesized the benzyl- and MPM-pro-
tected glycoserine derivatives by three stereoselective glycosyla-
tion processes using thioglycoside, N-phenyltrifluoroacetimidate
and fluoride methods. Selective cleavage of MPM groups followed
by conventional sulfation produced 3-O-sulfo- and 6-O-sulfo-tri-
saccharyl serine derivatives. A preliminary study illustrated that
selective debenzylation of the sulfated glycoserines was feasible
under optimized low-acidity TfOH conditions. Sulfated glycoserine
32 was successfully introduced into the solid-phase synthesis of
model glycopeptide 40. Through optimized deprotection, the de-
sired 40 was obtained as the major product in a 17% overall yield.
This study also demonstrates that sulfated glycoamino acid build-
ing blocks can be utilized in the usual SPPS without masking the
sulfate functionality. This benzyl-protection strategy is promising
for preparation of a variety of N- and O-glycopeptide samples car-
rying a sulfate motif for obtaining insight into the biological im-
portance of O-sulfation (Scheme 3).
3. Solid-phase synthesis of glycopeptide
Solid-phase synthesis was investigated targeting a model glyco-
peptide (40), which represents a mucin-like icosapeptide domain
(336–355: SRIVPTPTSPAIAPPTETMA) of human
a
-dystroglycan6b
with a pendent 3-O-sulfo-trisaccharide instead of the native sialo-
tetrasaccharide. Starting with commercial Fmoc-CLEAR amide
resin, 11 amino acids were assembled using an automated peptide
synthesizer under the Fastmoc program, where N-deprotection was
performed with 20% piperidine/1-methyl-2-pyrrolidinone (NMP),
and O-benzotriazol-1-yl-N,N,N0,N0-tetramethyluronium hexa-
fluorophosphate (HBTU) and 1-hydroxybenzotriazole (HOBt) were
used to activate Fmoc amino acids in NMP. Part of the machine-
made peptide resin (37, 27 mmol) was subjected to manual con-
densation with 32 (3 equiv) in a plastic tube. The reaction was
carried out at an elevated temperature (50 ꢁC) with DCC/HOBt in
NMP by stirring with a vortex mixer for 7 h. Then resin 38 was again
transferred to the synthesizer, by which the remaining N-terminal
octapeptide was elongated. The resulting resin (39) was treated
with reagent K (CF3CO2H/H2O/thioanisole/1,2-ethanedithiol/phenol)
for 1 h at 0 ꢁC to room temperature to split the synthetic glyco-
peptide from the resin. The product, precipitated by an addition of
ether and separated by centrifugation, was subjected to the low-
4. Experimental
4.1. General
Specific rotation values were determined with a Jasco DIP-370
polarimeter at 20ꢂ2 ꢁC for solutions in CHCl3. Column

K. Kawahira et al. / Tetrahedron 65 (2009) 8143–8153
8147
toluene/EtOAc). 1H NMR
d
: 7.57–7.55 (m, 2H, Ar-H), 7.38–7.16 (m,
20H, Ar-H), 6.83 (br d, 2H, J¼8.3 Hz, Ar-H), 4.95 (d, 1H, J¼11.2 Hz,
ArCH2–), 4.77 (d, 1H, J¼10.2 Hz, ArCH2–), 4.72 (d, 2H, J¼11.2 Hz,
2ꢃArCH2–), 4.68 (d,1H, J¼11.2 Hz, ArCH2–), 4.63 (d,1H, J¼9.8 Hz, H-
1), 4.58 (d, 1H, J¼11.2 Hz, ArCH2–), 4.40 (d, 1H, J¼11.2 Hz, ArCH2–),
4.34 (d, 1H, J¼11.2 Hz, ArCH2–), 3.96 (d, 1H, J¼2.4 Hz, H-4), 3.93
(t, 1H, J¼9.3 Hz, H-2), 3.74 (s, 3H, CH3O–), 3.63–3.55 (m, 4H, H-3, H-
5, 2ꢃH-6). Anal. Calcd for C41H42O6S: C, 74.29; H, 6.39; S, 4.84.
Found: C, 74.27; H, 6.43; S, 4.85.
4.1.2. t-Butyldiphenylsilyl 2,4,6-tri-O-benzyl-3-O-(4-methoxyphenyl)-
methyl-
chloroacetamido-
2,4,6-tri-O-benzyl-3-O-(4-methoxyphenyl)methyl-
anosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-trichloroacetamido-
b
-
D
-galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-tri-
-glucopyranoside and t-butyldiphenylsilyl
-galactopyr-
-glu-
b-D
7
a-D
b-D
copyranoside 8. A mixture of 3 (1.0 g, 1.5 mmol), 6 (750 mg,
1.0 mmol), TTBP (511 mg, 2.0 mmol), BSP (328 mg, 1.5 mmol), and
dried MS 4 Å (4.0 g) in anhydrous EtCN (51 ml) was cooled at ꢀ78 ꢁC
with stirring for 30 min. To the mixture was slowly added Tf2O
(190
m
l, 1.1 mmol). The mixture was stirred for 30 min at ꢀ78 ꢁC
before the reaction was quenched by adding excess satd NaHCO3 aq.
The insoluble materials were filtered off through Celite and washed
with EtOAc. The combined filtrate and washings were washed
successively with water and brine, dried over anhydrous Na2SO4,
and concentrated in vacuo. The crude product was chromato-
graphed on Bio-beads S X1 in toluene, and then on silica gel with
toluene/EtOAc (19:1) to afford 7 (935 mg, 71%) and less polar 8
(165 mg, 13%).
4.1.2.1. Compound 7. [
a
]D¼ꢀ0.8 (c, 1.1). Rf 0.28 (19:1 toluene/
EtOAc).1H NMR
d: 7.68–7.62 (m, 4H, Ar-H), 7.39–7.10 (m, 33H, Ar-H),
6.90–6.82 (m, 3H, TCANH, Ar-H), 4.95 (d, 1H, J¼10.2 Hz, ArCH2–),
4.94 (d, 1H, J¼11.2 Hz, ArCH2–), 4.89 (d, 1H, J¼7.3 Hz, H-1a), 4.73 (d,
1H, J¼10.7 Hz, ArCH2–), 4.67 (d, 1H, J¼10.7 Hz, ArCH2–), 4.64 (d, 1H,
J¼11.7 Hz, ArCH2–), 4.61 (d, 1H, J¼11.3 Hz, ArCH2–), 4.54 (d, 1H,
J¼11.2 Hz, ArCH2–), 4.51 (d, 1H, J¼11.7 Hz, ArCH2–), 4.43 (d, 1H,
J¼7.8 Hz, H-1b), 4.38 (d, 1H, J¼12.2 Hz, ArCH2–), 4.32 (d, 1H,
J¼11.7 Hz, ArCH2–), 4.22 (br d, 2H, J¼11.7 Hz, ArCH2–), 4.04 (t, 1H,
J¼8.3 Hz, H-4a), 3.86 (d, 1H, J¼3.4 Hz, H-4b), 3.83–3.74 (m, 5H, H-
3a, H-2a, CH3O–), 3.68 (dd, 1H, J¼7.8, 9.8 Hz, H-2b), 3.65 (dd, 1H,
J¼3.4, 11.2 Hz, H-6a), 3.48 (br t, 1H, J¼8.0 Hz, H-6b), 3.41–2.29 (m,
Scheme 3. Solid-phase synthesis of sulfated glycopeptide 40.
chromatography 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)]. For solutions in
CDCl3 the parts per million downfield chemical shifts are
expressed from the internal Me4Si signal. For assignment of the
signals of sugar residue in oligosaccharides, the reducing terminal,
the second, and the third residues are described as a, b and c,
respectively. MALDI TOF mass spectra were obtained with a Per-
Septive Voyager-DE PRO spectrometer (2,5-dihydroxybenzoic acid
4H, H-5b, H-6a, H-6b, H-3b), 3.08 (m, 1H, H-5a), 1.06 (s, 9H, tBu). 13
C
NMR:
d
102.8 (1JCH¼160.0 Hz, C-1b), 94.9 (1JCH¼160.0 Hz, C-1a).
Anal. Calcd for C73H78Cl3NO12Si: C, 67.66; H, 6.07; N, 1.08. Found: C,
67.50; H, 6.21; N, 1.05.
4.1.2.2. Compound 8. [
a
]D¼þ12.0 (c, 1.0). Rf 0.35 (19:1 toluene/
EtOAc).1H NMR
d: 7.71–7.65 (m, 4H, Ar-H), 7.38–7.12 (m, 33H, Ar-H),
6.87–6.80 (m, 3H, TCANH–, Ar-H), 5.34 (d, 1H, J¼3.9 Hz, H-1b), 4.87
(d, 1H, J¼11.7 Hz, ArCH2–), 4.82 (d, 1H, J¼7.3 Hz, H-1a), 4.72 (d, 1H,
J¼10.2 Hz, ArCH2–), 4.65 (d, 1H, J¼12.2 Hz, ArCH2–), 4.57 (d, 1H,
J¼12.0 Hz, ArCH2–), 4.55 (br s, 2H, ArCH2–), 4.52 (d, 1H, J¼12.2 Hz,
ArCH2–), 4.51 (d, 1H, J¼11.7 Hz, ArCH2–), 4.35–4.31 (m, 3H,
3ꢃArCH2–), 4.27 (d, 1H, J¼11.7 Hz, ArCH2–), 4.12–4.07 (m 2H, H-4a,
H-2a), 3.93 (dd,1H, J¼3.4,10.2 Hz, H-2b), 3.91 (br s,1H, H-4b), 3.89–
3.83 (m, 2H, H-3a, H-5b), 3.77 (s, 3H, CH3O–), 3.72 (dd, 1H, J¼2.4,
10.2 Hz, H-3b), 3.62 (dd,1H, J¼3.9,10.7 Hz, H-6a), 3.50–3.45 (m, 2H,
H-6b, H-6a), 3.41 (dd, 1H, J¼5.9, 8.8 Hz, H-6b), 3.32 (m, 1H, H-5a),
was used as
a matrix). High-resolution mass spectra were
obtained with an AccuTOF (JMS-T100LC) spectrometer. Micro-
wave irradiation was carried out with a CEM Discover microwave
reactor.
4.1.1. Phenyl 2,3,4-tri-O-benzyl-6-O-(4-methoxyphenyl)methyl-1-thio-
-galactopyranoside 5. To a stirred mixture of 4 (1.89 g, 3.48 mmol)
b-
D
and 60% NaH/mineral oil (0.28 g, 6.96 mmol) in anhydrous DMF
(35 ml) was added (4-methoxyphenyl)methyl chloride (0.95 ml,
7.01 mmol) at 0 ꢁC. Then the mixture was stirred at room temper-
ature for 2.5 h, diluted with EtOAc, successively washed with water
and brine, dried over anhydrous Na2SO4, and concentrated in
vacuo. The crude product was chromatographed on silica gel with
toluene/EtOAc (49:1) to give 5 (1.94 g, 84%). Mp 91.5–92 ꢁC
1.08 (s, 9H, tBu). 13C NMR:
d
97.61 (1JCH¼173.8 Hz, C-1b), 95.02
(1JCH¼158.9 Hz, C-1a). Anal. Calcd for C73H78Cl3NO12Si: C, 67.66; H,
6.07; N, 1.08. Found: C, 67.38; H, 6.20; N, 1.08.
4.1.3. t-Butyldiphenylsilyl 2,3,4-tri-O-benzyl-6-O-(4-methoxyphenyl)-
methyl-
chloroacetamido-
2,3,4-tri-O-benzyl-6-O-(4-methoxyphenyl)methyl-a-D -
b
-
D
-galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-tri-
b-D
-glucopyranoside and t-butyldiphenylsilyl
9
(recrystallized from hexane/EtOAc). [
a
]D¼ꢀ0.5 (c, 1.1). Rf 0.23 (49:1

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K. Kawahira et al. / Tetrahedron 65 (2009) 8143–8153
galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-trichloro-
acetamido- -glucopyranoside 10. Reaction of (178 mg, 0.27
8.3 Hz, H-6b), 3.16 (br, 1H, –OH). Anal. Calcd for C57H60Cl3NO12: C,
64.74; H, 5.72; N, 1.32. Found: C, 64.64; H, 5.79; N, 1.32.
b-D
5
mmol) and 6 (100 mg, 0.13 mmol) was performed using the same
procedure as described for 7. Chromatographic purification of the
crude product on Bio-beads and then on silica gel gave 9 (125 mg,
71%) and less polar 10 (25 mg, 15%).
4.1.5. 2,4,6-Tri-O-benzyl-3-O-(4-methoxyphenyl)methyl-
actopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-
-glucopyranosyl fluoride 12. To a stirred solution of 11 (149 mg,
0.14 mmol) in anhydrous THF (5.5 ml) was added DAST (37 l,
0.28 mmol) at 0 ꢁC. Then the mixture was stirred at room tem-
perature for 30 min before adding CH3OH to quench excess reagent,
and evaporating the solvent in vacuo. The residue was extracted
with EtOAc, washed successively with water and brine, dried over
anhydrous Na2SO4, and concentrated in vacuo. The crude product
was chromatographed on silica gel with toluene/EtOAc (10:1) to
b-D-gal-
a-D
m
4.1.3.1. Compound 9. [
a
]D¼ꢀ4.3 (c, 1.0). Rf 0.42 (2:1 hexane/EtOAc).
Mp 113.0–114.0 ꢁC (recrystallized from hexane/EtOAc). 1H NMR
d:
7.70–7.63 (m, 4H, Ar-H), 7.40–7.10 (m, 33H, Ar-H), 6.90 (d, 1H,
J¼7.3 Hz, TCANH–), 6.84–6.80 (m, 2H, Ar-H), 4.96 (d, 1H, J¼10.7 Hz,
ArCH2–), 4.93 (d, 1H, J¼11.2 Hz, ArCH2–), 4.89 (d, 1H, J¼7.3 Hz, H-
1a), 4.73 (d, 1H, J¼11.2 Hz, ArCH2–), 4.69 (br s, 2H, ArCH2–), 4.67 (d,
1H, J¼10.2 Hz, ArCH2–), 4.54 (d, 1H, J¼10.2 Hz, ArCH2–), 4.50 (d, 1H,
J¼11.2 Hz, ArCH2–), 4.44 (d, 1H, J¼7.8 Hz, H-1b), 4.38 (d, 1H,
J¼12.2 Hz, ArCH2–), 4.27 (d, 1H, J¼11.7 Hz, ArCH2–), 4.22 (d, 1H,
J¼11.7 Hz, ArCH2–), 4.17 (d, 1H, J¼11.2 Hz, ArCH2–), 4.06 (t, 1H,
J¼8.3 Hz, H-4a), 3.89 (d, 1H, J¼2.9 Hz, H-4b), 3.83 (dd, 1H, J¼7.8,
9.2 Hz, H-3a), 3.76 (s, 3H, CH3O–), 3.75 (br, 1H, H-2a), 3.70 (dd, 1H,
J¼7.8, 9.7, H-2b), 3.65 (dd, 1H, J¼3.4, 11.2 Hz, H-6a), 3.47 (br t, 1H,
afford 12 (135 mg, 91%) as
a
/
b
mixture (
a
/
b
¼7.4:1). Rf 0.49 (9:1
toluene/EtOAc). 1H NMR
d
: 7.32–7.14 (m, 27H, Ar-H), 6.85 (d, 2H,
J¼8.8 Hz, Ar-H), 6.63 (d, 1H, J¼8.3 Hz, TCANH–), [6.30 (d, 0.14H,
J¼7.3 Hz, TCANH–)], 5.75 (dd, 1H, J¼2.4, 53.6 Hz, H-1a), 5.03 (d, 1H,
J¼10.7 Hz, ArCH2–), 4.95 (d, 1H, J¼11.7 Hz, ArCH2–), 4.83 (d, 1H,
J¼11.2 Hz, ArCH2–), 4.74 (d, 1H, J¼11.2 Hz, ArCH2–), 4.67–4.60 (m,
3H, 3ꢃArCH2–), 4.55 (d, 1H, J¼11.7 Hz, ArCH2–), 4.52 (d, 1H,
J¼10.2 Hz, ArCH2–), 4.38 (d, 1H, J¼12.1 Hz, ArCH2–), 4.37 (d, 1H,
J¼7.8 Hz, H-1b), 4.33 (d, 1H, J¼11.7 Hz, ArCH2–), 4.24 (d, 1H,
J¼12.1 Hz, ArCH2–), 4.17–4.02 (m, 2H, H-4a, H-2a), 3.92–3.69 (m,
5H, H-5a, H-4b, H-2b, H-3a, H-6a), 3.80 (s, 3H, CH3O–), 3.56 (dd, 1H,
J¼1.4, 11.2 Hz, H-6a), 3.48–3.32 (m, 4H, H-6b, H-3b, H-6b, H-5b).
Anal. Calcd for C57H59Cl3FNO11: C, 64.62; H, 5.61; N, 1.32. Found: C,
64.65; H, 5.62; N, 1.41.
J¼8.3 Hz, H-6b), 3.45–3.37 (m, 2H, H-3b, H-5b), 3.32–3.29 (m, 2H,
t
H-6a, H-6b), 3.08 (m, 1H, H-5a), 1.05 (s, 9H, Bu). 13C NMR:
d 102.8
(1JCH¼159.7 Hz, C-1b), 94.9 (1JCH¼164.7 Hz, C-1a). Anal. Calcd for
C73H78Cl3NO12Si: C, 67.66; H, 6.07; N, 1.08. Found: C, 67.42; H, 6.10;
N, 1.07.
4.1.3.2. Compound 10. [
a
]D¼þ14.5 (c, 1.0). Rf 0.52 (2:1 hexane/
EtOAc). 1H NMR
d
: 7.70–7.62 (m, 4H, Ar-H), 7.38–7.11 (m, 33H, Ar-
H), 6.83–6.78 (m, 3H, TCANH–, Ar-H), 5.33 (d, 1H, J¼3.4 Hz, H-1b),
4.84 (d, 1H, J¼11.7 Hz, ArCH2–), 4.78 (d, 1H, J¼7.3 Hz, H-1a), 4.71
(d, 1H, J¼10.7 Hz, ArCH2–), 4.63 (d, 1H, J¼11.7 Hz, ArCH2–), 4.62 (br
s, 2H, ArCH2–), 4.59 (d, 1H, J¼11.2 Hz, ArCH2–), 4.57 (d, 1H,
J¼10.7 Hz, ArCH2–), 4.51 (d, 1H, J¼11.7 Hz, ArCH2–), 4.49 (d, 1H,
J¼11.2 Hz, ArCH2–), 4.33 (d, 1H, J¼12.2 Hz, ArCH2–), 4.28 (d, 1H,
J¼12.2 Hz, ArCH2–), 4.27 (d, 1H, J¼11.2 Hz, ArCH2–), 4.20 (d, 1H,
J¼11.2 Hz, ArCH2–), 4.13–4.07 (m, 2H, H-2a, H-4a), 3.96–3.93 (m,
2H, H-2b, H-4b), 3.87–3.83 (m, 2H, H-3a, H-5b), 3.76 (s, 3H, CH3O–),
3.72 (dd, 1H, J¼2.9, 10.2 Hz, H-3b), 3.58 (m, 1H, H-6a), 3.48–3.43
(m, 2H, H-6b, H-6a), 3.37 (br dd, 1H, H-6b), 3.34 (m, 1H, H-5a), 1.07
4.1.6. 2,3,4-Tri-O-benzyl-3-O-(4-methoxyphenyl)methyl-
actopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-
-glucopyranosyl (N-phenyl)-2,2,2-trifluoroacetimidate 13. A mixture
of 11 (438 mg, 0.42 mmol), N-phenyltrifluoroacetimidoyl chloride
(172 mg, 0.84 mmol), and K2CO3 (115 mg, 0.84 mmol) in acetone
(4.2 ml) was stirred at room temperature for 30 min. The insoluble
material was filtered off and the filtrate was concentrated in vacuo.
The residue was chromatographed on silica gel with toluene/EtOAc
b-D-gal-
D
(15:1) to afford 13 (506 mg, 99%) as a mixture of
a
/
b
and/or syn/anti
isomers. Rf 0.56 (9:1 toluene/EtOAc). 1H NMR:
d
7.35–7.16 (m, 29H,
Ar-H), 7.08 (m, 1H, Ar-H), 6.89–6.79 (m, 4H, Ar-H), 6.61 and 6.31 (2d,
1H, J¼7.8 Hz, H-1a), 5.01 (d, 1H, J¼11.2 Hz, ArCH2–), 4.97 (d, 1H,
J¼11.2 Hz, ArCH2–), 4.86 (d, 1H, J¼11.2 Hz, ArCH2–), 4.77 (d, 1H,
J¼11.2 Hz, ArCH2–), 4.68–4.52 (m, 5H, 5ꢃArCH2–), 4.42–4.23 (m,
4H, H-1b, 3ꢃArCH2–), 4.16–4.09 (m, 2H, H-2a, H-4a), 3.89 (d, 1H,
J¼2.4 Hz, H-4b), 3.86–3.72 (m, 4H, H-2b, H-3a, H-5a, H-6a), 3.80 (s,
3H, CH3O–), 3.55–3.32 (m, 5H, H-6a, H-5b, H-3b, 2ꢃH-6b). Anal.
Calcd for C65H64Cl3F3N2O12: C, 63.55; H, 5.25; N, 2.28. Found: C,
63.50; H, 5.38; N, 2.23.
(s, 9H, tBu). 13C NMR:
d
97.55 (1JCH¼173.8 Hz, C-1b), 95.1
(1JCH¼158.9 Hz, C-1a). MALDI TOF MS: calcd for C73H78Cl3NO12Si:
m/z 1316.4 (MþNa)þ. Found: m/z 1317.1. Anal. Calcd for
C73H78Cl3NO12Si: C, 67.66; H, 6.07; N, 1.08. Found: C, 67.63; H, 6.11;
N, 1.13.
4.1.4. 2,4,6-Tri-O-benzyl-3-O-(4-methoxyphenyl)methyl-
actopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-
-glucopyranose 11. A solution of 1 M TBAF/THF (3.2 ml, 3.2 mmol)
b-D-gal-
a-D
was added to a cold mixture of 7 (1.0 g, 0.8 mmol) and AcOH
(0.46 ml, 8.0 mmol) in freshly distilled THF (14 ml) on an ice-water
bath with stirring. The mixture was stirred for 2 days at room tem-
perature, then diluted with EtOAc, washed successively with water
and brine, dried over anhydrous Na2SO4, and concentrated in vacuo.
The residue was chromatographed on silica gel with toluene/EtOAc
4.1.7. 2,3,4-Tri-O-benzyl-6-O-(4-methoxyphenyl)methyl-
actopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-
-glucopyranose 14. Compound (212 mg, 0.16 mmol) was
b-D-gal-
a-D
9
desilylated with TBAF/AcOH in THF in a similar manner as de-
scribed for 11. The crude product was purified by chromatography
on silica gel with toluene/EtOAc (5:1) to give 14 (170 mg, 99%) as an
(5:1) to give 11 (0.85 g, 99%) as an
a-anomer rich mixture. Rf 0.15 (9:1
a
d
-anomer rich mixture. Rf 0.14 (9:1 toluene/EtOAc). 1H NMR:
7.32–7.11 (m, 26H, Ar-H), 6.86–6.80 (m, 3H, TCANH–, Ar-H), 5.33
toluene/EtOAc). 1H NMR
d: 7.32–7.10 (m, 27H, Ar-H), 6.88–6.83 (m,
3H, TCANH–, Ar-H), 5.37 (br t, 1H, J¼3.5 Hz, H-1a), 5.02 (d, 1H,
J¼11.2 Hz, ArCH2–), 4.94 (d, 1H, J¼11.2 Hz, ArCH2–), 4.83 (d, 1H,
J¼11.2 Hz, ArCH2–), 4.78 (d, 1H, J¼11.2 Hz, ArCH2–), 4.65 (d, 1H,
J¼11.2 Hz, ArCH2–), 4.61 (d, 1H, J¼11.2 Hz, ArCH2–), 4.52 (d, 1H,
J¼11.7 Hz, ArCH2–), 4.51 (d, 1H, J¼12.2 Hz, ArCH2–), 4.35 (d, 1H,
J¼7.3 Hz, H-1b), 4.34 (d, 1H, J¼12.2 Hz, ArCH2–), 4.30 (d, 1H,
J¼11.7 Hz, ArCH2–), 4.20 (d, 1H, J¼11.7 Hz, ArCH2–), 4.10 (m, 1H, H-
2a), 4.01–3.97 (m, 2H, H-5a, H-4a), 3.87 (d,1H, J¼2.4 Hz, H-4b), 3.86–
3.71 (m, 3H, H-2b, H-3a, H-6a), 3.79 (s, 3H, CH3O–), 3.60 (br d, 1H,
J¼9.7 Hz, H-6a), 3.44–3.33 (m, 3H, H-3b, H-5b, H-6b), 3.28 (dd, J¼4.9,
(br t, 1H, J¼3.4 Hz, H-1a), 5.02 (d, 1H, J¼10.7 Hz, ArCH2–), 4.93 (d,
1H, J¼11.2 Hz, ArCH2–), 4.82 (d, 1H, J¼11.2 Hz, ArCH2–), 4.78 (d, 1H,
J¼11.2 Hz, ArCH2–), 4.71 (d, 1H, J¼12.2 Hz, ArCH2–), 4.67 (d, 1H,
J¼11.7 Hz, ArCH2–), 4.59 (d, 1H, J¼10.7 Hz, ArCH2–), 4.51 (d, 1H,
J¼11.2 Hz, ArCH2–), 4.48 (d, 1H, J¼12.1 Hz, ArCH2–), 4.32 (d, 1H,
J¼8.3 Hz, H-1b), 4.30 (d, 1H, J¼12.7 Hz, ArCH2–), 4.23 (d, 1H,
J¼11.7 Hz, ArCH2–), 4.17 (d, 1H, J¼12.7 Hz, ArCH2–), 4.13 (m, 1H, H-
2a), 4.04–3.93 (m, 2H, H-5a, H-4a), 3.88 (d, 1H, J¼2.4 Hz, H-4b),
3.82–3.70 (m, 3H, H-6a, H-3a, H-2b), 3.77 (s, 3H, CH3O–), 3.63–3.58

K. Kawahira et al. / Tetrahedron 65 (2009) 8143–8153
8149
(m, 2H, –OH, H-6a), 3.42–3.32 (m, 2H, H-3b, H-5b), 3.26 (dd, 1H,
J¼4.9, 8.0 Hz, H-6b). Anal. Calcd for C57H60Cl3NO12: C, 64.74; H,
5.72; N, 1.32. Found: C, 64.61; H, 5.77; N, 1.31.
J¼9.3 Hz, H-4b), 3.85 (br t, 1H, J¼9.3 Hz, H-4a), 3.80 (d, 1H,
J¼2.4 Hz, H-4c), 3.77 (dd, 1H, H-3a), 3.72 (s, 3H, CH3O–), 3.71–3.65
(m, 3H, H-2c, H-6a, H-6b), 3.61–3.56 (m, 2H, H-6a, H-6b), 3.48–
3.43 (m, 2H, H-5b, H-6c), 3.35–3.25 (m, 4H, H-2b, H-3c, H-5c, H-
6c). 13C NMR:
d
102.7 (1JCH¼162.2 Hz, C-1c), 97.0 (1JCH¼164.7 Hz, C-
4.1.8. 2,3,4-Tri-O-benzyl-6-O-(4-methoxyphenyl)methyl-
b
-
D
-galactopyr-
1b), 86.2 (1JCH¼168.0 Hz, C-1a). MALDI TOF MS: calcd for
C90H92Cl3NO16S: m/z 1602.5 (MþNa)þ, 1618.5 (MþK)þ. Found: m/z
1602.5 and 1618.4. Anal. Calcd for C90H92Cl3NO16S: C, 68.32; H,
5.86; N, 0.89; Cl, 6.72; S, 2.03. Found: C, 68.15; H, 5.92; N, 0.95; Cl,
6.75; S, 2.04.
anosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-
D-glucopyr-
anosyl (N-phenyl)-2,2,2-trifluoroacetimidate 15. Compound 14 (158 mg,
0.15 mmol) was reacted with N-phenyltrifluoroacetimidoyl chlo-
ride (62 mg, 0.30 mmol), and K2CO3 (41 mg, 0.30 mmol) in acetone
(1.5 ml) as described for 13. The crude product was chromato-
graphed on silica gel with toluene/EtOAc (15:1) to give 15 (176 mg,
96%). Rf 0.63 (9:1 toluene/EtOAc). 1H NMR:
d
7.35–7.06 (m, 30H, Ar-
4.1.10. Phenyl 2,3,4-tri-O-benzyl-6-O-(4-methoxyphenyl)methyl-
actopyranosyl-(1/4)-3, 6-di-O-benzyl-2-deoxy-2-tri-
chloroacetamido- -glucopyranosyl-(1/2)-3,4,6-tri-O-benzyl-1-
thio- -mannnopyranoside 18. Disaccharyl donor 15 (0.97 g,
b-D-gal-
H), 6.84–6.75 (m, 4H, Ar-H), 6.62 and 6.29 (2d, 1H, J¼7.8 Hz, H-1a),
5.01 (d,1H, J¼10.7 Hz, ArCH2–), 4.94 (d, 1H, J¼11.2 Hz, ArCH2–), 4.85
(d, 1H, J¼11.2 Hz, ArCH2–), 4.77 (d, 1H, J¼11.2 Hz, ArCH2–), 4.73 (d,
1H, J¼12.2 Hz, ArCH2–), 4.68 (d, 1H, J¼11.7 Hz, ArCH2–), 4.64 (d, 1H,
J¼11.2 Hz, ArCH2–), 4.54 (d, 1H, J¼11.7 Hz, ArCH2–), 4.52 (d, 1H,
J¼11.7 Hz, ArCH2–), 4.38 (d, 1H, J¼11.2 Hz, ArCH2–), 4.37 (d, 1H,
J¼8.3 Hz, H–1b), 4.27 (d, 1H, J¼11.2 Hz, ArCH2–), 4.19 (d, 1H,
J¼11.7 Hz, ArCH2–), 4.25–4.09 (m, 2H, H-2a, H-4a), 3.90 (d, 1H,
J¼2.9 Hz, H-4b), 3.89–3.76 (m, 4H, H-6a, H-3a, H-2b, H-5a), 3.78 (s,
3H, CH3O–), 3.55 (br d, 1H, J¼10.7 Hz, H-6a), 3.45 (m, 1H, H-6b),
3.39–3.31 (m, 3H, H-5b, H-3b, H-6b). Anal. Calcd for
C65H64Cl3F3N2O12: C, 63.55; H, 5.25; N, 2.28. Found: C, 63.80; H,
5.34; N, 2.16.
b-D
a-D
0.79 mmol) and acceptor 16 (0.47 g, 0.87 mmol) were reacted in the
presence of TMSOTf (7.2
to the procedure A described for 17. The crude product was purified
m
l, 0.04 mmol) at ꢀ40 ꢁC for 3 h according
by chromatography on Bio-beads S X1 in toluene, and then on silica
gel with toluene/EtOAc (9:1) to give 18 (1.10 g, 83%). [
a
]D¼þ31.9 (c,
1.1). Rf 0.40 (9:1 toluene/EtOAc). 1H NMR:
d
7.45–7.42 (m, 2H, Ar-H),
7.35–7.10 (m, 46H, TCANH–, Ar-H), 6.83–6.81 (m, 2H, Ar-H), 5.53 (d,
1H, J¼1.5 Hz, H-1a), 5.22 (d, 1H, J¼8.3 Hz, H-1b), 5.06 (d, 1H,
J¼10.2 Hz, ArCH2–), 4.96 (d, 1H, J¼11.2 Hz, ArCH2–), 4.82 (d, 1H,
J¼10.7 Hz, ArCH2–), 4.79 (br s, 2H, ArCH2–), 4.76 (d, 1H, J¼10.7 Hz,
ArCH2–), 4.71 (br s, 2H, ArCH2–), 4.56–4.39 (m, 10H, 7ꢃArCH2–, H-
1c, H-2a, H-3b), 4.33 (d, 1H, J¼11.2 Hz, ArCH2–), 4.27–4.22 (m, 2H,
ArCH2–, H-5a), 4.18 (d,1H, J¼11.2 Hz, ArCH2–), 4.02 (br t,1H, J¼9.3 Hz,
H-4b), 3.92 (br t, 1H, J¼9.2 Hz, H-4a), 3.89 (d, 1H, J¼2.0 Hz, H-4c),
3.86–3.82 (m, 2H, H-3a, H-6b), 3.77 (s, 3H, CH3O–), 3.79–3.63 (m, 3H,
H-2c,H-6b,H-6a), 3.68–3.63(m, 2H,H-6a, H-6b), 3.54–3.49(m, 2H,H-
4.1.9. Phenyl 2,4,6-tri-O-benzyl-3-O-(4-methoxyphenyl)methyl-
actopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-
-glucopyranosyl-(1/2)-3,4,6-tri-O-benzyl-1-thio- -mannnopyr-
anoside 17.
b-D-gal-
b-
D
a-D
5b, H-6c), 3.42–3.34 (m, 4H, H-5c, H-3c, H-6c, H-2b).13C NMR:
d 102.7
(1JCH¼161.4 Hz, C-1c), 97.0(1JCH¼164.7 Hz, C-1b),86.1(1JCH¼168.0 Hz,
C-1a). Anal. Calcd for C90H92Cl3NO16S: C, 68.32; H, 5.86; N, 0.89; Cl,
6.72; S, 2.03. Found: C, 68.10; H, 5.89; N, 0.86; Cl, 6.62; S, 2.05.
4.1.9.1. Procedure A (reaction of 13 and 16). To a stirred mixture of
13 (109 mg, 88
(3 ml) was added TMSOTf (0.8
m
mol) and 16 (48 mg, 88
mmol) in anhydrous CH2Cl2
ml, 4.4
m
mol) at ꢀ78 ꢁC. Then the
temperature was raised to ꢀ40 ꢁC. The mixture was stirred for 2 h
before adding satd NaHCO3 aq to quench the reaction, diluted with
EtOAc, and filtrated through Celite. The combined filtrate and
washings (EtOAc) were successively washed with water and brine,
dried over anhydrous Na2SO4, and concentrated in vacuo. The
residue was chromatographed on Bio-beads S X1 in toluene, and
then on silica gel with toluene/EtOAc (9:1) to give 17 (127 mg, 91%).
4.1.11. Phenyl 2,4,6-tri-O-benzyl-3-O-(4-methoxyphenyl)methyl-
b
-
D
D
-
-
galactopyranosyl-(1/4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-
b
-
glucopyranosyl-(1/2)-3,4,6-tri-O-benzyl-1-thio-a-D-mannnopyr-
anoside 19. A mixture of 17 (564 mg, 0.36 mmol), powdered Zn
(3.5 g, 53.6 mmol), and AcOH (3.5 ml, 61.3 mmol) in EtOAc (35 ml)
was placed in a round-bottom flask equipped with a reflux con-
denser. The atmosphere was replaced with a balloon of Ar. The
reaction 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 EtOAc and filtered through Celite. The combined
filtrate and washings (EtOAc) were successively washed with satd
NaHCO3, water, and brine, dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue was chromatographed on silica
gel with toluene/EtOAc (4:1) to afford 19 (465 mg, 88%).
4.1.9.2. Procedure B (reaction of 12 and 16). A mixture of Cp2ZrCl2
(35 mg, 0.12 mmol), AgClO4 (25 mg, 0.12 mmol), and dried mo-
lecular sieves 4 Å powder (500 mg) in anhydrous CH2Cl2 (2 ml)
was stirred at room temperature under Ar for 30 min and then
cooled at ꢀ20 ꢁC. To the stirred mixture was added a mixture of 12
(114 mg, 0.11 mmol) and 16 (72 mg, 0.13 mmol) in anhydrous
CH2Cl2 (3 ml). Then the stirring was continued for 30 min, before
the reaction was quenched with aq NaHCO3. The mixture was
diluted with EtOAc and filtered through Celite. The combined fil-
trate and washings (EtOAc) were successively washed with satd
NaHCO3, water, and brine, dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue was chromatographed on silica
[
a
]D¼þ32.1 (c, 1.0). Rf 0.11 (7:1 toluene/EtOAc). 1H NMR:
d 7.44–
7.15 (m, 47H, Ar-H), 6.86–6.83 (m, 2H, Ar-H), 5.61 (d, 1H, J¼6.8 Hz,
AcNH–), 5.57 (d, 1H, J¼1.5 Hz, H-1a), 5.20 (d, 1H, J¼7.8 Hz, H-1b),
4.96 (d, 1H, J¼11.2 Hz, ArCH2–), 4.96 (d, 1H, J¼11.2 Hz, ArCH2–),
4.89 (d, 1H, J¼10.7 Hz, ArCH2–), 4.80–4.69 (m, 3H, 3ꢃArCH2–), 4.62
(d, 1H, J¼12.2 Hz, ArCH2–), 4.58 (d, 1H, J¼12.2 Hz, ArCH2–), 4.55–
4.35 (m, 10H, 8ꢃArCH2–, H-1c, H-3b), 4.29 (d, 1H, J¼12.2 Hz,
ArCH2–), 4.25 (d, 1H, J¼12.2 Hz, ArCH2–), 4.16 (m, 1H, H-5a), 4.03
(br t, 1H, J¼9.3 Hz, H-4a), 3.90 (br t, 1H, J¼9.3 Hz, H-4b), 3.86–3.69
(m, 4H, H-4c, H-3a, H-6b, H-6a), 3.80 (s, 3H, CH3O–), 3.63 (dd, 1H,
J¼1.5, 10.5 Hz, H-6a), 3.58 (m, 1H, H-5b), 3.52 (br t, 1H, J¼7.8 Hz, H-
6c), 3.42–3.35 (m, 3H, H-3c, H-5c, H-6c), 3.06 (m, 1H, H-2b), 1.63
(s, 3H, Ac). MALDI TOF MS: calcd for C90H95NO16S: m/z 1500.6
(MþNa)þ, 1516.6 (MþK)þ. Found: m/z 1500.5 and 1516.4. Anal.
gel with toluene/EtOAc (95:5) to give 17 (75 mg, 44%). [
a
]D¼þ32.1
(c, 1.0). Rf 0.39 (9:1 toluene/EtOAc). 1H NMR:
d
7.37–7.35 (m, 2H,
Ar-H), 7.25–7.02 (m, 46H, TCANH–, Ar-H), 6.78–6.76 (m, 2H, Ar-H),
5.46 (d, 1H, J¼2.0 Hz, H-1a), 5.15 (d, 1H, J¼8.3 Hz, H-1b), 4.98 (d,
1H, J¼10.2 Hz, ArCH2–), 4.98 (d, 1H, J¼11.7 Hz, ArCH2–), 4.75 (d, 1H,
J¼10.7 Hz, ArCH2–), 4.73 (d, 1H, J¼11.7 Hz, ArCH2–), 4.71 (d, 1H,
J¼11.7 Hz, ArCH2–), 4.66 (d, 1H, J¼10.7 Hz, ArCH2–), 4.59 (d, 1H,
J¼12.2 Hz, ArCH2–), 4.56 (d, 1H, J¼11.7 Hz, ArCH2–), 4.49–4.29 (m,
11H, 8ꢃArCH2–, H-1c, H-2a, H-3b), 4.20 (d, 1H, J¼11.2 Hz, ArCH2–),
4.17 (d, 1H, J¼11.2 Hz, ArCH2–), 4.15 (m, 1H, H-5a), 3.95 (br t, 1H,

8150
K. Kawahira et al. / Tetrahedron 65 (2009) 8143–8153
Calcd for C90H95NO16S: C, 73.10; H, 6.48; N, 0.95. Found: C, 73.00;
H, 6.57; N, 0.93.
(m, 1H, H-5b), 3.52 (m, 1H, H-6c), 3.43–3.37 (m, 3H, H-3c, H-5c, H-6c),
3.02 (m, 1H, H-2b), 1.63 (s, 3H, Ac). 13C NMR:
106.0 (JC–F¼220.9 Hz,
d
C-1a) 102.9 (C-1c), 97.7 (C-1b). MALDI TOF MS: calcd for C84H90FNO16
:
4.1.12. Phenyl 2,3,4-tri-O-benzyl-6-O-(4-methoxyphenyl)methyl-
b
-
D
D
-
-
m/z 1410.6 (MþNa)þ, 1426.6 (MþK)þ. Found: m/z 1410.6 and 1426.6.
Anal. Calcd for C84H90FNO16$2H2O: C, 70.82; H, 6.65; N, 0.98. Found:
C, 70.72; H, 6.47; N, 1.12.
galactopyranosyl-(1/4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-
b
-
glucopyranosyl-(1/2)-3,4,6-tri-O-benzyl-1-thio-a-D-mannnopyr-
anoside 20. Compound 18 (101 mg, 0.06 mmol) was dechlorinated
with Zn (630 mg) and AcOH (0.63 ml) in EtOAc under microwave
irradiation at 150 W for 1 h as described for 19. The crude product
was purified by column chromatography on silica gel with tolu-
4.1.14.2. Compound 22
b
. [
a
]D¼ꢀ8.0 (c, 1.0). Rf 0.12 (2:3 hexane/
EtOAc). 1H NMR:
d
7.30–7.13 (m, 42H, Ar-H), 6.83 (br d, 2H, J¼8.3 Hz,
Ar-H), 5.49 (d, 1H, J¼7.8 Hz, AcNH–), 5.30 (br d, 1H, JH–F¼51.2 Hz, H-
1a), 4.98 (d, 1H, J¼7.8 Hz, H-1b), 4.95 (d, 1H, J¼11.7 Hz, ArCH2–), 4.94
(d, 1H, J¼11.2 Hz, ArCH2–), 4.83–4.76 (m, 3H, 3ꢃArCH2–), 4.72 (d, 1H,
J¼11.2 Hz, ArCH2–), 4.64 (d, 1H, J¼11.2 Hz, ArCH2–), 4.61 (d, 1H,
J¼11.2 Hz, ArCH2–), 4.58 (d, 1H, J¼11.2 Hz, ArCH2–), 4.53 (d, 1H,
J¼11.2 Hz, ArCH2–), 4.52 (br s, 2H, ArCH2–), 4.46–4.33 (m, 5H,
4ꢃArCH2–, H-1c), 4.27–4.20 (m, 3H, 2ꢃArCH2–, H-2a), 4.11 (br t, 1H,
H-3b), 3.93 (br t, 1H, J¼8.8 Hz, H-4b), 3.86 (d, 1H, J¼2.9 Hz, H-4c), 3.79
(s, 3H, CH3O–), 3.82–3.64 (m, 7H, H-2c, H-3a, H-4a, 2ꢃH-6a, 2ꢃH-6b),
3.56 (m, 1H, H-5b), 3.50–3.43 (m, 2H, H-2b, H-6c), 3.42–3.36 (m, 3H,
ene/EtOAc (4:1) to give 20 (95 mg, 99%). [
a
]D¼þ33.8 (c, 1.0). Rf 0.11
(7:1 toluene/EtOAc). 1H NMR:
d
7.44–7.13 (m, 47H, Ar-H), 6.82 (br
d, 2H, J¼8.3 Hz, Ar-H), 5.59 (d, 1H, J¼1.5 Hz, H-1a), 5.58 (d, 1H,
J¼7.3 Hz, AcNH–), 5.21 (d, 1H, J¼8.3 Hz, H-1b), 4.98 (d, 1H,
J¼11.7 Hz, ArCH2–), 4.95 (d, 1H, J¼11.7 Hz, ArCH2–), 4.90 (d, 1H,
J¼10.7 Hz, ArCH2–), 4.81–4.76 (m, 3H, 3ꢃArCH2–), 4.71 (d, 1H,
J¼12.2 Hz, ArCH2–), 4.69 (d, 1H, J¼12.2 Hz, ArCH2–), 4.55–4.36 (m,
10H, 8ꢃArCH2–, H-1c, H-3b), 4.32 (d, 1H, J¼10.7 Hz, ArCH2–), 4.30
(d, 1H, J¼11.7 Hz, ArCH2–), 4.19 (d, 1H, J¼11.2 Hz, ArCH2–), 4.17 (m,
1H, H-5a), 4.02 (br t, 1H, J¼8.8 Hz, H-4a), 3.92 (br t, 1H, J¼8.8 Hz,
H-4b), 3.88 (d, 1H, J¼2.4 Hz, H-4c), 3.83 (dd, 1H, J¼2, 9, 10.7 Hz, H-
3a), 3.82–3.70 (m, 3H, H-2c, H-6b, H-6a), 3.76 (s, 3H, CH3O–), 3.63
(dd, 1H, J¼1.5, 10.7 Hz, H-6a), 3.58 (m, 1H, H-5b), 3.51 (m, 1H, H-
6c), 3.43–3.35 (m, 3H, H-3c, H-5c, H-6c), 3.07 (m, 1H, H-2b), 1.63
H-3c, H-5c, H-6c), 1.82 (s, 3H, Ac). 13C NMR:
d
106.4 (JC–H¼218.3 Hz, C-
1a), 102.9 (C-1c), 100.3 (C-1b). MALDI TOF MS: calcd for C84H90FNO16
:
m/z 1410.6 (MþNa)þ, 1426.6 (MþK)þ. Found: m/z 1410.8 and 1426.8.
Anal. Calcd for C84H90FNO16$2H2O: C, 70.82; H, 6.65; N, 0.98. Found: C,
70.85; H, 6.47; N, 1.06.
(s, 3H, Ac). 13C NMR:
d 102.9 (C-1c), 97.2 (C-1b), 85.1 (C-1a). Anal.
Calcd for C90H95NO16S: C, 73.10; H, 6.48; N, 0.95. Found: C, 73.03;
H, 6.66; N, 0.97.
4.1.15. 2,3,4-Tri-O-benzyl-6-O-(4-methoxyphenyl)methyl-
actopyranosyl-(1/4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-
copyranosyl-(1/2)-3,4,6-tri-O-benzyl- -mannopyranose
b
-
D
-gal-
-glu-
b-D
D
4.1.13. 2,4,6-Tri-O-benzyl-3-O-(4-methoxyphenyl)methyl-
actopyranosyl-(1/4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-
glucopyranosyl-(1/2)-3,4,6-tri-O-benzyl- -mannopyranose 21. To
b
-
D
-gal-
b-D-
23. According to the procedure described for 21, thioglycoside 20
(438 mg, 0.30 mmol) was converted to hemiacetal 23 by treatment
with NBS in aq acetone. The crude product was purified by chro-
matography on silica gel with hexane/EtOAc (3:7) to give 23
D
a stirred solution of 19 (484 mg, 0.33 mmol) in acetone/water (6:1,
6.6 ml) was added N-bromosuccimide (175 mg, 1.0 mmol). The
mixture was stirred at room temperature for 30 min, before
extracting with EtOAc. The extract was washed successively with
5% Na2S2O3 aq and brine, dried over anhydrous Na2SO4, and con-
centrated in vacuo. The residue was chromatographed on silica gel
with hexane/EtOAc (3:7) to give 21 (107 mg, 98%) as an anomeric
(375 mg, 91%). Rf 0.25 (2:3 hexane/EtOAc). 13C NMR:
d 103.0 and
102.5 (C-1c), 97.4 (C-1b), 92.4 and 91.7 (C-1a). MALDI TOF MS: calcd
for C84H91NO17: m/z 1408.6 (MþNa)þ, 1424.6 (MþK)þ. Found: m/z
1409.5 and 1424.6. HRMS: calcd for C84H91NNaO17: m/z 1408.6184.
Found: m/z 1408.6129.
mixture. Rf 0.26 (2:3 hexane/EtOAc). 13C NMR:
d
103.0 (C-1c), 102.5
4.1.16. 2,3,4-Tri-O-benzyl-6-O-(4-methoxyphenyl)methyl-
actopyranosyl-(1/4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-
copyranosyl-(1/2)-3,4,6-tri-O-benzyl- -mannopyranosyl
b
-
-
D
-gal-
-glu-
fluoride
and 97.5 (C-1a), 92.4 and 91.8 (C-1b). MALDI TOF MS: calcd for
C84H91NO17: m/z 1408.6 (MþNa)þ, 1424.6 (MþK)þ. Found: m/z
1409.4 and 1425.4. Anal. Calcd for C84H91NO17$1.5H2O: C, 71.37; H,
6.70; N, 0.99. Found: C, 71.14; H, 6.50; N, 1.02.
b
D
D
24. Hemiacetal 23 (375 mg, 0.27 mmol) was treated with DAST in
CH2Cl2, as described for 22. The crude product was chromato-
graphed on silica gel with toluene/EtOAc (3:1) to afford 24
a
4.1.14. 2,4,6-Tri-O-benzyl-3-O-(4-methoxyphenyl)methyl-
actopyranosyl-(1/4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-
glucopyranosyl-(1/2)-3,4,6-tri-O-benzyl- -mannopyranosyl
fluoride 22 ( ). To a stirred solution of 21 (556 mg, 0.4 mmol) in
anhydrous CH2Cl2 (11 ml) was added (diethylamino)sulfur tri-
fluoride (105
l, 0.8 mmol) at 0 ꢁC. The mixture was stirred for
30 min, before the reaction was quenched with aq NaHCO3. The
product was extracted with CHCl3, washed successively with
water and brine, dried over anhydrous Na2SO4, and concentrated
in vacuo. The crude product was chromatographed on silica gel
b
-
D
-gal-
b-D-
(310 mg, 83%) and 24 (56 mg, 15%).
b
a
/
b
-
D
4.1.16.1. Compound 24
a
. [
a
]D¼þ5.9 (c, 1.0). Rf 0.25 (2:3 hexane/
a
/
b
EtOAc). 1H NMR:
d
7.33–7.14 (m, 42H, Ar-H), 6.82 (br d, 2H, J¼8.8 Hz,
Ar-H), 5.59 (d, 1H, JH–F¼50.2 Hz, H-1a), 5.57 (d,1H, J¼6.8 Hz, AcNH–),
5.22 (d, 1H, J¼8.3 Hz, H-1b), 4.98 (d, 1H, J¼11.2 Hz, ArCH2–), 4.93 (d,
1H, J¼11.7 Hz, ArCH2–), 4.87 (d, 1H, J¼10.7 Hz, ArCH2–), 4.80 (br s,
2H, ArCH2–), 4.77 (d, 1H, J¼11.2 Hz, ArCH2–), 4.71 (br s, 2H, ArCH2–),
4.57–4.42 (m, 8H, 7ꢃArCH2–, H-1c), 4.35 (d,1H, J¼8.3 Hz, H-3b), 4.33
(d, 1H, J¼11.7 Hz, ArCH2–), 4.30 (d, 1H, J¼12.2 Hz, ArCH2–), 4.25 (d,
1H, H-2a), 4.20 (d, 1H, J¼11.2 Hz, ArCH2–), 4.04 (br t, 1H, J¼9.7 Hz, H-
4a), 3.92 (br t, 1H, J¼9.3 Hz, H-4b), 3.89–3.70 (m, 7H, H-4c, H-3a, H-
5a, H-6a, H-2c, 2ꢃH-6b), 3.76 (s, 3H, CH3O–), 3.65 (br d,1H, J¼9.3 Hz,
H-6a), 3.59 (m, 1H, H-5b), 3.51 (m,1H, H-6c), 3.43–3.38 (m, 3H, H-3c,
m
with toluene/EtOAc (3:1) to afford 22
a (466 mg, 83%) and 22b
(82 mg, 15%).
4.1.14.1. Compound 22
a
. [
a
]D¼þ11.6 (c, 1.0). Rf 0.32 (2:3 hexane/
EtOAc). 1H NMR:
d
7.34–7.17 (m, 42H, Ar-H), 6.86–6.82 (m, 2H, Ar-H),
H-5c, H-6c), 3.03 (m,1H, H-2b),1.64 (s, 3H, Ac). 13C NMR:
221.5 Hz, C-1a), 102.9 (C-1c), 97.7 (C-1b). Anal. Calcd for
C84H90FNO16$0.5H2O: C, 72.19; H, 6.56; N, 1.00. Found: C, 72.24; H,
6.64; N, 0.91.
d
106.0 (JC–F
¼
5.60 (br d, 1H, JH–F¼50.7 Hz, H-1a), 5.55 (d, 1H, J¼6.8 Hz, AcNH–),
5.22 (br d, 1H, J¼7.8 Hz, H-1b), 4.99 (d, 1H, J¼11.2 Hz, ArCH2–), 4.97
(d, 1H, J¼11.2 Hz, ArCH2–), 4.88 (d, 1H, J¼10.7 Hz, ArCH2–), 4.80–
4.75 (m, 3H, 3ꢃArCH2–), 4.64 (br s, 2H, ArCH2–), 4.58–4.24 (m, 12H,
H-1c, H-2a, H-3b, 9ꢃArCH2–), 4.04 (br t, 1H, J¼9.7 Hz, H-4a), 3.95–
3.80 (m, 5H, H-3a, H-4b, H-4c, H-5a, H-6b), 3.77 (s, 3H, CH3O–), 3.76–
3.72 (m, 3H, H-6a, H-6b, H-2c), 3.65 (br d, 1H, J¼10.2 Hz, H-6a), 3.60
4.1.16.2. Compound 24
EtOAc). 1H NMR:
2H, J¼8.8 Hz, Ar-H), 5.50 (d, 1H, J¼7.8 Hz, AcNH–), 5.30 (d, 1H,
b
. [
a
]D¼ꢀ12.3 (c, 1.0). Rf 0.11 (2:3 hexane/
d
7.33–7.31 (m, 42H, Ar-H), 6.82 (br d,

K. Kawahira et al. / Tetrahedron 65 (2009) 8143–8153
8151
JH–F¼51.2 Hz, H-1a), 4.99 (d, 1H, J¼8.3 Hz, H-1b), 4.95 (d, 1H,
J¼11.2 Hz, ArCH2–), 4.94 (d, 1H, J¼11.7 Hz, ArCH2–), 4.84–4.77
(m, 3H, 3ꢃArCH2–), 4.73 (d, 1H, J¼11.2 Hz, ArCH2–), 4.71 (d, 1H,
J¼11.7 Hz, ArCH2–), 4.68 (d, 1H, J¼11.7 Hz, ArCH2–), 4.59 (d, 1H,
J¼11.2 Hz, ArCH2–), 4.53–4.42 (m, 5H, 4ꢃArCH2–, H-1c), 4.39 (d,
1H, J¼11.7 Hz, ArCH2–), 4.34–4.18 (m, 5H, 4ꢃArCH2–, H-2a),
4.12 (br t, 1H, J¼9.3 Hz, H-2a), 3.94 (t, 1H, J¼8.8 Hz, H-4b), 3.89
(d, 1H, J¼2.4 Hz, H-4c), 3.78 (s, 3H, CH3O–), 3.79–3.65 (m, 6H,
H-2c, H-3a, 2ꢃH-6a, 2ꢃH-6b), 3.54 (m, 1H, H-5b), 3.53–3.46
(m, 2H, H-2b, H-6c), 3.43–3.35 (m, 3H, H-3c, H-5c, H-6c), 1.82
(s, 3H, Ac). HRMS: calcd for C84H90FNNaO16: m/z 1410.6141.
Found: m/z 1410.6120.
1835.6 and 1851.4. Anal. Calcd for C110H112N2O22: C, 72.83; H, 6.22;
N, 1.54. Found: C, 72.55; H, 6.30; N, 1.54.
4.1.17.4.
b
-Isomer. [
a
]D¼ꢀ18.2 (c, 1.4). Rf 0.34 (2:1 toluene/EtOAc).
1H NMR:
d
7.89 (br d, 2H, J¼7.3 Hz, Ar-H), 7.70 (br d, 2H, J¼6.3 Hz,
Ar-H), 7.62 (br, 2H, Ar-H), 7.36–7.10 (m, 49H, Ar-H), 6.83 (br d, 2H,
J¼8.8 Hz, Ar-H), 6.46 (d, 1H, J¼7.8 Hz, FmocNH–), 6.07 (d, 1H,
J¼8.3 Hz, AcNH–), 5.39 (br s, 2H, PhCOCH2–), 4.89 (d, 1H, J¼7.3 Hz,
H-1b), 4.96–4.87 (m, 3H, 3ꢃArCH2–), 4.76–4.69 (m, 4H, Ser-
a
H,
3ꢃArCH2–), 4.65–4.14 (m, 20H, 2ꢃSer-
bH, H-1c, H-1a, H-3b,
12ꢃArCH2–, Ar2CHCH2–, Ar2CH–), 3.90–3.21 (m, 17H, H-2b, H-4c,
H-4a, H-4b, H-2a, H-3a, H-2c, H-5a, H-5b, 2ꢃH-6a, 2ꢃH-6b, H-3c,
2ꢃH-6c, H-5c), 3.79 (s, 3H, CH3O–), 1.86 (s, 3H, Ac). 13C NMR:
d 103.1
4.1.17. N-(9-Fluorenylmethoxycarbonyl)-O-[2,4,6-tri-O-benzyl-3-
(1JC–H¼162.2 Hz, C-1c), 101.9 (1JC–H¼156.4 Hz, C-1a), 99.9 (1JC–
O-(4-methoxyphenyl)methyl-
amido-3,6-di-O-benzyl-2-deoxy- -glucopyranosyl-(1/2)-3,4,6-
tri-O-benzyl- -mannopyranosyl]-L-serine phenacyl ester 26.
b
-
D
-galactopyranosyl-(1/4)-2-acet-
¼158.1 Hz, C-1b). MALDI TOF MS: calcd for C110H112N2O22: m/z
H
b
-
D
1835.7 (MþNa)þ, 1851.7 (MþK)þ. Found: m/z 1836.1 and 1852.1.
HRMS: calcd for C110H112N2NaO22: m/z 1835.7604. Found: m/z
1835.7666.
a
-
D
4.1.17.1. Procedure A (reaction of 22 and 25). A mixture of Cp2ZrCl2
(160 mg, 0.55 mmol), AgClO4 (228 mg, 1.10 mmol), and dried mo-
lecular sieves 4 Å (3.3 g) in anhydrous CH2Cl2 (5 ml) was stirred at
room temperature under Ar for 30 min, and then cooled at ꢀ40 ꢁC.
To the mixture was added a mixture of 22 (378 mg, 0.27 mmol) and
25 (148 mg, 0.33 mmol) in anhydrous CH2Cl2 (6 ml). The reaction
mixture was stirred for 3 h by raising the temperature (ꢀ40 to 0 ꢁC).
The reaction was quenched by adding satd NaHCO3 aq. The mixture
was diluted with CHCl3 and filtered through Celite. The combined
filtrate and washings (CHCl3) were washed successively with satd
NaHCO3 aq, water and brine, dried over anhydrous Na2SO4, and
concentrated in vacuo. The crude product was chromatographed on
Bio-beads S X1 in toluene, and then on silica gel with toluene/EtOAc
4.1.18. N-(9-Fluorenylmethoxycarbonyl)-O-[2,4,6-tri-O-benzyl-
galactopyranosyl-(1/4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-
glucopyranosyl-(1/2)-3,4,6-tri-O-benzyl-
b
-
-
D
-
-
-
b
D
a
-
D
-mannopyranosyl]-
L
serine phenacyl ester 27. To a solution of 26 (340 mg, 0.19 mmol) in
CH2Cl2 (6.5 ml) was added 90% TFA aq (6.5 ml) at ꢀ10 ꢁC. The
mixture was stirred at the temperature for 30 min, and carefully
neutralized by addition of satd NaHCO3 aq. The mixture was
extracted with EtOAc. The extract was washed successively with
water and brine, dried over anhydrous Na2SO4, and concentrated in
vacuo. The residue was chromatographed on silica gel with tolu-
ene/EtOAc (3:1–1:1). The product was further purified by HPLC in
Mightysil (250ꢃ20 mm) with a program of recycled elution of 40%
(3:1) to give 26 (397 mg, 80%). In a less polar fraction the b-glyco-
side isomer was obtained (42 mg, 9%).
EtOAc/CHCl3 to give 27 (290 mg, 85%). [
a
]D¼þ2.5 (c,1.0). Rf 0.18 (5:3
toluene/EtOAc). 1H NMR:
d
7.86 (br d, 2H, J¼7.8 Hz, Ar-H), 7.73 (br d,
2H, J¼7.3 Hz, Ar-H), 7.59 (d, 2H, J¼7.3 Hz, Ar-H), 7.47 (br d, 1H,
J¼7.3 Hz, Ar-H), 7.39–7.17 (m, 45H, Ar-H), 5.89 (d, 1H, J¼8.8 Hz,
FmocNH–), 5.84 (d, 1H, J¼7.3 Hz, AcNH–), 5.53 (d, 1H, J¼16.6 Hz,
PhCOCH2–), 5.26 (d,1H, J¼16.1 Hz, PhCOCH2–), 5.17 (d,1H, J¼8.3 Hz,
H-1b), 4.96 (br s, 1H, H-1a), 4.95 (d, 1H, J¼11.2 Hz, PhCH2–), 4.83–
4.1.17.2. Procedure B (reaction of 19 and 25). A mixture of 19
(50 mg, 34 mol), 25 (18 mg, 40 mol), N-iodosuccimide (12 mg,
53
m
m
m
mol), and dried molecular sieves 3 Å (100 mg) in anhydrous
CH2Cl2 (1 ml) was stirred at ꢀ20 ꢁC under Ar for 30 min. Then TfOH
(0.45 ml, 5 mmol) was added to the mixture, which was stirred at
4.74 (m, 4H, 4ꢃPhCH2–), 4.68 (m, 1H, Ser-
aH), 4.60–4.21 (m, 17H,
ꢀ20 to 0 ꢁC for 4.5 h before adding satd NaHCO3 aq to quench the
reaction. The mixture was diluted with CHCl3 and filtered through
Celite. The filtrate and washings (CHCl3) were washed successively
with 10% Na2S2O3 aq and brine, dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue was chromatographed on Bio-
beads S X1 in toluene, and then on silica gel with toluene/EtOAc
11ꢃPhCH2–, H-1c, H-2a, H-3b, Ar2CHCH2–, Ar2CH–), 4.13 (m, 1H,
Ser- H), 4.03 (m, 4H, Ser- H, H-3a, H-4a, H-4b), 3.83–3.65 (m, 6H,
b
b
H-4c, H-5a, 2ꢃH-6a, 2ꢃH-6b), 3.58 (m, 1H, H-5b), 3.55–3.39 (m, 5H,
H-2c, H-3c, H-5c, 2ꢃH-6c), 3.27 (m, 1H, H-2b), 1.72 (s, 3H, Ac). 13C
NMR:
d 102.8 (C-1c), 99.2 (C-1a), 98.2 (C-1b). Anal. Calcd for
C102H104N2O21$0.5H2O: C, 71.94; H, 6.21; N, 1.65. Found: C, 71.90; H,
(3:1) to give 26 (25 mg, 41%). The b-isomer (18 mg, 29%) was also
6.31; N, 1.59.
obtained.
4.1.19. N-(9-Fluorenylmethoxycarbonyl)-O-[2,4,6-tri-O-benzyl-3-O-
4.1.17.3. Compound 26. [
a
]D¼þ4.7 (c, 1.0). Rf 0.26 (2:1 toluene/
sulfo-
deoxy-
anosyl]-
b
-
D
-galactopyranosyl-(1/4)-2-acetamido-3,6-di-O-benzyl-2-
-glucopyranosyl-(1/2)-3,4,6-tri-O-benzyl- -mannopyr-
-serine phenacyl ester 28. A mixture of 27 (277 mg,
EtOAc). 1H NMR:
d
7.87 (br d, 2H, J¼7.3 Hz, Ar-H), 7.73 (br d, 2H,
b
-D
a-D
J¼7.3 Hz, Ar-H), 7.59 (br d, 2H, J¼7.3 Hz, Ar-H), 7.47–7.16 (m, 49H,
Ar-H), 6.83 (br d, 2H, J¼8.8 Hz, Ar-H), 5.87 (d, 1H, J¼8.8 Hz,
FmocNH–), 5.79 (d, 1H, J¼7.3 Hz, AcNH–), 5.53 (d, 1H, J¼16.1 Hz,
PhCOCH2–), 5.26 (d,1H, J¼16.6 Hz, PhCOCH2–), 5.15 (d,1H, J¼7.8 Hz,
H-1b), 4.97–4.93 (m, 3H, H-1a, 2ꢃArCH2–), 4.81 (br d, 2H, J¼11.2 Hz,
2ꢃArCH2–), 4.74 (d, 1H, J¼11.2 Hz, ArCH2–), 4.71 (d, 1H, J¼11.2 Hz,
L
0.16 mmol) and Me3N$SO3 (453 mg, 3.2 mmol) in anhydrous DMF
(28 ml) was stirred at 60 ꢁC for 1 h under Ar. Most of the solvent
was evaporated with toluene under reduced pressure. The residue
was chromatographed on silica gel with CHCl3/MeOH/AcOH
(36:4:1) to afford 28 (282 mg, 97%). Rf 0.36 (36:4:1 CHCl3/MeOH/
AcOH). 1H NMR (CD3OD):
d
7.78 (br d, 2H, J¼7.3 Hz, Ar-H), 7.61 (br d,
ArCH2–), 4.69 (m, 1H, Ser-
(d, 1H, J¼11.2 Hz, ArCH2–), 4.56–4.22 (m, 16H, 10ꢃArCH2–, H-1c, H-
2a, H-3b, Ar2CHCH2–, Ar2CH–), 4.15 (m, 1H, Ser- H), 4.02 (m, 1H,
Ser- H), 3.98–3.64 (m, 10H, H-4b, H-3a, H-4c, H-2c, H-4a, H-5a,
a
H), 4.63 (d, 1H, J¼11.7 Hz, ArCH2–), 4.60
2H, J¼7.3 Hz, Ar-H), 7.51–7.41 (m, 4H, Ar-H), 7.32–7.00 (m, 45H, Ar-
H), 5.43 (d, 1H, J¼16.6 Hz, PhCOCH2–), 5.24 (d, 1H, J¼16.6 Hz,
PhCOCH2–), 5.03 (d,1H, J¼11.2 Hz, PhCH2–), 4.90–4.79 (m, 4H, H-1a,
H-1b, 2ꢃPhCH2–), 4.72–4.66 (m, 2H, 2ꢃPhCH2–), 4.60–4.51 (m, 3H,
b
b
2ꢃH-6a, 2ꢃH-6b), 3.79 (s, 3H, CH3O–), 3.58 (m,1H, H-5b), 3.51 (br t,
1H, J¼9.7 Hz, H-6c), 3.40–3.30 (m, 5H, H-2b, H-3c, H-5c, H-6c, H-
2b), 1.71 (s, 3H, Ac).
Ser-
a
H, 2ꢃPhCH2–), 4.43–3.35 (m, 6H, H-1c, H-3c, 4ꢃPhCH2–),
4.30–4.17 (m, 7H, H-2a, 4ꢃPhCH2–, Ar2CHCH2–), 4.17–3.97 (m, 4H,
PhCH2–, Ar2CH–, 2ꢃSer- H), 3.87–3.23 (m, 16H, H-3a, H-4a, H-5a,
2ꢃH-6a, H-2b, H-3b, H-4b, H-5b, 2ꢃH-6b, H-2c, H-4c, H-5c, 2ꢃH-
6c),1.78 (s, 3H, Ac). 13C NMR:
103.2 (C-1c), 99.9 (C-1b), 99.2 (C-1a).
13C NMR:
d
102.8 (1JC–H¼160.6 Hz, C-1c), 99.1 (1JC–H¼169.7 Hz,
b
C-1a), 98.4 (1JC–H¼162.2 Hz, C-1b). MALDI TOF MS: calcd for
C110H112N2O22: m/z 1835.7 (MþNa)þ, 1851.7 (MþK)þ. Found: m/z
d

8152
K. Kawahira et al. / Tetrahedron 65 (2009) 8143–8153
MALDI TOF MS: calcd for C102H103N2Na2O24S: m/z 1817.6, for
C102H103KN2NaO24S: m/z 1833.6. Found: m/z 1816.3 and 1832.3.
Anal. Calcd for C102H103N2NaO24S$H2O: C, 67.54; H, 5.83; N, 1.54.
Found: C, 67.81; H, 5.88; N, 1.55.
(m, 3H, H-3a, H-4b, H-4a), 3.81–3.64 (m, 7H, H-2c, H-4c, H-5a,
2ꢃH-6a, 2ꢃH-6b), 3.62–3.53 (m, 2H, H-5b, H-6c), 3.39 (dd, 1H,
J¼2.4, 9.8 Hz, H-3c), 3.30 (dd, 1H, J¼4.4, 11.2 Hz, H-6c), 3.23 (br d,
1H, J¼8.8 Hz, H-2b), 3.18 (dd, 1H, J¼4.4, 7.8 Hz, H-5c), 1.73 (s, 3H,
Ac). 13C NMR:
d 102.8 (C-1c), 99.1 (C-1a), 98.2 (C-1b). Anal. Calcd for
4.1.20. N-(9-Fluorenylmethoxycarbonyl)-O-[2,3,4-tri-O-benzyl-6-O-
C102H104N2O21$H2O: C, 71.56; H, 6.24; N, 1.64. Found: C, 71.52; H,
(4-methoxyphenyl)methyl-
amido-3,6-di-O-benzyl-2-deoxy-
tri-O-benzyl- -mannopyranosyl]-
similar procedure described for 26, fluoride 24 (276 mg,
0.20 mmol) and 25 (115 mg, 0.26 mmol) were reacted with
Cp2ZrCl2 (119 mg, 0.41 mmol), AgClO4 (166 mg, 0.80 mmol) and MS
4 Å (2.7 g) in anhydrous CH2Cl2 (8 ml) at ꢀ40 to 0 ꢁC for 2 h under
Ar. The crude product was chromatographed on Bio-beads S X1 in
toluene, and then on silica gel with toluene/EtOAc (2:1) to give 29
b
-
D
-galactopyranosyl-(1/4)-2-acet-
-glucopyranosyl-(1/2)-3,4,6-
-serine phenacyl ester 29. In
6.22; N, 1.74.
b-D
L
a-
D
4.1.22. N-(9-Fluorenylmethoxycarbonyl)-O-[2,3,4-tri-O-benzyl-6-O-
a
sulfo-
deoxy-
anosyl]-
b
-
D
-galactopyranosyl-(1/4)-2-acetamido-3,6-di-O-benzyl-2-
-glucopyranosyl-(1/2)-3,4,6-tri-O-benzyl- -mannopyr-
-serine phenacyl ester 31. In a similar manner as described
b
-D
a-D
L
for 28, compound 30 (170 mg, 0.10 mmol) was treated with
Me3N$SO3 (279 mg, 2.00 mmol) in DMF (17 ml), and the product
was purified by chromatography on silica gel to give 31 (125 mg,
76%). Rf 0.44 (38:2:1 CHCl3/MeOH/AcOH). 1H NMR (CDCl3–CD3OD):
(272 mg, 75%) and the b-isomer (18 mg, 5%).
d
7.86 (d, 2H, J¼8.3 Hz, Ar-H), 7.74 (d, 2H, J¼7.3 Hz, Ar-H), 7.68 (m,
4.1.20.1. Compound 29. [
a
]D¼þ2.8 (c, 1.0). Rf 0.25 (2:1 toluene/
2H, Ar-H), 7.61 (m, 2H), 7.45–7.10 (m, 45H, Ar-H), 5.55 (d, 1H,
J¼16.6 Hz, PhCOCH2–), 5.33 (d, 1H, J¼16.6 Hz, PhCOCH2–), 5.00 (br
s, 1H, H-1a), 4.97–4.92 (m, 2H, 2ꢃPhCH2–), 4.80 (d, 1H, J¼11.2 Hz,
EtOAc). 1H NMR:
d
7.87 (d, 2H, J¼7.8 Hz, Ar-H), 7.73 (d, 2H, J¼7.8 Hz,
Ar-H), 7.59 (d, 2H, J¼7.3 Hz, Ar-H), 7.46 (d, 2H, J¼7.3 Hz, Ar-H), 7.38–
7.12 (m, 47H, Ar-H), 6.82 (d, 2H, J¼8.8 Hz, Ar-H), 5.87 (d, 1H,
J¼8.8 Hz, FmocNH–), 5.79 (d, 1H, J¼7.3 Hz, AcNH–), 5.53 (d, 1H,
J¼16.1 Hz, PhCOCH2–), 5.26 (d, 1H, J¼16.6 Hz, PhCOCH2–), 5.16 (d,
1H, J¼7.8 Hz, H-1b), 4.98 (br s, 1H, H-1a), 4.96 (d, 1H, J¼11.2 Hz,
ArCH2–), 4.95 (d, 1H, J¼11.7 Hz, ArCH2–), 4.81 (br d, 2H, J¼10.7 Hz,
PhCH2–), 4.76–4.67 (m, 8H, 6ꢃPhCH2–, H-1b, Ser-
aH), 4.54–4.19 (m,
12H, 7ꢃPhCH2–, H-1c, H-2a, Ar2CHCH2–, H-6c), 4.12–3.35 (m, 19H,
Ar2CH–, 2ꢃSer-
bH, H-6c, H-3b, H-3a, H-2c, H-2b, H-4a, H-4b, H-4c,
H-5a, H-5b, H-5c, 2ꢃH-6a, 2ꢃH-6b, H-3c), 1.85 (s, 3H, Ac). 13C NMR:
d
102.1 (C-1c), 99.0 (C-1b), 97.9 (C-1a). MALDI TOF MS: calcd for
2ꢃArCH2–), 4.75–4.64 (m, 7H, 6ꢃArCH2–, Ser-
15H, 8ꢃArCH2–, H-1c, H-2a, H-3b, Ar2CHCH2–, Ar2CH–, Ser-
4.02–3.28 (m, 17H, Ser- H, H-4b, H-3a, H-4c, H-2c, H-4a, H-5a,
aH), 4.57–4.14 (m,
C102H103N2Na2O24S: m/z 1817.6, for C102H103KN2NaO24S: m/z
1833.6. Found: m/z 1818.2, 1834.0. HRMS: calcd for
C102H103N2Na2O24S: m/z 1817.6416. Found: m/z 1817.6411.
b
H),
b
2ꢃH-6a, 2ꢃH-6b, H-5b, H-5c, H-3c, H-2b, 2ꢃH-6c), 3.77 (s, 3H,
CH3O–), 1.70 (s, 3H, Ac). 13C NMR:
d
102.8 (1JC–H¼162.2 Hz, C-1c),
4.1.23. N-(9-Fluorenylmethoxycarbonyl)-O-[2,4,6-tri-O-benzyl-3-O-
99.1 (1JC–H¼169.7 Hz, C-1a), 98.4 (1JC–H¼163.0 Hz C-1b). Anal. Calcd
for C110H112N2O24$4H2O: C, 70.05; H, 6.41; N, 1.49. Found: C, 69.87;
H, 6.09; N, 1.58.
sulfo-
deoxy-
anosyl]-
b
-
D
-galactopyranosyl-(1/4)-2-acetamido-3,6-di-O-benzyl-2-
-glucopyranosyl-(1/2)-3,4,6-tri-O-benzyl- -mannopyr-
-serine 32. A mixture of 28 (217 mg, 0.12 mmol), pow-
b
-
D
a-D
L
dered Zn (1.85 g, 28.3 mmol), and AcOH (1.85 ml, 32.3 mmol) in
CH2Cl2 (17 ml) was stirred at room temperature for 5 h. Then the
insoluble material was filtered through Celite and washed with
CHCl3/MeOH (9:1). The filtrate and washings were concentrated
with toluene. The residue was chromatographed on silica gel with
CHCl3/MeOH/AcOH (36:4:1) to give 32 (196 mg, 97%). Rf 0.38
4.1.20.2.
b
-Isomer. [
a
]D¼ꢀ19.8 (c, 0.9). Rf 0.32 (2:1 toluene/EtOAc).
1H NMR:
d
7.88 (br d, 2H, J¼6.8 Hz, Ar-H), 7.70 (br d, 2H, J¼6.3 Hz,
Ar-H), 7.61 (br, 2H, Ar-H), 7.36–7.11 (m, 49H, Ar-H), 6.81 (br d, 2H,
J¼8.8 Hz, Ar-H), 6.45 (br d, 1H, J¼7.3 Hz, FmocNH–), 6.08 (br d, 1H,
J¼7.8 Hz, AcNH–), 5.39 (br s, 2H, PhCOCH2–), 4.99–4.84 (m, 4H, H-
1b, 3ꢃArCH2–), 4.81–4.63 (m, 6H, Ser-
a
H, 5ꢃArCH2–), 4.55–3.99
(18:2:1 CHCl3/MeOH/AcOH). 1H NMR (CD3OD):
d 7.72 (br d, 2H,
(m, 18H, 2ꢃSer-
b
H, H-1a, H-1c, H-3b, 10ꢃArCH2–, Ar2CHCH2–,
J¼7.3 Hz, Ar-H), 7.62–7.56 (m, 2H, Ar-H), 7.47 (br d, 2H, J¼7.3 Hz, Ar-
H), 7.37–7.10 (m, 42H, Ar-H), 5.13 (d, 1H, J¼11.7 Hz, PhCH2–), 5.00
(br d, 2H, J¼11.2 Hz, 2ꢃPhCH2–), 4.85–4.62 (m, 5H, H-1b, H-1a,
3ꢃPhCH2–), 4.56–4.42 (m, 6H, H-3c, H-1c, 4ꢃPhCH2–), 4.41–4.11
Ar2CH–), 3.75–3.17 (m, 16H, H-4b, H-3a, H-4c, H-2c, H-4a, H-5a,
2ꢃH-6a, 2ꢃH-6b, H-5b, H-5c, H-3c, H-2b, 2ꢃH-6c), 3.83 (s, 3H,
CH3O–), 1.87 (s, 3H, Ac). 13C NMR:
d
103.0 (1JC–H¼161.4 Hz, C-1c),
101.9 (1JC–H¼158.1 Hz, C-1a), 99.8 (1JC–H¼159.7 Hz, C-1b). MALDI
TOF MS: calcd for C110H112N2O22: m/z 1835.7 (MþNa)þ. Found: m/z
1836.4. HRMS: calcd for C110H112N2NaO22: m/z 1835.7604. Found:
m/z 1835.7672.
(m, 11H, Ser-
a
H, 6ꢃPhCH2–, ArCHCH2–, H-2a, Ar2CH–), 4.06–3.90
(m, 5H, Ser-
b
H, H-3b, H-3a, H-6a), 3.82–3.31 (m, 13H, H-4a, H-5a,
H-6a, H-2b, H-4b, H-5b, 2ꢃH-6b, H-2c, H-4c, H-5c, 2ꢃH-6c). 13C
NMR:
d 102.3 (C-1c), 98.9 and 98.0 (C-1a, C-1b). MALDI TOF MS:
calcd for C94H97N2Na2O23S: m/z 1699.6, for C94H97KN2NaO23S: m/z
1715.6. Found: m/z 1699.9, 1715.9. HRMS: calcd for
C94H97N2Na2O23S: m/z 1699.5998. Found: m/z 1699.5950.
4.1.21. N-(9-Fluorenylmethoxycarbonyl)-O-[2,3,4-tri-O-benzyl-
galactopyranosyl-(1/4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-
glucopyranosyl-(1/2)-3,4,6-tri-O-benzyl-
b
-
-
D
D
-
-
-
b
a
-
D
-mannopyranosyl]-
L
serine phenacyl ester 30. Compound 29 (340 mg, 0.19 mmol) was
stirred with 90% TFA aq (6.5 ml) in CH2Cl2 (6.5 ml) at ꢀ10 ꢁC for
30 min. The reaction was worked-up as described for 27, and the
crude product was purified by column chromatography and then by
4.1.24. N-(9-Fluorenylmethoxycarbonyl)-O-[2,3,4-tri-O-benzyl-6-O-
sulfo-
deoxy-
anosyl]-
b
-
D
-galactopyranosyl-(1/4)-2-acetamido-3,6-di-O-benzyl-2-
-glucopyranosyl-(1/2)-3,4,6-tri-O-benzyl- -mannopyr-
-serine 33. Compound 31 (125 mg, 0.07 mmol) was
b
-
D
a-D
L
HPLC to afford 30 (290 mg, 85%). [
a
]D¼þ7.7 (c, 1.0). Rf 0.18 (5:3
treated with Zn (1.05 g, 16.1 mmol) and AcOH (1.05 ml, 18.4 mmol)
in CH2Cl2 (10 ml) as described for 32. The crude product was
chromatographed on silica gel with CHCl3/MeOH/AcOH (38:2:1) to
afford 33 (95 mg, 82%). Rf 0.15 (38:2:1 CHCl3/MeOH/AcOH). 1H NMR
toluene/EtOAc). 1H NMR:
d
7.85 (d, 2H, J¼7.3 Hz, Ar-H), 7.71 (d, 2H,
J¼7.3 Hz, Ar-H), 7.59 (d, 2H, J¼7.3 Hz, Ar-H), 7.47–7.17 (m, 47H, Ar-
H), 5.90–5.86 (m, 2H, FmocNH–, AcNH–), 5.52 (d, 1H, J¼16.1 Hz,
PhCOCH2–), 5.24 (d,1H, J¼16.6 Hz, PhCOCH2–), 5.19 (d,1H, J¼7.8 Hz,
H-1b), 4.99–4.93 (m, 3H, H-1a, 2ꢃPhCH2–), 4.85–4.81 (m, 2H,
2ꢃPhCH2–), 4.76 (br s, 2H, PhCH2–), 4.71 (br s, 2H, PhCH2–), 4.68
(CD3OD/CDCl3):
7.37–7.16 (m, 36H, Ar-H), 4.96–4.63 (m, 14H, H-1a, H-1b, H-1c, Ser-
H, 10ꢃPhCH2–), 4.60–4.10 (m, 12H, 6ꢃPhCH2–, Ar2CHCH2–,
H, H-6c), 4.05–3.35 (m, 17H, H-2a, H-3a, H-4a, H-
d
7.74 (br d, 2H, J¼7.3 Hz, Ar-H), 7.62 (br, 2H, Ar-H),
a
(m, 1H, Ser-
a
H), 4.58–4.42 (m, 8H, 7ꢃPhCH2–, H-1c), 4.37–4.21 (m,
Ar2CH–, 2ꢃSer-
b
6H, PhCH2–, H-2a, H-3b, Ar2CHCH2–, Ar2CH–), 4.14 (br dd,1H, J¼3.0,
10.3 Hz, Ser- H), 3.96–3.89
H), 4.01 (br dd, 1H, J¼2.9, 10.3 Hz, Ser-
5a, 2ꢃH-6a, H-2b, H-3b, H-4b, H-5b, 2ꢃH-6b, H-2c, H-4c, H-5c, H-
b
b
6c), 3.40 (br d, 1H, J¼10.7 Hz, H-3c), 1.84 (s, 3H, Ac). 13C NMR:

K. Kawahira et al. / Tetrahedron 65 (2009) 8143–8153
8153
d
100.4 (C-1c), 97.2 and 96.8 (C-1a, C-1b). MALDI TOF MS: calcd for
based on the amino acid analysis). MALDI TOF MS (negative): calcd
for C109H182N25O46S2 [MꢀH]ꢀ: m/z 2641.2. Found: m/z 2642.0.
Amino acid analysis: Thr5.29 Ser2.58 Glu1.57 Pro6.24 Ala4.23 Val1.41
C94H97N2Na2O23S: m/z 1699.6, for C94H97KN2NaO23S: m/z 1715.6.
Found: m/z 1699.7 and 1715.7. HRMS: calcd for C94H97KN2NaO23S:
m/z 1715.5737. Found: m/z 1715.5745.
Met0.89 Ile2.28 Arg1.32
.
4.1.25. Debenzylation (for 34 and 35). Sulfo-glycoserine 34/35
Acknowledgements
(5.0 mg, 2.8
(5:3:1, 54
mmol) was dissolved in a mixture of TFA/DMS/m-cresol
m
l) and the mixture was stirred at ꢀ15 ꢁC. On the other
This work was supported by a grant-in-aid for creative scientific
research (17GS0420) from the Japan Society for the Promotion of
Science, and in part by a grant-in-aid for scientific research from the
Ministry of Education, Sports, Science and Technology of Japan
(20380069). We thank the chemical analysis team at Riken for the
combustion analyses, and Prof. J. Tamura, Tottori University, for the
helpful discussion on the preparation of sulfated samples. We also
thank Tokai University for its support via a grant-in-aid for high
technology research.
hand, a mixture of TFA/DMS/m-cresol (5:3:1) containing TfOH (10%
v/v) was also cooled at ꢀ15 ꢁC. Then the TfOH mixture (30
ml,
34 mmol) was added to the reactant mixture. The resultant mixture
was stirred for 7 h at ꢀ15 ꢁC, before the reaction was quenched by
adding cold (ꢀ80 ꢁC) ether (180
ml) containing pyridine (2.8 ml,
35 mmol). The mixture diluted with additional cold ether was vig-
orously stirred with a vortex mixer. The precipitate was separated
by centrifugation, the ethereal layer was decanted, and the residual
precipitate was washed further five times with ether by vortex
mixing, centrifugation, and decantation. The crude product was
dried under vacuum, dissolved in 50% aq CH3CN, and analyzed by
HPLC using a reversed column (Inertsil ODS-SP, 4.6ꢃ150 mm) with
a gradient elution of aq CH3CN containing 0.1% TFA (Fig. 2).
References and notes
1. (a) Yamamoto, N.; Ohmori, Y.; Sakakibara, T.; Sasaki, K.; Juneja, L. R.; Kajihara, Y.
Angew. Chem., Int. Ed. 2003, 42, 2537–2540; (b) Ichiyanagi, T.; Takatani, M.;
Sakamoto, K.; Nakahara, Y.; Ito, Y.; Hojo, H.; Nakahara, Y. Tetrahedron Lett. 2002,
43, 3297–3300 and the cited references therein for the earlier studies.
2. Nakahara, Y.; Nakahara, Y.; Ito, Y.; Ogawa, T. Tetrahedron Lett. 1997, 38,
7211–7214.
4.1.26. Glycopeptide 40. Commercial Fmoc-CLEAR amide resin
(521 mg, 0.25 mmol) was subjected to an automated synthesis of
the peptide to produce an undecapeptide (PAIAPPTETMA)-resin by
the Fastmoc program of the synthesizer, using 20% piperidine/NMP
for N-deprotection and HBTU/HOBt as the condensing agent. t-Bu
group was employed for the protection of Thr and Glu, respectively.
3. (a) Ando, S.; Nakahara, Y.; Ito, Y.; Ogawa, T.; Nakahara, Y. Carbohydr. Res. 2000,
329, 773–780; (b) Takano, Y.; Habiro, M.; Someya, M.; Hojo, H.; Nakahara, Y.
Tetrahedron Lett. 2002, 43, 8395–8399; (c) Hojo, H.; Haginoya, E.; Matsumoto,
Y.; Nakahara, Y.; Nabeshima, K.; Toole, B. P.; Watanabe, Y. Tetrahedron Lett. 2003,
44, 2961–2964; (d) Takano, Y.; Kojima, N.; Nakahara, Y.; Hojo, H.; Nakahara, Y.
Tetrahedron 2003, 59, 8415–8427; (e) Takano, Y.; Hojo, H.; Kojima, N.; Nakahara,
Y. Org. Lett. 2004, 6, 3135–3138; (f) Hojo, H.; Matsumoto, Y.; Nakahara, Y.; Ito, E.;
Suzuki, Y.; Suzuki, M.; Suzuki, A.; Nakahara, Y. J. Am. Chem. Soc. 2005, 127,
13720–13725; (g) Tanaka, E.; Nakahara, Y.; Kuroda, Y.; Takano, Y.; Kojima, N.;
Hojo, H.; Nakahara, Y. Biosci. Biotechnol. Biochem. 2006, 70, 2515–2522; (h)
Nakahara, Y.; Ozawa, C.; Tanaka, E.; Ohtsuka, K.; Takano, Y.; Hojo, H.; Nakahara,
Y. Tetrahedron 2007, 63, 2161–2169; (i) Ueki, A.; Nakahara, Y.; Hojo, H.; Naka-
hara, Y. Tetrahedron 2007, 63, 2170–2181; (j) Ozawa, C.; Hojo, H.; Nakahara, Y.;
Katayama, H.; Nabeshima, K.; Akahane, T.; Nakahara, Y. Tetrahedron 2007, 63,
9685–9690; (k) Ozawa, C.; Katayama, H.; Hojo, H.; Nakahara, Y.; Nakahara, Y.
Org. Lett. 2008, 10, 3531–3533.
4. Yuen, C.-T.; Lawson, A. M.; Chai, W.; Larkin, M.; Stoll, M. S.; Stuart, A. C.;
Sullivan, F. X.; Ahern, T. J.; Feizi, T. Biochemistry 1992, 31, 9126–9131.
5. Chemical and enzymatic O-sulfations of Tyr residues in the PSGL-1 related
glycopeptides at a final step of synthesis have been reported: (a) Koeller, K. M.;
Smith, M. E. B.; Huang, R.-F.; Wong, C.-H. J. Am. Chem. Soc. 2000, 122, 4241–
4242; (b) Baumann, K.; Kowalczyk, D.; Gutjahr, T.; Pieczyk, M.; Jones, C.; Wild,
M. K.; Vestweber, D.; Kunz, H. Angew. Chem., Int. Ed. 2009, 48, 3174–3178.
6. (a) Chiba, A.; Matsumura, K.; Yamada, H.; Inazu, T.; Shimizu, T.; Kusunoki, S.;
Kanazawa, I.; Kobata, A.; Endo, T. J. Biol. Chem. 1997, 272, 2156–2162; (b) Manya,
H.; Suzuki, T.; Akasaka-Manya, K.; Ishida, H.; Mizuno, M.; Suzuki, Y.; Inazu, T.;
Dohmae, N.; Endo, T. J. Biol. Chem. 2007, 282, 20200–20206.
A part of the peptide resin (84 mg, 27
a polypropylene test tube, to which a solution of 32 (90 mg,
80 mol) in NMP (1.4 ml), 0.1 M HOBt/NMP (81 l, 81 mol), and
0.1 M DCC/NMP (81 l, 81 mol) were added. The mixture was
mmol) was transferred into
m
m
m
m
m
stirred for 7 h with a vortex mixer at 50 ꢁC in an oven, and filtered.
The resin was washed several times with NMP and CH2Cl2, and
again submitted to the automated procedure using the synthesizer
to complete the icosapeptide chain. To the resin dried in vacuo, was
added an ice-cooled solution of reagent K (TFA/phenol/water/thi-
oanisole/ethanedithiol, 33:2:2:2:1, 1.5 ml). The mixture was stirred
with the vortex mixer at 0 ꢁC to room temperature for 1 h. Then the
resin was filtered off, and the volatile materials in the mixture 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, to which was added
a mixture of TFA/DMS/m-cresol (5:3:1, 288
m
l), and the mixture was
cooled at ꢀ15 ꢁC. TfOH (32
ml, 0.36 mmol) was added to the mixture
7. Ueki, A.; Hirota, M.; Kobayashi, Y.; Komatsu, K.; Takano, Y.; Iwaoka, M.; Naka-
hara, Y.; Hojo, H.; Nakahara, Y. Tetrahedron 2008, 64, 2611–2618.
8. Kanie, O.; Ito, Y.; Ogawa, T. Tetrahedron Lett. 1996, 37, 4551–4554.
9. Ziegler, T.; Dettmann, R.; Ariffadhillah; Zettl, U. J. Carbohydr. Chem. 1999, 18,
1079–1095.
10. Zang, Y.-M.; Mallet, J.-M.; Sinay¨, P. Carbohydr. Res. 1992, 236, 73–88.
11. Suzuki, K.; Maeta, H.; Matsumoto, T. Tetrahedron Lett. 1989, 30, 4853–4856.
12. Tamura, K.; Mizukami, H.; Maeda, K.; Watanabe, H.; Uneyama, K. J. Org. Chem.
1993, 58, 32–35.
and the plastic vessel was shaken in the cooling bath. The reaction
mixture was left at ꢀ15 ꢁC for 7 h, before the reaction was termi-
nated by the addition of ether cooled at ꢀ80 ꢁC. The mixture was
centrifuged to separate the debenzylated product, which was
washed three times with ether and centrifuged as mentioned above
to give a precipitate. The crude product was dissolved in 50% CH3CN
aq water, and purified by preparative HPLC. The major fraction was
collected and lyophilized to afford 40 (16.6 mg, 17% overall yield
13. Yu, B.; Tao, H. Tetrahedron Lett. 2001, 42, 2405–2407.
14. Luening, B.; Norberg, T.; Tejbrant, J. Glycoconjugate J. 1989, 6, 5–19.