Solid-phase synthesis of glycopeptide carrying a tetra-N-acetyllactosamine-containing core 2 decasaccharide

Article abstract of DOI:10.1016/j.tet.2009.12.031

A novel synthesis of tetralactosaminyl O-glycoamino acid is described. The stereoselective assemblage of a lactosaminyl unit was performed by 2-trichloroacetamido group-assisted β-glycosylation. Initial investigation into the synthesis of decasaccharyl threonine 2 showed limited success because of the low yield in the step concerning the removal of 4-O-chloroacetyl groups. In contrast, 4-O-benzylated decasaccharyl threonine 50 was efficiently synthesized from key LacNAc derivative 35 carrying a 3-O-allyl protecting group at the Gal residue by reiterative glycosylation using the (N-phenyl)trifluoroacetimidate method. Decasaccharide 50 was used as a building block in the solid-phase synthesis of a MUC1-related glycopeptide. Synthetic glycopeptide was obtained through two acidic processes: cleavage from resin with reagent K at a lowered temperature and debenzylation with a diluted cocktail of low-acidity TfOH. Desired glycopeptide 54 was isolated as the major product, while a series of the saccharide-shortened minor products were generated due to the acid-labile property of the β-GlcNAc glycosidic linkages.

Full text of DOI:10.1016/j.tet.2009.12.031

Tetrahedron 66 (2010) 1742–1759
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Tetrahedron
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Solid-phase synthesis of glycopeptide carrying a tetra-N-acetyllactosamine-
containing core 2 decasaccharide
*
*
Akiharu Ueki, Yutaka Takano, Akiko Kobayashi, Yuko Nakahara, Hironobu Hojo , Yoshiaki Nakahara
Department of Applied Biochemistry, Institute of Glycoscience, Tokai University, 1117 Kitakaname, Hiratsuka-shi, 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:
A novel synthesis of tetralactosaminyl O-glycoamino acid is described. The stereoselective assemblage of
a lactosaminyl unit was performed by 2-trichloroacetamido group-assisted -glycosylation. Initial in-
Received 11 November 2009
Received in revised form 9 December 2009
Accepted 9 December 2009
Available online 22 December 2009
b
vestigation into the synthesis of decasaccharyl threonine 2 showed limited success because of the low
yield in the step concerning the removal of 4-O-chloroacetyl groups. In contrast, 4-O-benzylated deca-
saccharyl threonine 50 was efficiently synthesized from key LacNAc derivative 35 carrying a 3-O-allyl
protecting group at the Gal residue by reiterative glycosylation using the (N-phenyl)trifluoroacetimidate
method. Decasaccharide 50 was used as a building block in the solid-phase synthesis of a MUC1-related
glycopeptide. Synthetic glycopeptide was obtained through two acidic processes: cleavage from resin
with reagent K at a lowered temperature and debenzylation with a diluted cocktail of low-acidity TfOH.
Desired glycopeptide 54 was isolated as the major product, while a series of the saccharide-shortened
minor products were generated due to the acid-labile property of the b-GlcNAc glycosidic linkages.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
and/or benzyl-protected N-trichloroacetyllactosaminyl fluorides
with high b-selectivity. The oligosaccharides thus prepared were
N-Acetyllactosamine, [
b
-
D
-Gal-(1/4)-
b
-
D
-GlcNAc], is a common
successfully introduced into the glycopeptides through solid-phase
peptide synthesis (SPPS) followed by debenzylation under the
conditions of low-acidity trifluoromethanesulfonic acid (TfOH). The
high potencies of the N-trichloroacetyllactosaminyl fluoride in
disaccharide component of the N- and O-linked oligosaccharides of
glycoproteins, and serves as a scaffold for LeX and SLex epitopes of
biological significance. In addition, several numbers of the di-
saccharide units are often assembled linearly to form a so-called
poly-N-acetyllactosamine structure. It has been reported that
N-acetyllactosamine is an essential structural unit recognized by the
galectins of an animal lectin family and that the recognitions are
enhanced based on galectin species by the multivalent N-acetyl-
lactosamines present in branched N-glycan or repeated N-acetyl-
lactosamine chains.1 In order to gain better insight into such
carbohydrate-mediated biological mechanisms and to tackle such
inaccessible complex structures, efforts have been made to obtain
synthetic homogeneous samples. Syntheses of oligosaccharides
carrying repeated N-acetyllactosamines have previously been
reported by several groups, where the N-phthaloylated,2–8 N-tet-
rachlorophthaloylated9 or N-trichloroethoxycarbonylated10 lacto-
saminyl derivatives were used as glycosyl donors to achieve
terms of reactivity and b-selectivity prompted us to synthesize this
complex molecule as core 2 O-glycan with a tetrameric N-ace-
tyllactosamine motif (2), which would allow us to apply in the solid-
phase glycopeptide synthesis. It should be noted that the related
decasaccharide has already been synthesized in an earlier study, but
the most difficult task concerning deprotection of the oligosaccha-
ride was not realized.4b Apart from the chemical approach, a che-
moenzymatic synthesis of the same structural framework was
recently reported.12a We wish to describe here a novel synthesis of
the tetra-N-acetyllactosamine-containing core 2 O-glycan and the
solid-phase synthesis of a glycopeptide carrying decasaccharide.
2. Synthesis of decasaccharyl threonine building block
b
-selective glycosylation.11,12 In contrast, we have recently de-
veloped new synthetic procedures for the core 2,13 core 3,14 core 4,15
In our initial synthesis planning shown in Scheme 1, the route to
the properly protected decasaccharyl threonine 2 was designed to
couple tetralactosaminyl intermediate 3 and known disaccharyl
threonine 413a,b on the basis of our benzyl-protection strategy.13–15
It has been established that glycosylation of 4 and the related
4,6-dihydroxy disaccharide preferentially occur at the more re-
active 6-O-position.13a,b,16 We envisioned that the lactosamine
and core 614 O-glycans of glycoprotein, employing benzylidene-
* Corresponding authors. Tel.: þ81 463 50 2075.
E-mail addresses: hojo@keyaki.cc.u-tokai.ac.jp (H. Hojo), yonak@key-
aki.cc.u-tokai.ac.jp (Y. Nakahara).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.12.031

A. Ueki et al. / Tetrahedron 66 (2010) 1742–1759
1743
Scheme 1. Retrosynthetic scheme to the tetralactosaminyl O-glycothreonine in the initial synthesis planning.
tetramer could be obtained by coupling di-lactosaminyl donor 5
and acceptor 6, which could be prepared from known disaccharide
716 or 813b (see Scheme 1).
obtained only in 25% yield. Hexasaccharide 27 [(MþNa)þ: m/z
2840.35, 23%] and a trace amount of tetrasaccharide isomer
[(MþNa)þ: m/z 1974.56] were also found in the reaction mixture.
The structure of 25 was confirmed by conversion to 4-O-chloro-
acetate 26, which exhibited a characteristic lower-field shifted
proton signal for H-4b at 5.48 ppm. We recently observed similar
Isopropylidenation of 7 with acetone and an acid catalyst
afforded a mixture of 9 (70%) and 10 (24%), and the structures of
which were determined by the NMR data of corresponding diac-
etate 11 and 12. The shifted proton signals for H-2b of 11 appeared
at 4.93 ppm, while H-2b and H-3b of 12 were observed at 5.27 and
4.75 ppm, respectively. The major isomer 9 was benzylated to 13
(84%), which was heated with ethylenediamine in n-BuOH to give
19 (90%). Compound 19 was alternatively prepared from 8 in
a comparable overall yield. Isomers 14 (60%) and 15 (18%) were
obtained by reacting 8 with acetone. The structures of 14 and 15
were also determined after acetylation to 16 and 17. Benzylation of
14 followed by reduction of the azido group with Zn and AcOH
yielded 19 (85% in two steps). Subsequently, 2-amino sugar 19 was
then trichloroacetylated to give key disaccharide 20 (97%). Desily-
lation of 20 with tetra-n-butylammonium fluoride (TBAF)/THF in
the presence of AcOH was followed by treatment with dieth-
ylaminosulfur trifluoride (DAST) to afford glycosyl donor 22 (83% in
two steps). Glycosyl acceptor 23 was obtained via the deisopropyl-
idenation of 20. Regioselective glycosylation of diol 23 (1.2 equiv)
with glycosyl donor 22 was examined under the Cp2Zr(ClO4)2/
CH2Cl2 conditions.17 The donor was consumed at ꢀ15 ꢁC within 2 h.
However, desired tetrasaccharide 25 [(MþNa)þ: m/z 1973.54] was
insufficient regioselectivity, when glycosylation of
protected galactose derivative was performed with a highly re-
active glycosyl donor carrying a 2-trichloroacetamido group.15
a 3,4-un-
A
modified procedure, employing the slow addition of 22 by a syringe
pump into the more diluted reaction mixture containing excess
acceptor 23 (1.5 equiv), improved the yield of 25 (72%). However,
the byproducts were also generated in 5–7% yields. To avoid the
formation these byproduct, 4-O-chloroacetate 24 was prepared by
regioselective cleavage of the corresponding 3,4-O-orthoester and
reacted with 22. The reaction proceeded smoothly to give tetra-
saccharide 26 in 85% yield. Glycosyl donor 5 was then prepared
from 26 by desilylation and fluorination (79%), while glycosyl
acceptor 6 was obtained through deisopropylidenation and 4-O-
chloroacetylation (65%).
The coupling reaction of 5 and 6 under similar conditions was
successful in producing octasaccharide 30 in 73% yield. Conversion
of 30 into glycosyl fluoride 3 was achieved straightforwardly by
desilylation and fluorination. Glycosylation of 4 with 3 using
a zirconium promoter resulted in the formation of 32 (86%) as the

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A. Ueki et al. / Tetrahedron 66 (2010) 1742–1759
sole decasaccharide product. The decasaccharide structure was
evident from 1H-, 13C NMR and mass spectral data [(MþNa)þ: m/z
4700.3]. Having the necessary decasaccharide framework, we
attempted to manipulate the protecting groups to suite the SPPS. A
conventional dechloroacetylation condition consisting of heating
with thiourea at 70 ꢁC in DMF was examined using 32. However,
the reaction produced the desired 33 in only 36% yield, and the
remaining part of 32 was converted into complex materials with
high polarity. The use of other dechloroacetyl agents such as aq
pyridine or 1-selenocarbamoylpiperidine18 did not offer any im-
provement. Although the optimal conditions were not attained for
the selective removal of chloroacetyl groups, the study was
Figure 1. Intermediates in the synthesis of tetralactosaminyl O-glycothreonine 2.

A. Ueki et al. / Tetrahedron 66 (2010) 1742–1759
1745
progressed to achieve necessary deprotection. Upon Zn reduction
accelerated by microwave irradiation15 and subsequent acetyla-
tion, compound 33 was converted to 34 (86%), which was then
treated with Pd(PPh3)4 and dimedone in THF to quantitatively
produce 2. Our observation of the structure of 2 was supported by
the NMR and mass spectral data.
migration followed by hydrolysis to give glycosyl acceptor 39 (97%).
The coupling reaction of 37 and 39 with Cp2Zr(ClO4)2 in CH2Cl2
gave a complex mixture of products, from which the desired tet-
rasaccharide 40 [(MþNa)þ: m/z 2153.97] was isolated in only 29%
yield. A byproduct with larger molecular weight corresponding to
hexasaccharide 43 [(MþNa)þ: m/z 3078.97] was also generated in
16% yield. This result presents a remarkable contrast to the high-
yielding case of 26. The 4-O-benzylated Gal residue may enhance
the armed nature of GlcNTCA residue, and thus strong Lewis acid
may permit the departure of the t-butyldiphenylsilyloxy group
from initially formed 40 to form a tetrasaccharyl glycosyl donor
leading to 43. Regeneration of 35 (9%) in the byproducts partly
supported the occurrence of the cleavage of the t-butyldiphe-
nylsilyloxy group. We have previously observed that reactivity of
LacNTCA fluoride is considerably influenced by the protecting
groups at the Gal residue.15 The LacNTCA fluoride carrying 2,3,4,6-
tetra-O-benzyl Gal was more reactive than that carrying 2,3-di-O-
benzyl-4,6-O-benzylidene Gal. In order to reduce such side
reactions, glycosylation that could be promoted by a catalytic Lewis
acid was exploited. Thus, we selected the glycosyl imidate method
reported by Yu and Tao.20 By treatment with (N-phenyl)-
trifluoroacetimidoyl chloride21 and K2CO3, hemiacetal 36 was
As only limited success was achieved from the above mentioned
procedure, we explored an alternative route to the (LacNAc)4-
containing core 2 O-glycoamino acid by omitting 4-O-chloroacetyl
protection. We recently reported the facile synthesis of allyl and
benzyl-protected LacNAc derivative 35 by propionitrile-mediated
b
-selective galactosylation.19 The disaccharide 35 was considered
optimal for the preparation of a series of (LacNAc)n glycosyl donors
and acceptors. In addition, we found that the 3-O-allyl protecting
group was removable when exposed to the low-acidity TfOH con-
ditions used for the ultimate deprotection of synthetic glycopep-
tides. Accordingly, 3-O-allyl-protected oligosaccharides are usable
in our benzyl-based protocol of solid-phase glycopeptide synthesis
without specific deallylative processing. By desilylation and sub-
sequent fluorination, the disaccharyl fluoride 37 was obtained in
96% yield (two steps). In contrast, selective removal of the allyl
group of 35 was readily performed by iridium-catalyzed olefin
Figure 2. Compounds in the second synthesis of tetralactosaminyl O-glycothreonine 50.

1746
A. Ueki et al. / Tetrahedron 66 (2010) 1742–1759
readily converted to a stable (N-Phenyl)trifluoroacetimidate 38
(94%), which was reacted with 39 in the presence of TMSOTf
(0.05 equiv) to give an excellent yield of 40 (93%). The tetra-
saccharide was then transformed into the subsequent coupling
partners for octasaccharide. Desilylation of 40, followed by imidate
formation afforded glycosyl donor 42 (94% in two steps), while
deallylation of 40 gave glycosyl acceptor 44 (97%). The coupling of
42 and 44 was smoothly promoted by catalytic TMSOTf to afford 45
in 93% yield, which was converted to glycosyl imidate 47 (98%) in an
analogous manner. Reacting 4 and 47 under the same glycosylation
conditions, the desired tetralactosaminyl O-glycothreonine 48 was
obtained in 85% yield. Expeditious reduction of the azido group and
dehalogenation of the trichloroacetyl group proceeded by reaction
with Zn and AcOH under microwave irradiation within 1 h, and
subsequent acetylation of the generated 2-aminogalactosyl moiety
readily produced penta-acetamido decasaccharide 49 (88%). Allyl
ester was selectively cleaved by Pd(0)-catalyzed reaction to provide
the desired 50 (83%). The tetralactosaminyl core 2 glycothreonine in
a suitably protected form for solid-phase glycopeptide synthesis
was thus obtained in better overall yield than that by the initial
procedure (2) (Fig. 2).
Before applying 50 to the solid-phase synthesis, we examined
the deprotection of such large oligosaccharide covered by 24 benzyl
groups and an allyl group using our protocol of low-acidity TfOH. A
limited amount of TfOH was used to optimize the deprotection
reaction, since it is known that the LacNAc glycosides tend to split
under debenzylation conditions with excess TfOH.13b,15 Sample 50
was dissolved in a mixture of trifluoroacetic acid (TFA)/dimethyl
sulfide (DMS)/m-cresol [5:3:1] and cooled at ꢀ15 ꢁC. The mixture
was treated with TfOH (6 equiv/benzyl and allyl groups) for 4 h at
ꢀ15 ꢁC, and the product was precipitated from diethyl ether. HPLC
analysis of the product is shown in Figure 3. The major fraction
(peak 1) corresponds to desired oligosaccharide 1 [(MþNa)þ: m/z
2189.4]. This result shows that the allyl group in the decasaccharide
was sufficiently susceptible to the acidic deprotection. A LacNAc-
deleted octasaccaride [peak 2, (MþNa)þ: m/z 1824.2] and other
degraded oligosaccharides were also detected in the less mobile
fractions. Attempts to suppress the LacNAc cleavage by lowering
the temperature or by reducing the amount of TfOH resulted in
incomplete deprotection. Decasaccharide 1 was isolated by re-
versed-phase preparative HPLC in 19% yield.
Figure 3. HPLC profile of the products by treatment of 50 with low-acidity TfOH. Peak
1 and 2 correspond to desired 1 and the LacNAc-deleted analog, respectively. Condi-
tions: column, Mightysil KANTO RP-18, 4.6ꢂ150 (5
mm); eluent A, distilled water
containing 0.1% TFA, eluent B, acetonitrile containing 0.1% TFA; flow rate, 1 ml/min.
4-Methoxytrityl (Mmt) was used as a protective group for histi-
dine. After completion of the peptide assembly, the synthesized
glycopeptide 53 was cleaved from the resin. To avoid undesired
scission of the labile LacNAc glycosidic linkages, the resin was
treated with reagent K (TFA/H2O/thioanisole/1,2-ethanedithiol/
phenol)22 at a lowered temperature (0 ꢁC) for 6 h. The product
precipitated from diethyl ether was collected by centrifugation,
and then subjected to the debenzylation reaction without iso-
lation of the intermediates. The final deprotection was performed
at ꢀ15 ꢁC for 15 h with a reduced amount of TfOH (calculated
1.5 equiv/benzyl and allyl groups) in a more diluted cocktail [TFA/
DMS/m-cresol/TfOH (5:3:1:0.3)] than for the deprotection of 50
(vide supra), since excess TfOH brought about uncontrolled
cleavage of the LacNAc glycosides and benzyl ethers. An HPLC
elution profile of the crude product is shown in Figure 4. The
chromatogram was more complicated than that in the model ex-
periment with 50. The major peak (1) corresponds to desired
glycopeptide 54 [(MþH)þ: m/z 3255.1], which was separated by
reversed-phase preparative HPLC. Purity of isolated 54 was sup-
ported by HPLC (Fig. 4b) and the mass spectrum. The yield of 54
was estimated as 4.8% based on the value of Gly in the amino acid-
analysis data of the acid-hydrolyzed sample. The accompanying
byproducts were derived by cleavage predominantly at the
3. Solid-phase synthesis of glycopeptide
With the suitably protected glyco-threonine 50 in hand, we next
studied solid-phase glycopeptide synthesis and deprotection of the
product with low-acidity TfOH. A glycosylated (*) MUC1 fragment
(HGVT*SAPDTRPA) 54 was chosen as a synthetic model. An octa-
peptide (SAPDTRPA) was synthesized on commercial Fmoc-Sieber
amide resin using a peptide synthesizer under a tailor-made program
(FastMoc) for Fmoc chemistry. Each Fmoc amino acid was activated
with a mixture of O-benzotriazol-1-yl-N, N, N0, N0-tetramethyluro-
nium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBt),
and diisopropylethylamine (DIEA) in 1-methyl-2-pyrrolidinone
(NMP), and then condensed with the growing peptide on resin, while
N-deprotection was performed with 20% piperidine/NMP before the
condensation (Scheme 2).
To protect side-chain functional groups, 2,2,4,6,7-pentam-
ethyldihydrobenzofuran-5-sulfonyl (Pbf) group was employed for
arginine, and a t-butyl group for threonine, serine and aspartic
acid, respectively. A part of the machine-made peptide resin 51
was subjected to manual condensation with 50 (2 equiv). The re-
action was performed at an elevated temperature (50 ꢁC) with N,
N’-dicyclohexylcarbodiimide (DCC)/HOBt in NMP using a vortex
mixer. Subsequently, the three N-terminal amino acids were
introduced with DCC/HOBt in NMP by manual operation.
b
-LacNAc linkages. Mass spectral data of the following fractions
exhibited side-production of a series of dodecapeptides carrying
octasaccharide [peak 2: (MþH)þ m/z 2889.68; 54-LacNAc], hex-
asaccharide [peak 3: (MþH)þ m/z 2524.98; 54d(LacNAc)2],
tetrasaccharide [peak 4: (MþH)þ m/z 2159.82; 54–(LacNAc)3], and
disaccharide [peak 5: (MþH)þ m/z 1794.39; 54–(LacNAc)4]. A trace
amount of the non-glycosylated dodecapeptide [peak 7: (MþH)þ
m/z 1429.66] was also observed in the products. In contrast, peak 6

A. Ueki et al. / Tetrahedron 66 (2010) 1742–1759
1747
Scheme 2. Solid-phase synthesis of glycopeptide 54.
demonstrated a mass spectral value (m/z 1019.47) coinciding with
an unusual DCC adduct to the C-terminal octapeptide, probably
generated by the forced conditions (50 ꢁC, 5 h) in the step to in-
troduce 50. Amino-acid analysis supported its octapeptidyl
structure. No other byproducts corresponding to the shorter
peptides were detected. It was therefore clear that building block
50 was introduced into the resin-supported peptide with high
efficiency and that the bulk of the settled decasaccharide posed
little obstacle to further elongation of the peptide chain.
group-assisted
b
-stereoselective glycosylation and constructed
a core 2 O-linked decasaccharyl threonine with the (LacNAc)4
substituent in an N-Fmoc-, O-benzyl- and O-allyl-protected form.
The bulky glycothreonine was successfully introduced into
a MUC1-related dodecapeptide, even when a reduced amount
(2 equiv) of the building block was used in the solid-phase syn-
thesis. Both acidic conditions for releasing the synthetic glyco-
peptide from resin with reagent K and for its deprotection with
low-acidity TfOH were carefully examined to suppress scission of
the acid-labile glycosidic linkages. The reagent K reaction at a lower
reaction temperature needed a longer reaction period than usual to
In conclusion, we have synthesized a linear tetralactosaminyl
oligosaccharide by taking advantage of the 2-trichloroacetamido

1748
A. Ueki et al. / Tetrahedron 66 (2010) 1742–1759
Figure 4. HPLC profile of synthetic glycopeptide 54 (a-1) and isolated 54 (b) Conditions: column, Mightysil KANTO RP-18, 4.6ꢂ150 (5
mm); eluent A, distilled water containing 0.1%
TFA, eluent B, acetonitrile containing 0.1% TFA; flow rate 1 ml/min.
complete removal of the peptide protecting groups. The benzyl and
allyl groups in the oligosaccharide part were finally cleaved by
treatment with a reduced amount of TfOH in the cleavage cocktail
at ꢀ15 ꢁC by extending the reaction time. Since considerable
damage to the oligosaccharide moiety could not be eliminated after
the optimization studies, such heavily benzylated glycopeptides
containing the accumulated acid-labile linkages as 53 may lie vir-
tually within the limitations of deprotection when the low-acidity
TfOH method is employed.
performed in Mightysil RP-18 (4.6ꢂ150 mm for analysis,
10ꢂ250 mm and 20ꢂ250 mm for preparation, Kanto Chemical Co.).
Fmoc-Sieber-amide resin was purchased from NOVAbiochem. The
yield of glycopeptide was determined by amino acid analysis after
the samples were hydrolyzed in a sealed tube with 20% HCl and 0.5%
phenol at 150 ꢁC for 2 h. Amino acids were analyzed by a Hitachi
L-8500 amino acid analyzer, in which Val was separated with GlcNAc
(1/4 sensitivity).
4.1.1. tert-Butyldiphenylsilyl
anosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-D
3,4-O-isopropylidene-
b
-
D
-galactopyr-
-gluco-
4. Experimental
4.1. General
pyranoside 9. To a solution of 7 (423 mg, 0.48 mmol) in anhydrous
acetone (250 ml) were added dried CuSO4 (1.47 g) and p-TsOH$H2O
(71 mg). The mixture was stirred at room temperature for 5 h. Then
the reaction mixture was neutralized with satd NaHCO3 aq, and the
precipitate was filtered off through Celite. The filtrate was concen-
trated in vacuo, and the residual product was extracted with EtOAc.
The extract was successively washed with satd NaHCO3 aq, water,
and brine, dried over Na2SO4, and concentrated in vacuo. The crude
product was chromatographed on silica gel with toluene–EtOAc
(1:1) to give 9 (311 mg, 70%) and 10 (107 mg, 24%). Compound 9:
Optical rotation values were determined with a Jasco DIP-370
polarimeter at 20ꢃ2 ꢁC for solutions in CHCl3, unless noted other-
wise. Column chromatography was performed on silica gel PSQ 100B
(Fuji Silysia), while 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 at 400 MHz and 13C at 100 MHz). Chemical
shifts are expressed in ppm downfield from the signal for internal
Me4Si for solutions in CDCl3. For description of the NMR data, each
sugar residue in oligosaccharide is indicated by alphabetical mark as
shown in Figure 1 MALDI TOF mass spectra were obtained with
a PerSeptive Voyager-DE PRO spectrometer (2,5-dihydroxybenzoic
acid was used as a matrix) Automated solid-phase peptide synthesis
was performed with an Applied Biosystems 433A peptide synthe-
sizer. Manual solid-phase reactions were undertaken in capped
polypropylene test tubes equipped with a filter and three-way
stopcock by stirring on an EYELA CM-1000 vortex mixer. HPLC was
[a]
D þ7.4 (c 1). Rf 0.53 (1:1 toluene–EtOAc). 1H NMR:
d 7.67–6.86 (m,
24H, Ar), 5.18 (m, 1H, H-1a), 4.81 (d, 1H, J¼12.0 Hz, –CH2Ph), 4.61 (d,
1H, J¼12.2 Hz, –CH2Ph), 4.45 (d, 1H, J¼12.0 Hz, –CH2Ph), 4.44 (d, 1H,
J¼8.0 Hz, H-1b), 4.38 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.32–4.26 (m, 2H,
H-2a, H-3a), 4.08 (m, 1H, H-4a), 4.02 (dd, 1H, J¼1.7, 5.6 Hz, H-4b),
3.97 (dd, 1H, J¼5.6, 6.8 Hz, H-3b), 3.76 (dd, 1H, J¼3.2, 12.5 Hz, H-6a),
3.70 (m, 1H, H-6b), 3.66–3.63 (m, 2H, H-5b, –OH), 3.51–3.45 (m, 2H,
H-6a, H-2b), 3.25 (br d, 1H, J¼10.0 Hz, H-5a), 2.92 (d, 1H, J¼2.5 Hz,
t
–OH), 1.48 (s, 3H, –CH3), 1.31 (s, 3H, –CH3), 0.88 (s, 9H, Bu). MALDI

A. Ueki et al. / Tetrahedron 66 (2010) 1742–1759
1749
TOF MS: calcd for C53H59NO12Si (MþNa)þ m/z 952.38. Found:
3.42 (br d, 1H, J¼10.7 Hz, H-6a), 3.32 (br t, 1H, J¼7.5 Hz, H-2b), 3.18
(br d, 1H, J¼8.8 Hz, H-5a), 1.34 (s, 3H, –CH3), 1.32 (s, 3H, –CH3), 0.89
952.26. Anal. calcd for C53H59NO12Si: C, 68.44; H, 6.39; N, 1.51.
t
Found: C, 68.56; H, 6.46; N, 1.35. Compound 10: [
a
]
D þ44.6 (c 1). Rf
(s, 9H, Bu.). MALDI TOF MS: calcd for C67H71NO12Si (MþNa)þ m/z
0.16 (1:1 toluene–EtOAc). 1H NMR:
d
7.78–6.84 (m, 24H, Ar), 5.17 (d,
1132.47. Found: 1132.70. Anal. calcd for C67H71NO12Si: C, 72.47; H,
6.45; N, 1.26. Found: C, 72.64; H, 6.55; N, 1.12.
1H, J¼7.8 Hz, H-1a), 4.85 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.62 (d, 1H,
J¼12.2 Hz, –CH2Ph), 4.54 (d, 1H, J¼7.3 Hz, H-1b), 4.49 (d, 1H,
J¼12.2 Hz, –CH2Ph), 4.45 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.36 (dd, 1H,
J¼8.3, 10.5 Hz, H-3a), 4.29 (dd, 1H, J¼7.8, 10.5 Hz, H-2a), 4.12 (m, 1H,
H-4a), 4.01 (br d, 1H, J¼2.9 Hz, H-4b), 3.89 (dd, 1H, J¼2.9, 12.0 Hz, H-
6a), 3.83 (dd, 1H, J¼2.2, 12.7 Hz, H-6b), 3.75 (dd, 1H, J¼1.2, 12.7 Hz,
H-6b), 3.62–3.68 (m, 2H, H-2b, –OH), 3.47–3.43 (m, 2H, H-6a, H-3b),
3.24 (br d, 1H, J¼10.0 Hz, H-5a), 2.94 (br s, 1H, H-5b), 2.44 (d, 1H,
J¼8.3 Hz, –OH), 1.38 (s, 3H, –CH3), 1.34 (s, 3H, –CH3), 0.88 (s, 9H, tBu).
MALDI TOF MS: calcd for C53H59NO12Si (MþNa)þ m/z 952.38. Found:
952.53. Anal. calcd for C53H59NO12Si: C, 68.44; H, 6.39; N, 1.51.
Found: C, 68.34; H, 6.46; N, 1.47.
4.1.5. tert-Butyldiphenylsilyl 3,4-O-isopropylidene- -galactopyr-
b
-D
anosyl-(1/4)-2-azido-3,6-di-O-benzyl-2-deoxy-b-D-glucopyrano-
side 14. To a solution of 8 (1.69 g, 2.15 mmol) in anhydrous
acetone (300 ml) were added well-dried CuSO4 (8.0 g) and p-
TsOH$H2O (70 mg). The mixture was stirred at room temperature
for 3 h. Then the reaction mixture was neutralized with satd
NaHCO3, and the precipitate was filtered off through Celite. The
filtrate was concentrated in vacuo, and the residual product was
extracted with EtOAc. The extract was successively washed with
satd NaHCO3, water, and brine, dried over Na2SO4, and concen-
trated in vacuo. The crude product was chromatographed on silica
gel with toluene–EtOAc (1: 1) to give 14 (1.12 g, 60%) and 15
4.1.2. tert-Butyldiphenylsilyl 2,6-di-O-acetyl-3,4-O-isopropylidene-
-galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-
-glucopyranoside 11. Compound was stirred with Ac2O
b-
D
(0.35 g, 18%). Compound 14: [
a
]
þ6.5 (c 1). Rf 0.46 (1:1 hexane–
D
b
-D
9
EtOAc). 1H NMR:
d
7.73–7.68, 7.52–7.05 (m, 20H, Ar), 4.88 (d, 1H,
(10 equiv) in pyridine at room temperature overnight. The crude
product was chromatographed on silica gel with toluene–EtOAc (7:3)
J¼10.5 Hz, –CH2Ph), 4.78 (d, 1H, J¼10.7 Hz, –CH2Ph), 4.49 (d, 1H,
J¼12.0 Hz, –CH2Ph), 4.35–4.29 (m, 3H, H-1a, H-1b, –CH2Ph), 3.98–
3.90 (m, 3H, H-4a, H-4b, H-3b), 3.65–3.56 (m, 3H, H-6a, H-6b, H-
6b), 3.52–3.47 (m, 2H, H-2a, H-5b), 3.43 (m, 1H, H-2b), 3.32–3.26
(m, 2H, H-3a, H-6a), 2.96–2.92 (m, 2H, H-5a, –OH), 1.49 (s, 3H,
–CH3), 1.31 (s, 3H, –CH3), 1.12 (s, 9H, tBu). MALDI TOF MS: calcd for
C45H55N3O10Si (MþNa)þ m/z 848.35. Found: 848.13. Anal. calcd for
C45H55N3O10Si: C, 65.43; H, 6.71; N, 5.09. Found: C, 65.53; H, 6.74;
to give 11. 1H NMR:
d
7.77–6.78 (m, 24H, Ar), 5.18 (d, 1H, J¼8.0 Hz, H-
1a), 4.93 (dd,1H, J¼7.6, 8.3 Hz, H-2b), 4.76 (d,1H, J¼12.5 Hz, –CH2Ph),
4.67 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.49 (d, 1H, J¼8.3 Hz, H-1b), 4.41 (d,
1H, J¼12.2 Hz, –CH2Ph), 4.39 (d, 1H, J¼12.5 Hz, –CH2Ph), 4.30–4.22
(m, 2H, H-6b, H-2a), 4.19 (dd, 1H, J¼4.6, 11.7 Hz, H-6b), 4.14 (dd, 1H,
J¼8.5, 10.7 Hz, H-3a), 4.09–3.97 (m, 3H, H-4a, H-3b, H-4b), 3.84 (m,
1H, H-5b), 3.67 (dd, 1H, J¼3.2, 11.2 Hz, H-6a), 3.44 (dd, 1H, J¼1.5,
11.2 Hz, H-6a), 3.16 (br d, 1H, J¼9.8 Hz, H-5a), 2.07 (s, 3H, Ac), 2.02 (s,
3H, Ac), 1.50 (s, 3H, –CH3), 1.30 (s, 3H, –CH3), 0.88 (s, 9H, tBu).
N, 5.03. Compound 15: [
a
]
D ꢀ6.2 (c 1). Rf 0.18 (1:1 hexane–EtOAc).
1H NMR:
d
7.72–7.67, 7.43–7.15 (m, 20H, Ar), 4.96 (d, 1H, J¼11.2 Hz,
–CH2Ph), 4.87 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.51 (d, 1H, J¼12.2 Hz,
–CH2Ph), 4.47 (d, 1H, J¼7.8 Hz, H-1b), 4.35 (d, 1H, J¼7.8 Hz, H-1a),
4.32 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.00 (t, 1H, J¼9.3 Hz, H-4a), 3.98
(d, 1H, J¼4.4 Hz, H-4b), 3.78–3.74 (m, 2H, H-6a, H-6b), 3.67 (dd,
1H, J¼1.5, 12.7 Hz, H-6b), 3.57–3.47 (m, 3H, –OH, H-2a, H-2b), 3.40
(dt, 1H, J¼3.9, 8.3 Hz, H-3b), 3.35 (t, 1H, J¼9.8 Hz, H-3a), 3.26 (dd,
1H, J¼1.9, 12.1 Hz, H-6a), 2.93 (m, 1H, H-5a), 2.76 (br s, 1H, H-5b),
2.42 (d, 1H, J¼8.3 Hz, –OH), 1.39 (s, 3H, –CH3), 1.38 (s, 3H, –CH3),
1.11 (s, 9H, tBu). MALDI TOF MS: calcd for C45H55N3O10Si (MþNa)þ
m/z 848.36. Found: 848.36. Anal. calcd for C45H55N3O10Si: C,
65.43; H, 6.71; N, 5.09. Found: C, 65.29; H, 6.73; N, 5.02.
4.1.3. tert-Butyldiphenylsilyl 2,3-di-O-acetyl-4,6-O-isopropylidene-
-galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-
-glucopyranoside 12. Compound 10 was acetylated as described
for 11 to give 12. 1H NMR:
7.66–6.79 (m, 24H, Ar), 5.27 (dd, 1H,
b-
D
b-D
d
J¼8.1, 10.3 Hz, H-2b), 5.18 (d, 1H, J¼7.8 Hz, H-1a), 4.91 (d,
1H, J¼12.5 Hz, –CH2Ph), 4.75 (dd, 1H, J¼3.7, 10.5 Hz, H-3b), 4.63 (d,
1H, J¼8.0 Hz, H-1b), 4.63 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.50 (d, 1H,
J¼12.5 Hz, –CH2Ph), 4.38 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.27–4.11 (m,
3H, H-2a, H-3a, H-4b), 4.06 (br t, 1H, J¼9.0 Hz, H-4a), 3.90 (m, 2H,
H-6bꢂ2), 3.66 (dd, 1H, J¼3.3, 11.2 Hz, H-6a), 3.44 (br d, 1H,
J¼11.0 Hz, H-6a), 3.17–3.14 (m, 2H, H-5a, H-5b), 2.05 (s, 3H, Ac),1.98
4.1.6. tert-Butyldiphenylsilyl 2,6-di-O-acetyl-3,4-O-isopropylidene-
b
-
t
(s, 3H, Ac), 1.31 (s, 3H, –CH3), 1.29 (s, 3H, –CH3), 0.87 (s, 9H, Bu).
D
-galactopyranosyl-(1/4)-2-azido-3,6-di-O-benzyl-2-deoxy-b-D
-
glucopyranoside 16. Compound 14 was acetylated with Ac2O in
pyridine to give 16. 1H NMR:
7.74–7.68 (m, 4H, Ar), 7.43–7.20 (m,
4.1.4. tert-Butyldiphenylsilyl 2,6-di-O-benzyl-3,4-O-isopropylidene-
d
b
-
D
-galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-phthali-
mido- -glucopyranoside 13. To a stirred mixture of 9 (201 mg,
0.22 mmol) and 60% NaH/mineral oil (35 mg, 0.87 mmol) in anhy-
drous DMF (15 ml) was added benzyl bromide (103 l, 0.87 mmol)
16H, Ar), 4.95 (d, 1H, J¼10.7 Hz, –CH2Ph), 4.89 (br t, 1H, J¼7.9 Hz, H-
2b), 4.68 (d, 1H, J¼10.7 Hz, –CH2Ph), 4.55 (d, 1H, J¼12.2 Hz,
–CH2Ph), 4.44 (d, 1H, J¼8.3 Hz, H-1b), 4.33 (d, 1H, J¼7.8 Hz, H-1a),
4.28 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.23 (br t, 1H, J¼6.4 Hz, H-6b), 4.13
(dd, 1H, J¼7.0, 11.7 Hz, H-6b), 4.04 (m, 1H, H-4b), 3.98–3.93 (m, 2H,
H-4a, H-3b), 3.74 (m, 1H, H-5b), 3.56 (dd, 1H, J¼2.9, 11.3 Hz, H-6a),
3.45 (dd, 1H, J¼7.8, 9.7 Hz, H-2a), 3.28–3.24 (m, 2H, H-3a, H-6a),
2.85 (br d, 1H, J¼9.7 Hz, H-5a), 1.99 (s, 3H, Ac), 1.95 (s, 3H, Ac), 1.51
b-D
m
at 0 ꢁC under Ar. Then, the cooling bath was removed and the
mixture was stirred at room temperature for 4 h. The reaction was
quenched with a careful addition of a few piece of ice. The mixture
was diluted with ether–EtOAc (1:1), successively washed with
water and brine, dried over Na2SO4, and concentrated in vacuo. The
crude product was chromatographed on silica gel with toluene–
t
(s, 3H, –CH3), 1.30 (s, 3H, –CH3), 1.10 (s, 9H, Bu).
EtOAc (9:1) to afford 13 (201 mg, 84%). [
a
]
D þ37.4 (c 1). Rf 0.46 (9:1
4.1.7. tert-Butyldiphenylsilyl 2,3-di-O-acetyl-4,6-O-isopropylidene-
b
-
toluene–EtOAc). 1H NMR:
d
7.80–6.82 (m, 34H, Ar), 5.19 (d, 1H,
D
-galactopyranosyl-(1/4)-2-azido-3,6-di-O-benzyl-2-deoxy-b-D
-
J¼8.0 Hz, H-1a), 4.78 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.76 (d, 1H,
J¼11.7 Hz, –CH2Ph), 4.68 (d, 1H, J¼11.7 Hz, –CH2Ph), 4.57 (d, 1H,
J¼12.0 Hz, –CH2Ph), 4.50 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.46 (d,
1H, J¼8.0 Hz, H-1b), 4.42 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.40 (d, 1H,
J¼12.0 Hz, –CH2Ph), 4.36 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.31 (dd,
1H, J¼8.1, 10.8 Hz, H-2a), 4.21 (dd, 1H, J¼8.3, 10.7 Hz, H-3a), 4.14–
4.03 (m, 3H, H-4a, H-3b, H-4b), 3.78–3.75 (m, 2H, H-6a, H-5b), 3.68
(dd, 1H, J¼5.9, 10.0 Hz, H-6b), 3.60 (dd, 1H, J¼6.5, 10.0 Hz, H-6b),
glucopyranoside 17. Compound 15 was acetylated with Ac2O in
pyridine to give 17. 1H NMR:
7.73–7.68 (m, 4H, Ar), 7.51 (br d, 2H,
d
J¼7.0 Hz, Ar), 7.42–7.18 (m, 14H, Ar), 5.22 (dd, 1H, J¼8.0, 10.5 Hz, H-
2b), 5.06 (d,1H, J¼10.5 Hz, –CH2Ph), 4.74 (d,1H, J¼10.5 Hz, –CH2Ph),
4.70 (dd, 1H, J¼3.9, 7.8 Hz, H-3b), 4.58 (d, 1H, J¼8.0 Hz, H-1b), 4.51
(d, 1H, J¼12.2 Hz, –CH2Ph), 4.32 (d, 1H, J¼7.8 Hz, H-1a), 4.25 (d, 1H,
J¼12.0 Hz, –CH2Ph), 4.21 (d, 1H, J¼3.6 Hz, H-4b), 3.96 (br t, 1H,
J¼9.3 Hz, H-4a), 3.84–3.79 (m, 2H, H-6b), 3.57 (dd, 1H, J¼3.1,

1750
A. Ueki et al. / Tetrahedron 66 (2010) 1742–1759
11.4 Hz, H-6a), 3.45 (dd, 1H, J¼8.0, 9.7 Hz, H-2a), 3.31–3.25 (m, 2H,
H-3a, H-6a), 3.02 (br s, 1H, H-5b), 2.84 (br d, 1H, J¼8.5 Hz, H-5a),
2.04 (s, 3H, Ac), 1.91 (s, 3H, Ac), 1.40 (s, 3H, –CH3), 1.34 (s, 3H, –CH3),
1.10 (s, 9H, tBu).
trichloroacetamido-
19 (130 mg, 0.35 mmol) in pyridine (5 ml) was added tri-
chloroacetyl chloride (45
l, 0.41 mmol) at 0 ꢁC. The mixture was
stirred at 0 ꢁC for 2 h to complete the reaction, and concentrated in
vacuo. The residual product was extracted with ether–EtOAc (1:1),
successively washed with water and brine, dried over Na2SO4, and
concentrated in vacuo. The crude product was chromatographed on
b-D-glucopyranoside 20. To a stirred solution of
m
4.1.8. tert-Butyldiphenylsilyl 2,6-di-O-benzyl-3,4-O-isopropylidene-
b-D-galactopyranosyl-(1/4)-2-azido-3,6-di-O-benzyl-2-deoxy-b-D-
glucopyranoside 18. To a stirred mixture of 14 (1.12 g, 1.36 mmol)
and 60% NaH/mineral oil (217 mg, 5.43 mmol) in anhydrous THF
(50 ml) was added benzyl bromide (0.66 ml 5.42 mmol) under Ar.
The stirred mixture was heated at 60 ꢁC for 6 h and then cooled to
ambient temperature. A few piece of ice was carefully added to the
mixture, which was concentrated in vacuo. The residual product
was extracted with ether–EtOAc (1:1), successively washed with
water and brine, dried over Na2SO4, and concentrated in vacuo. The
crude product was chromatographed on silica gel with toluene–
silica gel with hexane–EtOAc (3:1) to afford 20 (150 mg, 97%). [a]
D
þ9.8 (c 1). Rf 0.43 (3:1 hexane–EtOAc). 1H NMR:
d 7.70–7.63 (m, 4H,
Ar), 7.40–7.14 (m, 26H, Ar), 6.90 (d, 1H, J¼7.8 Hz, –NH), 4.91 (d, 1H,
J¼7.3 Hz, H-1a), 4.84 (d, 1H, J¼10.3 Hz, –CH2Ph), 4.72 (d,
1H, J¼11.9 Hz, –CH2Ph), 4.63 (d, 1H, J¼11.9 Hz, –CH2Ph), 4.57 (d, 1H,
J¼10.3 Hz, –CH2Ph), 4.48 (d, 1H, J¼12.0 Hz, –CH2Ph), 4.42 (d,
1H, J¼7.8 Hz, H-1b), 4.41 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.30 (d, 1H,
J¼11.9 Hz, –CH2Ph), 4.25 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.14–4.02 (m,
3H, H-4a, H-3b, H-4b), 3.88 (t, 1H, J¼9.5 Hz, H-3a), 3.76–3.62 (m,
4H, H-2a, H-6a, H-5b, H-6b), 3.51 (dd, 1H, J¼6.5, 9.6 Hz, H-6b),
3.34–3.28 (m, 2H, H-2b, H-6a), 3.10 (br d,1H, J¼9.0 Hz, H-5a),1.34 (s,
3H, –CH3), 1.33 (s, 3H, –CH3), 1.06 (s, 9H, tBu). MALDI TOF MS: calcd
for C61H68Cl3NO11Si (MþNa)þ m/z 1146.35. Found: 1146.53. Anal.
calcd for C61H68Cl3NO11Si: C, 65.09; H, 6.09; N, 1.24; Cl, 9.45. Found:
C, 64.84; H, 6.08; N, 1.21; Cl, 9.53.
EtOAc (9:1) to afford 18 (1.18 g, 86%). [
a
]
þ2.4 (c 1). Rf 0.53 (4:1
D
hexane–EtOAc). 1H NMR:
d
7.73–7.69, 7.44–7.14 (m, 30H, Ar), 4.92
(d, 1H, J¼10..0 Hz, –CH2Ph), 4.73 (d, 1H, J¼11.7 Hz, –CH2Ph), 4.65 (d,
1H, J¼10.3 Hz, –CH2Ph), 4.61 (d, 1H, J¼11.7 Hz, –CH2Ph), 4.51 (d, 1H,
J¼12.2 Hz, –CH2Ph), 4.42 (d, 1H, J¼12.0 Hz, –CH2Ph), 4.40 (d, 1H,
J¼8.1 Hz, H-1b), 4.31 (d, 1H, J¼7.6 Hz, H-1a), 4.27–4.23 (m, 2H,
–CH2Phꢂ2), 4.12 (dd, 1H, J¼1.9, 5.4 Hz, H-4b), 4.09–3.97 (m, 2H, H-
3a, H-4a), 3.74–3.62 (m, 3H, H-5b, H-6a, H-6b), 3.53 (dd, 1H, J¼5.9,
9.3 Hz, H-6b), 3.38–3.20 (m, 4H, H-2a, H-2b, H-6a, H-3a), 2.87 (m,
4.1.11. 2,6-Di-O-benzyl-3,4-O-isopropylidene-
(1/4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-a/b-D
b
-
D
-galactopyranosyl-
-gluco-
t
1H, H-5a), 1.38 (s, 3H, –CH3), 1.34 (s, 3H, –CH3), 1.10 (s, 9H, Bu).
pyranose 21. To a stirred mixture of 20 (407 mg, 0.36 mmol) and
AcOH (0.21 ml, 3.62 mmol) in distilled THF (5 ml) was added 1 M
TBAF/THF (1.45 ml. 1.45 mmol) on an ice-water bath. The mixture
was allowed to stir overnight at room temperature. Then the
mixture was diluted with enough volume of CHCl3, washed suc-
cessively with water and brine, dried over Na2SO4, and concen-
trated in vacuo. The crude product was chromatographed on silica
MALDI TOF MS: calcd for C59H67N3O10Si (MþNa)þ m/z 1028.45.
Found: 1028.51. Anal. calcd for C59H67N3O10Si: C, 70.42; H, 6.71; N,
4.18. Found: C, 70.22; H, 6.79; N, 4.08.
4.1.9. tert-Butyldiphenylsilyl 2,6-di-O-benzyl-3,4-O-isopropylidene-
b-D-galactopyranosyl-(1/4)-2-amino-3,6-di-O-benzyl-2-deoxy-b-D-
glucopyranoside 19. Procedure A (dephthaloylation of 13). A mixture
of 13 (1.91 g, 1.73 mmol) and ethylenediamine (5.19 ml, 77.6 mmol)
in n-BuOH (90 ml) was stirred at 90 ꢁC under Ar for 46 h, before
being concentrated in vacuo. The residual product 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–EtOAc (19:1) to give
gel with toluene–EtOAc (4:1) to give 21 (304 mg, 95%,
a
/
b
¼>10). Rf
0.31 ( ) and 0.15 ( 7.34–7.24 (m,
a
b
) (4:1 toluene–EtOAc). 1H NMR:
d
20H, Ar), 6.83 (d, 1H, J¼8.6 Hz, –NH), 5.38 (br s, 1H, H-1a), 4.91 (d,
1H, J¼10.7 Hz, –CH2Ph), 4.80 (d, 1H, J¼11.7 Hz, –CH2Ph), 4.69 (d, 1H,
J¼11.7 Hz, –CH2Ph), 4.64 (d, 1H, J¼10.7 Hz, –CH2Ph), 4.56 (d,
1H, J¼12.0 Hz, –CH2Ph), 4.46 (d,1H, J¼12.2 Hz, –CH2Ph), 4.39 (d, 1H,
J¼12.0 Hz, –CH2Ph), 4.31 (d, 1H, J¼8.1 Hz, H-1b), 4.29 (d,
1H, J¼12.2 Hz, –CH2Ph), 1.40 (s, 3H, –CH3), 1.34 (s, 3H, –CH3). MALDI
TOF MS: calcd for C45H50Cl3NO11 (MþNa)þ, m/z 908.24. Found:
908.59. Anal. calcd for C45H50Cl3NO11: C, 60.92; H, 5.68; N, 1.58;
Found: C, 60.87; H, 5.68; N, 1.53.
19 (1.56 g, 90%). [
a
]
þ2.5 (c 1). Rf 0.36 (9:1 toluene–EtOAc). 1H
D
NMR:
d
7.72–7.67 (m, 4H, Ar), 7.39–7.06 (m, 26H, Ar), 5.06 (d,
1H, J¼10.5 Hz, –CH2Ph), 4.75 (d, 1H, J¼11.7 Hz, –CH2Ph), 4.65 (d, 1H,
J¼11.7 Hz, –CH2Ph), 4.54 (d, 1H, J¼12.0 Hz, –CH2Ph), 4.52 (d,
1H, J¼10.5 Hz, –CH2Ph), 4.43 (d, 1H, J¼12.0 Hz, –CH2Ph), 4.43 (d, 1H,
J¼8.0 Hz, H-1a), 4.34 (d, 1H, J¼12.0 Hz, –CH2Ph), 4.20 (d,
1H, J¼12.0 Hz, –CH2Ph), 4.11 (dd, 1H, J¼1.7, 5.7 Hz, H-4b), 4.06 (t,
1H, J¼9.3 Hz, H-4a), 4.03 (dd, 1H, J¼5.7, 6.6 Hz, H-3b), 3.74 (dt, 1H,
J¼1.7, 6.3 Hz, H-5b), 3.70–3.66 (m, 2H, H-6a, H-6b), 3.57 (dd,
1H, J¼6.1, 9.5 Hz, H-6b), 3.22 (dd,1H, J¼7.1, 8.0 Hz, H-2a), 3.28 (t,1H,
J¼9.5 Hz, H-3a), 3.27 (dd, 1H, J¼1.3, 11.5 Hz, H-6a), 2.96 (m, 1H, H-
5a), 2.95 (dd, 1H, J¼7.8, 9.8 Hz, H-2b), 1.34 (s, 3H, –CH3), 1.33 (s, 3H,
–CH3), 1.09 (s, 9H, tBu). MALDI TOF MS: calcd for C59H69NO10Si
(MþNa)þ m/z 1002.46. Found: 1002.53. Anal. calcd for C59H69NO10Si:
C, 72.29; H, 7.09; N, 1.43. Found: C, 72.24; H, 7.07; N, 1.35.
4.1.12. 2,6-Di-O-benzyl-3,4-O-isopropylidene-
(1/4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-
pyranosyl fluoride 22. To stirred solution of 21 (357 mg,
b
-
D
-galactopyranosyl-
a/b-D
-gluco-
a
0.40 mmol) in distilled THF (10 ml) was added DAST (0.11 ml,
0.80 mmol) on an ice-water bath under Ar. The mixture was stirred
at 0 ꢁC for 1 h, before the reaction was quenched by addition of
a few drops of CH3OH. The mixture was concentrated in vacuo to
the residue, which was dissolved in CHCl3. The extract was washed
successively with water and brine, dried over Na2SO4, and con-
centrated in vacuo. The crude product was chromatographed on
Procedure
B
(reduction of 18).
A
mixture of 18 (1.18 g,
silica gel with toluene–EtOAc (9:1) to give 22 (312 mg, 87%,
a/
1.17 mmol), AcOH (1.41 ml, 23.4 mmol), and Zn (3 g, 45.9 mmol)
in CH2Cl2 (30 ml) was stirred at room temperature for 3 h. Then
the mixture was filtered through Celite and the filtrate was
concentrated with toluene in vacuo. The residual product was
extracted with CHCl3, successively washed with water and brine,
dried over Na2SO4, and concentrated in vacuo. Chromatography
of the crude product on silica gel with CHCl3–EtOAc (19:1) to give
19 (1.13 g, 99%).
b
¼19). Rf 0.68 ( ) and 0.67 ( 7.36–
a
b
) (7:3 toluene–EtOAc). 1H NMR:
d
7.24 (m, 20H, Ar), 6.62 (d, 1H, J¼7.8 Hz, –NH), 5.76 (dd, 1H, J¼2.7,
53.7 Hz, H-1a), 4.92 (d, 1H, J¼11.0 Hz, –CH2Ph), 4.80 (d, 1H,
J¼11.7 Hz, –CH2Ph), 4.68 (d, 1H, J¼12.0 Hz, –CH2Ph), 4.67 (d,
1H, J¼11.0 Hz, –CH2Ph), 4.59 (d, 1H, J¼11.9 Hz, –CH2Ph), 4.49 (d, 1H,
J¼12.2 Hz, –CH2Ph), 4.41 (d, 1H, J¼12.0 Hz, –CH2Ph), 4.34 (d, 1H,
J¼7.8 Hz, H-1b), 4.32 (d, 1H, J¼12.0 Hz, –CH2Ph), 4.17 (br t,
1H, J¼9.5 Hz, H-4a), 4.11–4.05 (m, 2H, H-2a, H-4b), 4.00 (br t, 1H,
J¼6.2 Hz, H-3b), 3.94 (dd, 1H, J¼2.9, 11.0 Hz, H-6b), 3.99 (m, 1H,
H-5a), 3.75 (dd,1H, J¼9.0,10.7 Hz, H-3a), 3.71–5.59 (m, 3H, H-6a, H-
5b, H-6b), 3.52 (dd, 1H, J¼6.4, 9.8 Hz, H-6a), 3.35 (dd, 1H, J¼6.8,
4.1.10. tert-Butyldiphenylsilyl 2,6-di-O-benzyl-3,4-O-isopropylidene-
b-D-galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-

A. Ueki et al. / Tetrahedron 66 (2010) 1742–1759
1751
8.0 Hz, H-2b), 1.40 (s, 3H, –CH3), 1.34 (s, 3H, –CH3). MALDI TOF MS:
calcd for C45H49Cl3FNO10 (MþNa)þ m/z 910.23. Found: 911.67. Anal.
calcd for C45H49Cl3FNO10: C, 60.78; H, 5.55; N,1.58; F, 2.14. Found: C,
60.87; H, 5.68; N, 1.53; F, 2.17.
was washed successively with water and brine, dried over
Na2SO4, and concentrated in vacuo. The crude product was
chromatographed on silica gel with toluene–EtOAc (4:1) to give
27 (52 mg, 23%) and 25 (41 mg, 25%). Further chromatography of
the less mobile fraction by preparative TLC gave isomer of 25
4.1.13. tert-Butyldiphenylsilyl 2,6-di-O-benzyl-
(1/4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-
anoside 23. A solution of 20 (365 mg, 0.32 mmol) in CH2Cl2
(10 ml) was stirred with 80% TFA aq (2 ml) at 0 ꢁC for 1 h. The
mixture was concentrated with toluene in vacuo to the residue,
which was chromatographed on silica gel with toluene–EtOAc
b
-
D
-galactopyranosyl-
(5 mg. 3%). Compound 25: [
a
]
þ6.1 (c 1). Rf 0.40 (4:1 toluene–
D
b-
D-glucopyr-
EtOAc). 1H NMR:
d
7.66 (br d, 2H, J¼7.3 Hz, Ar), 7.62 (br d, 2H,
J¼7.3 Hz, Ar), 7.39–7.11 (m, 46H, Ar), 6.89 (d, 1H, J¼8.0 Hz, –NH),
6.84 (d, 1H, J¼7.6 Hz, –NH), 5.10 (d, 1H, J¼7.4 Hz, H-1c), 4.90 (d,
1H, J¼10.2 Hz, –CH2Ph), 4.86 (d, 1H, J¼7.4 Hz, H-1a), 4.79 (d, 1H,
J¼11.2 Hz, –CH2Ph), 4.69 (d, 1H, J¼11.5 Hz, –CH2Ph), 4.60 (br s,
2H, –CH2Phꢂ2), 4.56–4.46 (m, 3H, –CH2Phꢂ3), 4.43–4.36 (m, 4H,
H-1b, H-1d, –CH2Phꢂ2), 4.19 (d, 1H, J¼12.2 Hz, –CH2Ph), 2.92 (br
(7:3) to afford 23 (303 mg, 86%). [
a
]
þ7.4 (c 1). Rf 0.35 (7:3
D
toluene–EtOAc). 1H NMR:
d
7.69 (br d, 2H, J¼7.9 Hz, Ar), 7.63 (m,
2H, Ar), 7.39–7.13 (m, 26H, Ar), 6.89 (d, 1H, J¼8.0 Hz, –NH), 4.94
(d, 1H, J¼11.7 Hz, –CH2Ph), 4.91 (d, 1H, J¼8.0 Hz, H-1a), 4.73 (d,
1H, J¼11.5 Hz, –CH2Ph), 4.60 (d, 1H, J¼10.5 Hz, –CH2Ph), 4.59 (d,
1H, J¼11.5 Hz, –CH2Ph), 4.45 (d, 1H, J¼7.3 Hz, H-1b), 4.42 (d, 1H,
J¼11.7 Hz, –CH2Ph), 4.41 (d, 1H, J¼11.9 Hz, –CH2Ph), 4.36 (d, 1H,
J¼12.0 Hz, –CH2Ph), 4.26 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.10 (br t,
1H, J¼8.5 Hz, H-4a), 3.94 (br d, 1H, J¼2.5 Hz, H-4b), 3.89 (br t, 1H,
J¼9.0 Hz, H-3a), 3.76 (dd, 1H, J¼7.8, 9.5 Hz, H-2a), 3.65 (dd, 1H,
J¼3.2, 11.2 Hz, H-6a), 3.59 (dd, 1H, J¼6.3, 10.0 Hz, H-6b), 3.37 (m,
3H, H-2b, H-5b, H-6b), 3.33 (dd, 1H, J¼2.2, 11.2 Hz, H-6a), 3.06
(m, 1H, H-5a), 1.06 (s, 9H, tBu). MALDI TOF MS: calcd for
C58H64Cl3NO11Si (MþNa)þ m/z 1106.33. Found: 1106.18. Anal.
calcd for C58H64Cl3NO11Si: C, 64.17; H, 5.94; N, 1.29; Cl, 9.80.
Found: C, 63.87; H, 5.91; N, 1.28; Cl, 9.87.
d, 1H, J¼9.1 Hz, H-5a), 1.37 (s, 3H, –CH3), 1.34 (s, 3H, –CH3), 1.04
t
(s, 9H, Bu). 13C NMR:
d
92.1 and 92.4 (–COCCl3ꢂ2), 94.7 (C-1a),
99.0 (C-1c), 102.1 (C-1b, C-1d), 109.8 [(CH3)2C<]. MALDI TOF MS:
calcd for C103H112Cl6N2O21Si (MþNa)þ m/z 1973.57. Found
1973.54. Anal. calcd for C103H112Cl6N2O21Si: C, 63.29; H, 5.77; N,
1.43. Found: C, 63.27; H, 5.76; N, 1.27. Compound 27: Rf 0.55 (4:1
toluene–EtOAc). 1H NMR:
d
5.24 (d, 1H, J¼8.3 Hz, GlcNTCA H-1),
5.03 (d, 1H, J¼7.8 Hz, GlcNTCA H-1), 4.81 (d, 1H, J¼7.6 Hz,
GlcNTCA H-1). 13C NMR:
d
92.1, 92.6, and 92.9 (–COCCl3ꢂ2), 94.9
(C-1a), 100.1, and 100.2 (C-1c and C-1e), 102.0, 102.2, and 102.6
(C-1b, C-1d, and C-1f). MALDI TOF MS: calcd for
C148H160Cl9N3O31Si (MþNa)þ m/z 2840.79. Found 2840.35. Iso-
mer of 25: Rf 0.28 (4:1 toluene–EtOAc). 1H NMR:
d 7.07 (d, 1H,
J¼7.8 Hz, –NH), 6.92 (d, 1H, J¼7.8 Hz, –NH), 5.13 (d, 1H, J¼7.8 Hz,
H-1c), 4.92 (d, 1H, J¼7.3 Hz, H-1a). MALDI TOF MS: Found
1974.56.
4.1.14. tert-Butyldiphenylsilyl 2,6-di-O-benzyl-4-O-chloroacetyl-
galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-tri-
chloroacetamido- -glucopyranoside 24. A mixture of 20 (332 mg,
0.31 mmol), triethyl orthochloroacetate (291 l, 1.53 mmol), and
b-D-
Procedure B: A mixture of 23 (211 mg, 0.20 mmol), Cp2ZrCl2
(76 mg, 0.26 mmol), AgClO4 (108 mg, 0.52 mmol), and dried MS
4A (550 mg) in anhydrous CH2Cl2 (16 ml) was stirred at ꢀ5 ꢁC
under Ar for 30 min. To the mixture was added a solution of 22
(115 mg, 0.13 mmol) in anhydrous CH2Cl2 (6 ml) dropwise over
a period of 1 h using a syringe pump. The mixture was allowed to
stir for another 4 h, and worked up as mentioned above. Chro-
matography of the crude product on silica gel with toluene–
EtOAc (4:1) afforded 27 (19 mg, 5%) and 25 (183 mg, 72%). From
the less mobile fraction, the tetrasaccharide isomer (17 mg, 7%)
was obtained.
b-D
m
a catalytic amount of p-TsOH$H2O in CH2Cl2 (5 ml) was stirred at
room temperature for 30 min. The reaction was quenched by
adding satd NaHCO3 aq and the mixture was extracted with CHCl3.
The extract was washed successively with water and brine, dried
over Na2SO4, and concentrated in vacuo. The residue was dissolved
in 80% AcOH aq (10 ml) and stirred at room temperature overnight
before being concentrated in vacuo. The crude product was chro-
matographed on silica gel with toluene–EtOAc (4:1) to give 24
(348 mg, 99%). [
NMR:
a
]
ꢀ12.7 (c 1). Rf 0.39 (4:1 toluene–EtOAc). 1H
D
d
7.70–7.13 (m, 30H, Ar), 6.89 (d, 1H, J¼7.8 Hz, –NH), 5.40 (d,
4.1.16. tert-Butyldiphenylsilyl 2,6-di-O-benzyl-3,4-O-isopropylidene-
1H, J¼2.9 Hz, H-4b), 4.95 (d, 1H, J¼7.8 Hz, H-1a), 4.92 (d, 1H,
J¼12.5 Hz, –CH2Ph), 4.71 (d, 1H, J¼11.5 Hz, –CH2Ph), 4.57 (d, 2H,
J¼11.2 Hz, –CH2Phꢂ2), 4.49 (d, 1H, J¼7.8 Hz, H-1b), 4.45–4.42 (m,
2H, –CH2Phꢂ2), 4.26 (d, 1H, J¼12.0 Hz, –CH2Ph), 4.25 (d, 1H,
J¼12.0 Hz, –CH2Ph), 4.10 (br t, 1H, J¼8.3 Hz, H-4a), 3.95–3.82 (m,
3H, H-3a, CH2Cl), 3.68 (m, 1H, H-2a), 3.70–3.63 (m, 2H, H-6a, H-6b),
3.59–3.56 (m, 1H, H-3b), 3.35–3.26 (m, 4H, H-6a, H-2b, H-5b, H-6b),
3.05–3.08 (m, 1H, H-5a), 1.06 (s, 9H, tBu). MALDI TOF MS: calcd for
C60H65Cl4NO12Si (MþNa)þ m/z 1182.29. Found 1182.51. Anal. calcd
for C60H65Cl4NO12Si: C, 62.01; H, 5.64; N, 1.21. Found: C, 61.67; H,
5.37; N, 1.21.
b
-
D
-galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-tri-
chloroacetamido- -glucopyranosyl-(1/3)-2,6-di-O-benzyl-4-O-
chloroacetyl- -galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-
2-trichloroacetamido- -glucopyranoside 26. By chloroacetylation
of 25: To a stirred solution of 25 (62 mg, 32 mol) in a mixture of
anhydrous CH2Cl2 (5 ml) and pyridine (3 ml) was added chlor-
oacetyl chloride (35
l, 0.42 mmol) at 0 ꢁC. The mixture was stirred
b-D
b-D
b-D
m
m
at 0 ꢁC to room temperature for 6 h, before adding satd NaHCO3 aq
to quench the reaction. The mixture was extracted with CHCl3. The
extract was successively washed with water and brine, dried over
Na2SO4, and concentrated in vacuo. The residue was chromato-
graphed on silica gel with toluene–EtOAc (4:1) to give 26 (59 mg,
4.1.15. tert-Butyldiphenylsilyl 2,6-di-O-benzyl-3,4-O-isopropylidene-
91%). [
a
]
ꢀ0.8 (c 1). Rf 0.20 (9:1 toluene–EtOAc). 1H NMR:
d 7.66
D
b
-
D
-galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-tri-
chloroacetamido- -glucopyranosyl-(1/3)-2,6-di-O-benzyl-
galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-tri-
chloroacetamido- -glucopyranoside 25. Procedure A: A mixture
(m, 4H, Ar), 7.63 (m, 2H, Ar), 7.38–7.10 (m, 46H, Ar), 6.88 (d, 1H,
J¼8.1 Hz, –NH), 6.67 (d, 1H, J¼8.0 Hz, –NH), 5.48 (d, 1H, J¼3.4 Hz, H-
4b), 5.02 (d, 1H, J¼7.3 Hz, H-1c), 4.87 (d, 1H, J¼7.8 Hz, H-1a), 1.37 (s,
b
-
D
b-D-
b-
D
3H, –CH3), 1.33 (s, 3H, –CH3), 1.04 (s, 9H, tBu). 13C NMR:
d 92.3 and
of 23 (107 mg, 0.10 mmol), Cp2ZrCl2 (69 mg, 0.23 mmol), AgClO4
(98 mg, 0.47 mmol), and dried MS 4A (300 mg) in anhydrous
CH2Cl2 (5 ml) was stirred at ꢀ15 ꢁC under Ar for 30 min. Then,
a solution of 22 (73 mg, 0.08 mmol) in anhydrous CH2Cl2 (3 ml)
was added to the mixture. The mixture was stirred for 2 h, before
the reaction was quenched with satd NaHCO3 aq. The mixture
was diluted with CHCl3 and filtered through Celite. The filtrate
92.5 (–COCCl3), 94.7 (C-1a), 99.1 (C-1c), 102.0, and 102.1 (C-1b
and C-1d), 109.7 [(CH3)2C<]. MALDI TOF MS: calcd for
C105H113Cl7N2O22Si (MþNa)þ m/z 2049.52. Found 2050.30. Anal.
calcd for C105H113Cl7N2O22Si: C, 62.09; H, 5.61; N, 1.38. Found: C,
62.07; H, 5.61; N, 1.38.
By coupling of 22 and 24: As described for 25 (procedure A),
a solution of 22 (226 mg, 0.25 mmol) in anhydrous CH2Cl2 (4 ml)

1752
A. Ueki et al. / Tetrahedron 66 (2010) 1742–1759
was added to a stirred mixture of 24 (348 mg, 0.30 mmol),
Cp2ZrCl2 (148 mg, 0.51 mmol), AgClO4 (210 mg, 1.01 mmol), and
dried MS 4A (500 mg) in anhydrous CH2Cl2 (4 ml) at ꢀ15 ꢁC under
Ar. The mixture was stirred at ꢀ15 ꢁC for 1 h to complete the re-
action, and worked up. Chromatography of the crude product on
silica gel with toluene–EtOAc (9:1) gave 26 (435 mg, 85%).
J¼7.6 Hz, H-1c), 1.05 (s, 9H, tBu). 13C NMR:
d 92.1 and 92.3
(–COCCl3), 94.5 (C-1a), 98.8 (C-1c), 101.8 and 102.5 (C-1b and C-1d).
MALDI TOF MS: calcd for C102H109Cl7N2O22Si (MþNa)þ m/z
2009.49. Found 2010.04. Anal. calcd for C102H109Cl7N2O22Si: C,
61.52; H, 5.52; N, 1.41. Found: C, 61.25; H, 5.47; N, 1.37.
4.1.20. tert-Butyldiphenylsilyl 2,6-di-O-benzyl-4-O-chloroacetyl-
galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-tri-
chloroacetamido- -glucopyranosyl-(1/3)-2,6-di-O-benzyl-4-O-
chloroacetyl- -galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-
2-trichloroacetamido- -glucopyranoside 6. As described for 24,
compound 29 (85 mg, 42 mol) was converted to a 3,4-O-cyclic
orthoester by transesterification with triethyl orthochloroacetate
(40 l, 212 mol) and p-TsOH$H2O (cat.) in CH2Cl2 (5 ml). The
b-D-
4.1.17. 2,6-Di-O-benzyl-3,4-O-isopropylidene-
(1/4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-
anosyl-(1/3)-2,6-di-O-benzyl-4-O-chloroacetyl-
anosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-(
glucopyranose 28. To a stirred mixture of 26 (120 mg, 59 mol) and
AcOH (34 l, 0.59 mmol) in freshly distilled THF (3 ml) was added
1 M TBAF/THF (236 l, 0.24 mmol) under Ar. The mixture was
b
-
D
-galactopyranosyl-
-glucopyr-
-galactopyr-
)-
b
-D
b-D
b
-
D
b-D
a
D
-
b-D
m
m
m
m
m
m
stirred at room temperature for 30 h. THF was co-evaporated with
toluene to the residue, which was extracted with EtOAc, washed
successively with water and brine, dried over Na2SO4, and con-
centrated in vacuo. The crude product was chromatographed on
orthoester was treated with 80% AcOH aq (5 ml) overnight. The
product was purified by chromatography on silica gel with toluene–
EtOAc (4:1) to give 6 (67 mg, 76%). [
a
]
ꢀ0.5 (c 1). Rf 0.34 (4:1
D
toluene–EtOAc).1H NMR:
d
7.68–7.61 (m, 4H, Ar), 7.38–7.11 (m, 46H,
silica gel with toluene–EtOAc (1:1) to afford 28 as an
mixture (103 mg, 97%). Rf 0.48 (1:1 toluene–EtOAc). 1H NMR:
7.51–7.04 (m, 40H, Ar), 6.81 (d, 1H, J¼8.5 Hz, –NH), 6.70 (d, 1H,
J¼8.1 Hz, –NH), 5.48 (d, 1H, J¼3.4 Hz, H-4b), 5.35 (d, J¼3.7 Hz, H-
1a
), 5.04 (d, 1H, J¼7.1 Hz, H-1c), 1.38 (s, 3H, –CH3), 1.34 (s, 3H,
–CH3). 13C NMR:
90.7 (C-1a), 92.1, and 92.2 (–COCCl3), 99.0 (C-1c),
102.0, and 102.1 (C-1b and C-1d), 109.5 [(CH3)2C<]. MALDI TOF MS:
calcd for C89H95Cl7N2O22 (MþNa)þ m/z 1811.40. Found 1811.23.
Anal. calcd for C89H95Cl7N2O22$4H2O: C, 57.32; H, 5.57; N, 1.50.
Found: C, 57.40; H, 5.47; N, 1.34.
a
-isomer rich
Ar), 6.92 (d, 1H, J¼7.8 Hz, –NH), 6.72 (d, 1H, J¼8.1 Hz, –NH), 5.49 (d,
1H, J¼3.1 Hz), and 5.39 (d, 1H, J¼2.9 Hz) (H-4b and H-4d), 5.06 (d,
d
1H, J¼7.4 Hz, H-1c), 1.08 (s, 9H, tBu). 13C NMR:
d 92.1 and 92.3
(–COCCl3), 94.5 (C-1a), 98.7 (C-1c), 101.9 and 102.3 (C-1b and C-1d).
MALDI TOF MS: calcd for C104H110Cl8N2O23Si (MþNa)þ m/z 2085.47.
Found 2086.28. Anal. calcd for C104H110Cl8N2O23Si: C, 60.41; H, 5.36;
N, 1.35. Found: C, 61.13; H, 5.36; N, 1.35.
a
d
4.1.21. tert-Butyldiphenylsilyl 2,6-di-O-benzyl-3,4-O-isopropylidene-
b
-
D
-galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-tri-
chloroacetamido- -glucopyranosyl-(1/3)-2,6-di-O-benzyl-4-O-
chloroacetyl- -galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-
2-trichloroacetamido- -glucopyranosyl-(1/3)-2,6-di-O-benzyl-4-
O-chloroacetyl- -galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-de-
oxy-2-trichloroacetamido- -glucopyranosyl-(1/3)-2,6-di-O-ben-
zyl-4-O-chloroacetyl- -galactopyranosyl-(1/4)-3,6-di-O-benzyl-
2-deoxy-2-trichloroacetamido-
reaction of 6 (96 mg, 47 mol) and 5 (77 mg, 43
moted using Cp2ZrCl2 (25 mg, 86
b-D
4.1.18. 2,6-Di-O-benzyl-3,4-O-isopropylidene-
(1/4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-
anosyl-(1/3)-2,6-di-O-benzyl-4-O-chloroacetyl-
anosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-(
glucopyranosyl fluoride 5. To a stirred solution of 28 (119 mg,
0.07 mmol) was added DAST (17
l, 0.13 mmol) at 0 ꢁC. The mixture
b
-
D
-galactopyranosyl-
-glucopyr-
-galactopyr-
)-
b-D
b
-D
b-D
b
-
D
b-D
a
D
-
b-D
b-D
m
b-
D-glucopyranoside
30. Coupling
was stirred for 1 h before adding a few drops of CH3OH to destroy
excess reagent. The mixture was concentrated in vacuo to the res-
idue, which was dissolved in CHCl3. The extract was washed suc-
cessively with water and brine, dried over Na2SO4, and
concentrated in vacuo. The crude product was purified by gel-
permeation chromatography on LH-60 with CHCl3–CH3OH (3:2) to
m
mmol) was pro-
mmol), AgClO4 (36 mg, 172 mmol)
and MS 4A (300 mg) in anhydrous CH2Cl2 (6 ml) at ꢀ15 ꢁC as
describe above for 25 (procedure A). The mixture was stirred for
4 h and worked up. The product was chromatographed using
a column of LH-60 with CHCl3–CH3OH (3:2) to give 30 (120 mg,
give 5 as an
a
-isomer rich mixture (96 mg, 81%,
a
/
b
¼10/1). Rf 0.62
73%). [
a
]
D ꢀ3.4 (c 1). Rf 0.72 (7:3 toluene–EtOAc). 1H NMR:
d 7.67–
(7:3 toluene–EtOAc). 1H NMR:
d
7.66–7.04 (m, 40H, Ar), 6.75 (d, 1H,
7.61 (m, 4H, Ar), 7.40–7.09 (m, 86H, Ar), 6.87 (d, 1H, J¼8.1 Hz,
–NH), 6.69–6.63 (m, 3H, -NHꢂ3), 5.51–5.46 (m, 3H, H-4b, H-4d,
and H-4f), 5.05–4.98 (m, 3H, H-1c, H-1e, H-1 g), 4.87 (d, 1H,
J¼7.6 Hz, H-1a), 1.38 (s, 3H, –CH3), 1.34 (s, 3H, –CH3), 1.05 (s, 9H,
J¼8.1 Hz, –NH), 6.60 (d, 1H, J¼8.1 Hz, –NH), 5.72 (dd, 1H, J¼2.0,
53.7 Hz, H-1a), 5.49 (d, 1H, J¼2.7 Hz, H-4b), 5.05 (d, 1H, J¼7.1 Hz, H-
1c), 1.37 (s, 3H, –CH3), 1.34(s, 3H, –CH3). 13C NMR:
d 91.8 and 92.0
(–COCCl3), 99.0 (C-1c), 101.8 and 102.0 (C-1b and C-1d) 105.3 (d,
JCF¼221.7 Hz, C-1a), 109.5 [(CH3)2C<]. MALDI TOF MS: calcd for
C89H94Cl7FN2O21 (MþNa)þ m/z 1813.40. Found 1814.48. Anal. calcd
for C89H94Cl7FN2O21: C, 59.56; H, 5.28; N, 1.56. Found: C, 59.62; H,
5.45; N, 1.51.
tBu). 13C NMR:
d
92.0, 92.1, and 92.3 (–COCCl3), 94.5
(1JCH¼168.2 Hz, C-1a), 98.8 and 99.0 (1JCH¼165.9 Hz, C-1c, C-1e
and C-1 g), 101.8, 101.9, 102.0, and 102.1 (1JCH¼163.4 Hz, C-1b, C-
1d, C-1f, C-1 h), 109.5 [(CH3)2C<]. MALDI TOF MS: calcd for
C193H203Cl15N4O44Si [average, (MþNa)þ] m/z 3865.87. Found
3865.57. Anal. calcd for C193H203Cl15N4O44Si: C, 60.33; H, 5.32; N,
1.46. Found: C, 60.18; H, 5.35; N, 1.41.
4.1.19. tert-Butyldiphenylsilyl 2,6-di-O-benzyl-
(1/4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-
anosyl-(1/3)-2,6-di-O-benzyl-4-O-chloroacetyl-
anosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-
b
-
D
-galactopyranosyl-
-glucopyr-
-galactopyr-
b-D
D
b
-
4.1.22. 2,6-Di-O-benzyl-3,4-O-isopropylidene-
(1/4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-
anosyl-(1/3)-2,6-di-O-benzyl-4-O-chloroacetyl-
anosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-
glucopyranosyl-(1/3)-2,6-di-O-benzyl-4-O-chloroacetyl- -gal-
actopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-tri-
chloroacetamido- -glucopyranosyl-(1/3)-2,6-di-O-benzyl-4-O-
chloroacetyl- -galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-
2-trichloroacetamido-( )- -glucopyranose 31. Desilylation of octa-
saccharide 30 (162 mg, 42 mol) was performed for three days in
b
-
D
-galactopyranosyl-
-glucopyr-
-galactopyr-
b
-
D
-
b-D
b-D
glucopyranoside 29. A solution of 26 (214 mg, 0.11 mmol) in CH2Cl2
(2 ml) was stirred with 80% TFA aq (4 ml) at 0 ꢁC for 1 h. The
mixture was neutralized with satd NaHCO3 aq and extracted with
CHCl3. The extract was washed successively with satd NaHCO3 aq,
water and brine, dried over Na2SO4, and concentrated in vacuo. The
residue was chromatographed on silica gel with toluene–EtOAc
b-D-
b-D
b-D
b-D
(2:1) to afford 29 (185 mg, 86%). [
a
]
þ3.8 (c 1). Rf 0.57 (1:1 tolu-
a D
D
ene–EtOAc). 1H NMR:
d
7.68–7.61 (m, 4H, Ar), 7.49–7.10 (m, 46H,
m
Ar), 6.88 (d, 1H, J¼8.1 Hz, –NH), 6.66 (d, 1H, J¼8.1 Hz, –NH), 5.50 (d,
1H, J¼3.7 Hz, H-4b), 5.02 (d, 1H, J¼7.3 Hz, H-1a), 4.87 (d, 1H,
a similar manner described for 24. The crude product was purified
through a column of LH-20 with CHCl3–CH3OH (3:2) to give 31

A. Ueki et al. / Tetrahedron 66 (2010) 1742–1759
1753
(134 mg, 89%). Rf 0.27 (4:1 toluene–EtOAc). 1H NMR:
d
7.49–7.11 (m,
[average, (MþNa)þ] m/z 4700.33. Found 4700.31. Anal. calcd for
C239H249Cl15N8O57: C, 61.37; H, 5.37; N, 2.40. Found: C, 61.39; H,
5.37; N, 2.24.
80H, Ar), 6.81 (d, 1H, J¼8.5 Hz, –NH), 6.69–6.65 (m, 3H, -NHꢂ3),
5.51 (d, 1H, J¼3.4 Hz), 5.49 (d, 1H, J¼3.2 Hz), and 5.45 (d, 1H,
J¼3.1 Hz) (H-4b, H-4d, and H-4f), 5.33 (d, 1H, J¼2.9 Hz, H-1a), 5.04
(d, 1H, J¼7.1 Hz), 5.01 (d, 1H, J¼7.1 Hz), and 4.99 (d, 1H, J¼6.8 Hz)
(H-1c, H-1e, H-1g), 1.37 (s, 3H, –CH3), 1.34 (s, 3H, –CH3). 13C NMR:
4.1.25. N-(9-Fluorenylmethoxycarbonyl)-O-{2,6-di-O-benzyl-3,4-O-
isopropylidene-
oxy-2-trichloroacetamido-
zyl- -galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-tri-
chloroacetamido- -glucopyranosyl-(1/3)-2,6-di-O-benzyl-
galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-tri-
chloroacetamido- -glucopyranosyl-(1/3)-2,6-di-O-benzyl-
galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-tri-
chloroacetamido- -glucopyranosyl-(1/6)-[2,3,4,6-tetra-O-ben-
zyl- -galactopyranosyl-(1/3)]-2-azido-2-deoxy- -galactopyr-
anosyl}- -threonine allyl ester 33. A mixture of 32 (68 mg, 15 mol)
and thiourea (17 mg, 218 mol) in anhydrous DMF (3 ml) was
b
-
D
-galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-de-
d
90.6 (C-1a), 92.1, and 92.3 (–COCCl3), 98.9, and 99.0 (C-1c, C-1e,
b-D
-glucopyranosyl-(1/3)-2,6-di-O-ben-
and C-1), 101.9 and 102.1 (C-1b, C-1d, C-1f, and C-1h), 109.5
[(CH3)2C<]. MALDI TOF MS: calcd for C177H185Cl15N4O44 [average,
(MþNa)þ] m/z 3627.14. Found 3626.94. Anal. calcd for
C177H185Cl15N4O44: C, 58.98; H, 5.17; N, 1.55. Found: C, 59.03; H,
5.35; N, 1.47.
b-D
b
-
D
b-D-
b
-
D
b-D-
b-D
4.1.23. 2,6-Di-O-benzyl-3,4-O-isopropylidene-
(1/4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-
anosyl-(1/3)-2,6-di-O-benzyl-4-O-chloroacetyl-
anosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-
glucopyranosyl-(1/3)-2,6-di-O-benzyl-4-O-chloroacetylꢀ -gal-
actopyranosyl-(1/4)-3, 6-di-O-benzyl-2-deoxy-2-tri-
chloroacetamido- -glucopyranosyl-(1/3)-2,6-di-O-benzyl-4-O-
chloroacetyl- -galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-
2-trichloroacetamido-( )- -glucopyranosyl fluoride 3. Compound 31
b
-
D
-galactopyranosyl-
-glucopyr-
-galactopyr-
b
-
D
a-D
b-
D
L
m
b
-
D
m
b
-
D
-
heated with stirring under Ar at 70 ꢁC overnight. The mixture
was concentrated in vacuo to remove most DMF, and the residue
was dissolved in ether–EtOAc (1:1). The extract was washed
successively with water and brine, dried over Na2SO4, and con-
centrated in vacuo. The residue was chromatographed on silica
b-D
b-D
b-D
a
D
gel with toluene–EtOAc (7:3) to give 33 (25 mg, 36%). [
a
]
þ19.1
D
(92 mg, 0.03 mmol) was fluorinated with DAST in THF and purified
(c 0.9). Rf 0.37 (7:3 toluene–EtOAc). 1H NMR:
d
7.74 (d, 2H,
by gel-permeation chromatography as described for 5 to afford 3 as
J¼7.3 Hz, Ar), 7.59 (m, 2H, Ar), 7.40–7.22 (m, 104H, Ar), 6.98 (d,
1H, J¼6.8 Hz, TCANH), 6.94–6.89 (m, 3H, TCANHꢂ3), 5.91 (m, 1H,
–CH]CH2), 5.66 (d, 1H, J¼10.0 Hz, FmocNH), 5.33 (d, 1H,
J¼17.1 Hz, –CH]CH2), 5.22 (d, 1H, J¼10.3 Hz, –CH]CH2), 5.13 (d,
1H, J¼7.1 Hz), 5.09 (d, 1H, J¼7.3 Hz) and 5.07 (d, 1H, J¼7.3 Hz) (H-
1d, H-1f, and H-1h), 4.94 (d, 1H, J¼3.7 Hz, H-1a), 1.38 (s, 3H,
a mixture of
a
and
b
anomers (88 mg, 94%,
7.37–7.18 (m, 80H, Ar), 6.77–6.66 (m,
), 5.51 (d, 1H,
a
/
b
¼5). Rf 0.66 (7:3
toluene–EtOAc). 1H NMR:
d
3H, –NHꢂ3), 5.71 (dd, 1H, J¼2.9, 55.9 Hz, H-1a
a
J¼3.4 Hz), 5.49 (d, 1H, J¼3.4 Hz), and 5.48 (d, 1H, J¼3.4 Hz) (H-4b,
H-4d, and H-4f), 5.04 (d, 1H, J¼7.0 Hz), and 5.01 (d, 2H, J¼7.2 Hz)
(H-1c, H-1e, and H-1g), 1.38 (s, 3H, –CH3), 1.34 (s, 3H, –CH3). 13C
–CH3), 1.35 (s, 3H, –CH3), 1.30 (d, 3H, J¼6.3 Hz, Thr-
g
H). 13C NMR:
NMR:
d
92.0 and 92.3 (–COCCl3), 99.0, 99.1, and 99.2 (C-1c, C-1e, and
d 92.0 (–COCCl3), 98.7, and 98.8 (C-1d, C-1f, and C-1h), 99.5 (C-
C-1g), 102.0, 102.1, and 102.3 (C-1b, C-1d, C-1f, and C-1h), 105.2
(1JCF¼221.0 Hz, C-1a), 109.7 [(CH3)2C<]. MALDI TOF MS: calcd for
C177H184Cl15FN4O43 [average, (MþNa)þ] m/z 3629.13. Found
3629.19. Anal. calcd for C177H184Cl15FN4O43: C, 58.95; H, 5.14; N,
1.55. Found: C, 59.07; H, 5.31; N, 1.53.
1a), 99.6 (C-1b), 102.2, 102.4, and 102.6 (C-1c, C-1e, C-1g, and C-
1i), 103.7 (C-1j), 109.7 [(CH3)2C<]. MALDI TOF MS: calcd for
C233H246Cl12N8O54 [average, (MþNa)þ] m/z 4448.90. Found
4449.30. Anal. calcd for C233H246Cl12N8O54: C, 62.92; H, 5.57; N,
2.52. Found: C, 62.81; H, 5.68; N, 2.24.
4.1.24. N-(9-Fluorenylmethoxycarbonyl)-O-{2,6-di-O-benzyl-3,4-O-
4.1.26. N-(9-Fluorenylmethoxycarbonyl)-O-{2,6-di-O-benzyl-3,4-O-
isopropylidene-
oxy-2-trichloroacetamido-
zyl-4-O-chloroacetyl- -galactopyranosyl-(1/4)-3,6-di-O-benzyl-
2-deox-2-trichloroacetamido- -glucopyranosyl-(1/3)-2,6-di-O-
benzyl-4-O-chloroacetyl- -galactopyranosyl-(1/4)-3,6-di-O-ben-
zyl-2-deoxy-2-trichloroacetamido- -glucopyranosyl-(1/3)-2,6-
di-O-benzyl-4-O-chloroacetyl- -galactopyranosyl-(1/4)-3,6-di-
O-benzyl-2-deoxy-2-trichloroacetamido- -glucopyranosyl-(1/6)-
[2,3,4,6-tetra-O-benzyl- -galactopyranosyl-(1/3)]-2-azido-2-de-
oxy- -galactopyranosyl}- -threonine allyl ester 32. Coupling re-
action of 3 (84 mg, 23 mol) and 4 (31 mg, 28 mol) was promoted
using Cp2ZrCl2 (13 mg, 46 mol), AgClO4 (19 mg, 92 mol) and MS
b
-
D
-galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-de-
isopropylidene-
benzyl-2-deoxy-
actopyranosyl-(1/4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-
glucopyranosyl-(1/3)-2,6-di-O-benzyl- -galactopyranosyl-(1/
4)-2-acetamido-3,6-di-O-benzyl-2-deoxy- -glucopyranosyl-(1/
3)-2,6-di-O-benzyl- -galactopyranosyl-(1/4)-2-acetamido-3,6-
di-O-benzyl-2-deoxy- -glucopyranosyl-(1/6)-[2,3,4,6-tetra-O-
benzyl- -galactopyranosyl-(1/3)]-2-acetamido-2-deoxy- -gal-
actopyranosyl}- -threonine allyl ester 34. A mixture of 33 (17 mg,
3.9 mol), powdered Zn (76 mg, 1.2 mmol), and AcOH (77 l,
1.4 mmol) in EtOAc (1 ml) was placed in a round-bottom flask
equipped with a reflux condenser. The atmosphere was replaced
with a balloon of Ar. The reaction mixture was stirred under mi-
crowave irradiation at 150 W for 1 h. The microwave machine was
controlled so as to continuously irradiate the flask during this
period. The mixture was diluted with EtOAc, filtered through
Celite, and the filtrate was concentrated in vacuo. The residue was
dissolved in EtOAc, successively washed with satd NaHCO3 aq,
water, and brine, dried over Na2SO4, and concentrated in vacuo.
The residue was dissolved in a mixture of CH2Cl2 (0.4 ml) and
b
-
-
D
-galactopyranosyl-(1/4)-2-acetamido-3,6-di-O-
b
-
D
-glucopyranosyl-(1/3)-2,6-di-O-ben-
b
D
-glucopyranosyl-(1/3)-2,6-di-O-benzyl- -gal-
b-D
b
-
D
b-D-
b
-
D
b-D
b-D
b
-
D
b
-
D
b-D
b-D
b
-
D
b
-D
b
-
D
a-D
b
-
D
L
a-D
L
m
m
m
m
m
m
4A (500 mg) in anhydrous CH2Cl2 (3.5 ml) at ꢀ15 ꢁC as describe
above for 25 (procedure A). The mixture was stirred for 5 h before
adding satd NaHCO3 aq to quench the reaction, and worked up.
The product was chromatographed using a column of LH-60 with
CHCl3–CH3OH (3:2) to give 32 (92 mg, 86%). [
a
]
þ11.9 (c 1). Rf
D
0.63 (7:3 toluene–EtOAc). 1H NMR:
d
7.74 (br d, 2H, J¼7.6 Hz, Ar),
7.58 (m, 2H, Ar), 7.41–7.20 (m, 104H, Ar), 6.95 (d, 1H, J¼7.0 Hz,
TCANH), 6.68–6.65 (m, 3H, TCANHꢂ3), 5.91 (m, 1H, –CH]CH2),
5.65 (d, 1H, J¼9.3 Hz, FmocNH), 5.50–5.47 (m, 3H, H-4c, H-4e, H-
4 g), 5.33 (br d, 1H, J¼17.3 Hz, –CH2]CH2), 5.22 (br d, 1H,
J¼10.3 Hz, –CH2]CH2), 5.05–4.99 (m, 4H, H-1d, H-1f, H-1 h,
–CH2Ph), 4.96 (d, 1H, J¼2.9 Hz, H-1a), 1.38 (s, 3H, –CH3), 1.34 (s, 3H,
MeOH (0.1 ml), and stirred with Ac2O (40 ml) at room temperature
for 2 h. The mixture was concentrated in vacuo to the residue,
which was chromatographed on Bio-beads S-X1 with toluene–
EtOAc (1:1) and then by recycled HPLC [JAIGEL-2H with CHCl3] to
–CH3), 1.31 (d, 3H, J¼5.9 Hz, Thr-
g
H). 13C NMR:
d
92.1 and 92.3
afford 34 (13.5 mg, 86%). [
a
]
þ24.0 (c 0.7). Rf 0.21 (1:9 toluene–
D
(–COCCl3), 98.8 and 99.0 (C-1d, C-1f, and C-1h), 99.4 (C-1a), 99.5
(C-1b), 102.0, and 102.1 (C-1c, C-1e, C-1g, and C-1i), 103.7 (C-1j),
109.5 [(CH3)2C<]. MALDI TOF MS: calcd for C239H249Cl15N8O57
EtOAc). 1H NMR:
d
7.75 (d, 2H, J¼7.4 Hz, Ar), 7.61 (br d, 2H,
J¼7.3 Hz, Ar), 7.40–7.21 (m, 104H, Ar), 5.82 (m, 1H, –CH]CH2), 5.71
(d, 1H, J¼9.3 Hz, –NH), 5.66 (d, 1H, J¼9.3 Hz, –NH), 5.61 (d, 1H,

1754
A. Ueki et al. / Tetrahedron 66 (2010) 1742–1759
J¼8.3 Hz, –NH), 5.30 (d, 1H, J¼17.6 Hz, –CH]CH2), 1.81, 1.66, 1.47,
and 1.25 (br s, 15H, –COCH3ꢂ5), 1.39 (s, 3H, –CH3), 1.34 (s, 3H,
53.7 Hz, H-1a), 5.34 (dd, 1H, J¼1.0, 17.2 Hz, –CH]CH2), 5.19 (dd, 1H,
J¼1.0, 10.7 Hz, –CH]CH2), 5.04 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.95 (d,
1H, J¼11.2 Hz, –CH2Ph), 4.82 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.72 (d, 1H,
J¼11.2 Hz, –CH2Ph), 4.64 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.55 (d, 1H,
J¼12.2 Hz, –CH2Ph), 4.53 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.38 (d, 1H,
J¼12.2 Hz, –CH2Ph), 4.37 (d, 1H, J¼7.8 Hz, H-1b), 4.33 (d, 1H,
J¼12.2 Hz, –CH2Ph), 4.24 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.18–4.07 (m,
4H, –CH2CH]CH2, H-4a, H-2a), 3.91 (dd, 1H, J¼2.4, 10.7 Hz, H-6a),
3.86 (br d, 1H, J¼2.4 Hz, H-4b), 3.84 (m, 1H, H-5a), 3.73 (br t, 1H,
J¼9.8 Hz, H-3a), 3.72 (dd, 1H, J¼7.8, 9.3 Hz, H-2b), 3.57 (br d, 1H,
J¼10.2 Hz, H-6a), 3.47 (br t, 1H, J¼6.6 Hz, H-6b), 3.39–3.32 (m, 2H,
–CH3), 1.14 (br s, 3H, Thr-gH). MALDI TOF MS: calcd for
C235H262N6O55 [average, (MþNa)þ and (MþK)þ] m/z 4073.59 and
4089.70. Found 4071.66 and 4087.46.
4.1.27. N-(9-Fluorenylmethoxycarbonyl)-O-{2,6-di-O-benzyl-3,4-O-
isopropylidene-
benzyl-2-deoxy-
actopyranosyl-(1/4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-
glucopyranosyl-(1/3)-2,6-di-O-benzyl- -galactopyranosyl-(1/
4)-2-acetamido-3,6-di-O-benzyl-2-deoxy- -glucopyranosyl-(1/
3)-2,6-di-O-benzyl- -galactopyranosyl-(1/4)-2-acetamido-3,6-
di-O-benzyl-2-deoxy- -glucopyranosyl-(1/6)-[2,3,4,6-tetra-O-
benzyl- -galactopyranosyl-(1/3)]-2-acetamido-2-deoxy- -gal-
actopyranosyl}- -threonine 2. A mixture of 34 (13 mg, 3.2 mol),
Pd(PPh3)4 (1 mg, 0.8 mol), and dimedone (5,5-dimethyl-1,3-
b
-
D
-galactopyranosyl-(1/4)-2-acetamido-3,6-di-O-
b
-D
-glucopyranosyl-(1/3)-2,6-di-O-benzyl- -gal-
b-D
b-D-
b-D
b
-
D
H-5b, H-6b), 3.28 (dd, 1H, J¼2.4, 9.3 Hz, H-3b). 13C NMR:
d 161.9
b
-
D
(Cl3CCONH), 134.9 (–CH]CH2), 116.5 (–CH]CH2), 105.5 (d,
b
-
D
1JCF¼222.7 Hz, C-1a,
a-F), 102.7 (C-1b), 92.1 (–CCl3), 82.2 (C-3b),
b
-D
a
-
D
79.7 (C-2b), 76.4 (C-3a), 75.5 (C-4a), 75.4 (–CH2Ph), 74.6 (–CH2Ph),
74.5 (–CH2Ph), 73.4 (C-4b), 73.4 (C-5a), 73.4 (–CH2Ph), 73.1
(–CH2Ph), 73.1 (C-5b), 71.4 (–CH2CH]CH2), 68.1 (C-6b), 66.9 (C-6a),
L
m
m
2
cyclohexanedione, 9 mg, 0.06 mmol) in anhydrous THF (2 ml) was
stirred at room temperature under Ar for 30 min, and the con-
centrated in vacuo. The residue was subjected to gel-permeation
chromatography on LH-20 with CHCl3–CH3OH (3:2) to afford 2
54.5 (d, JCF¼24.8 Hz, C-2a). MALDI TOF MS: calcd for
C52H55NO10Cl3F (MþNa)þ m/z 1000.28. Found: 1000.25. Anal. calcd
for C52H55NO10Cl3F: C, 63.77; H, 5.66; N, 1.43. Found: C, 63.81; H,
5.72; N, 1.49.
quantitatively. [
a
]
d
þ28.0 (c 0.3). Rf 0.48 (90:10:1 CHCl3–CH3OH–
D
AcOH). 1H NMR:
1.47, 1.38, 1.34, and 1.25 (br s, 21H, –COCH3ꢂ5,
4.1.30. 3-O-Allyl-2,4,6-tri-O-benzyl-
3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-D
b
-
D
-galactopyranosyl-(1/4)-
-glucopyranosyl (N-
–CH3ꢂ2), 0.85 (d, 3H, J¼6.3 Hz, Thr-
gH). MALDI TOF MS: calcd for
C232H258N6O55 [average, (MþNa)þ and (MþK)þ] m/z 4033.53 and
phenyl)-2,2,2-trifluoroacetimidate 38. A suspension of 36
(108.3 mg, 0.11 mmol), (N-Phenyl)-2,2,2-trifluoroacetimidoyl
chloride (46 mg, 0.22 mmol), and K2CO3 (30.6 mg, 0.22 mmol) in
acetone (2 ml) was stirred at room temperature for 3 h. The re-
action mixture was filtrated and concentrated in vacuo. The crude
product was chromatographed on silica gel with toluene–EtOAc
(19:1) to afford 38 (119.2 mg, 94%). Rf 0.51 (7:1 toluene–EtOAc). 1H
4049.63. Found 4031.95 and 4046.96.
4.1.28. 3-O-Allyl-2,4,6-tri-O-benzyl-
3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-
36. Compound 35 (92.7 mg, 76 mol) was desilylated with 1 M
TBAF/THF (0.31 ml, 0.31 mmol) and AcOH (44 l, 0.76 mmol) in
b
-
D
-galactopyranosyl-(1/4)-
D
-glucopyranose
m
m
freshly distilled THF (1 ml) as described for 21. The crude product
was chromatographed on silica gel with toluene–EtOAc (7:1–4:1) to
afford 36 (73.9 mg, 99%). Mp 125.5–127.0 ꢁC (recrystallized from
NMR:
d
7.36–7.16 (m, 27H, Ar), 7.08 (br t, 1H, J¼7.6 Hz, ]NPh), 6.76
(d, 2H, J¼7.8 Hz, ]NPh), 6.61 (d, 1H, J¼7.3 Hz, –NH), 6.45 (br s, 1H,
H-1a), 5.94 (m, 1H, –CH]CH2), 5.34 (dd, 1H, J¼1.5, 17.1 Hz,
–CH]CH2), 5.19 (dd, 1H, J¼1.5, 10.7 Hz, –CH]CH2), 5.00 (d,
1H, J¼11.2 Hz, –CH2Ph), 4.95 (d, 1H, J¼11.7 Hz, –CH2Ph), 4.84 (d,
1H, J¼10.7 Hz, –CH2Ph), 4.74 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.64
(d, 1H, J¼11.2 Hz, –CH2Ph), 4.53 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.53 (d,
1H, J¼11.2 Hz, –CH2Ph), 4.39 (d, 1H, J¼11.7 Hz, –CH2Ph), 4.38 (d,
1H, J¼7.8 Hz, H-1b), 4.34 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.26 (d, 1H,
J¼12.2 Hz, –CH2Ph), 4.22–4.16 (m, 3H, H-2a, –CH2CH]CH2), 4.12
(br t, 1H, J¼9.0 Hz, H-4a), 3.87 (dd, 1H, J¼3.4, 10.7 Hz, H-6a), 3.86
(br d, 1H, J¼2.9 Hz, H-4b), 3.83–3.77 (m, 2H, H-3a, H-5a), 3.73 (dd,
1H, J¼7.8, 9.8 Hz, H-2b), 3.56 (br d, 1H, J¼10.7 Hz, H-6a), 3.48 (br t,
1H, J¼6.0 Hz, H-6b), 3.40–3.34 (m, 2H, H-5b, H-6b), 3.30 (dd, 1H,
hexane–EtOAc). Rf 0.42 (4:1 toluene–EtOAc). 1H NMR:
d 7.34–7.12
(m, 25H, Ar), 6.84 (d, 1H, J¼8.3 Hz, –NH), 5.93 (m, 1H, –CH]CH2),
5.38 (br t, 1H, J¼3.7 Hz, H-1a), 5.32 (dd, 1H, J¼1.5, 17.1 Hz,
–CH]CH2), 5.18 (dd, 1H, J¼1.5, 10.2 Hz, –CH]CH2), 5.02 (d, 1H,
J¼10.7 Hz, –CH2Ph), 4.94 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.82 (d,
1H, J¼11.2 Hz, –CH2Ph), 4.76 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.60 (d, 1H,
J¼10.7 Hz, –CH2Ph), 4.52 (d, 1H, J¼11.7 Hz, –CH2Ph), 4.51 (d,
1H, J¼12.2 Hz, –CH2Ph), 4.34 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.34 (d,1H,
J¼7.8 Hz, H-1b), 4.30 (d, 1H, J¼11.7 Hz, –CH2Ph), 4.20 (d, 1H,
J¼11.7 Hz, –CH2Ph), 4.16 (dd, 2H, J¼1.5, 5.4 Hz, –CH2CH]CH2), 4.12
(m, 1H, H-2a), 4.03–3.94 (m, 2H, H-5a, H-4a), 3.84 (br d,
1H, J¼2.9 Hz, H-4b), 3.81 (m, 1H, H-6a), 3.79 (dd, 1H, J¼7.8, 10.2 Hz,
H-3a), 3.71 (dd,1H, J¼7.8, 9.3 Hz, H-2b), 3.60 (br d,1H, J¼10.2 Hz, H-
6a), 3.43 (br t, 1H, J¼8.0 Hz, H-6b), 3.37–3.25 (m, 3H, H-5b, H-6b, H-
J¼2.9, 9.8 Hz, H-3b). 13C NMR:
d 161.8 (Cl3CCONH), 143.0
(>C]NPh), 134.9 (–CH]CH2), 124.6 (]NPh), 119.3 (]NPh), 116.5
(–CH]CH2), 102.8 (C-1b), 92.1 (–CCl3), 82.3 (C-3b), 79.7 (C-2b),
76.4 (C-3a), 75.6 (C-4a), 75.4 (–CH2Ph), 74.5 (–CH2Ph), 74.4
(–CH2Ph), 73.9 (C-5a), 73.4 (–CH2Ph), 73.4 (C-4b), 73.2 (–CH2Ph),
73.2 (C-5b), 71.5 (–CH2CH]CH2), 68.2 (C-6b), 67.2 (C-6a), 53.6 (C-
2a). Anal. calcd for C60H60N2O11Cl3F3: C, 62.75; H, 5.27; N, 2.44.
Found: C, 62.80; H, 5.33; N, 2.36.
3b), 2.95 (dd, 1H, J¼1.5, 3.4 Hz, –OH). 13C NMR:
d 161.7 (Cl3CCONH),
134.9 (–CH]CH2), 116.4 (–CH]CH2), 103.0 (C-1b), 92.5 (C-1a), 90.8
(-CCl3), 82.2 (C-3b), 79.8 (C-2b), 77.3 (C-3a), 77.0 (C-4a), 75.3
(–CH2Ph), 74.5 (–CH2Ph), 74.5 (–CH2Ph), 73.5 (C-4b), 73.4 (–CH2Ph),
73.1 (–CH2Ph), 73.1 (C-5b), 71.5 (–CH2CH]CH2), 70.9 (C-5a), 68.1
(C-6b), 68.1 (C-6a), 54.6 (C-2a). MALDI TOF MS: calcd for
C52H56NO11Cl3 (MþNa)þ, m/z 998.28. Found: 998.17. Anal. calcd for
C52H56NO11Cl3: C, 63.90; H, 5.78; N, 1.43. Found: C, 63.90; H, 5.80;
N, 1.44.
4.1.31. tert-Butyldiphenylsilyl
anosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-
glucopyranoside 39. A mixture of Ir(COD)(PMe2Ph)2PF6 (5 mg,
mol) in freshly distilled THF (5 ml) was stirred at room
2,4,6-tri-O-benzyl-b-D-galactopyr-
b-D-
6
m
4.1.29. 3-O-Allyl-2,4,6-tri-O-benzyl-
3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-
fluoride 37. Compound 36 (91.3 mg, 93 mol) was fluorinated with
DAST (25 l, 0.19 mmol) in THF (2 ml) as described for 22. The crude
product was chromatographed on silica gel with hexane–EtOAc (7:
1) to give 37 as a mixture of anomers (88.1 mg, 97%,
¼>9). Rf
0.46 (2:1 hexane–EtOAc). 1H NMR:
7.34–7.14 (m, 25H, Ar), 6.66 (d,
1H, J¼7.8 Hz, –NH), 5.94 (m, 1H, –CH]CH2), 5.75 (dd, 1H, J¼2.4,
b
-
D
-galactopyranosyl-(1/4)-
temperature for 15 min under H2, and the atmosphere was
replaced by Ar. To the mixture of activated Ir complex in THF
was added a solution of 35 (250 mg, 0.21 mmol) in THF (5 ml)
under Ar. The mixture was stirred for 30 min before adding
water (2 ml) and iodine (104 mg, 0.41 mmol), and stirred for
10 min. The reaction mixture was diluted EtOAc, successively
washed with satd Na2S2O3 aq, water, and brine, dried over
Na2SO4, and concentrated in vacuo. The crude product was
D
-glucopyranosyl
m
m
a/b
d

A. Ueki et al. / Tetrahedron 66 (2010) 1742–1759
1755
chromatographed on silica gel with toluene–EtOAc (19:1–9:1)
6a, H-5c, Gal H-6ꢂ2, Gal H-5), 3.38–3.25 (m, 4H, Gal H-5, Gal H-3,
Gal H-6ꢂ2), 3.19 (br d, 1H, J¼11.2 Hz, H-6a), 2.87 (br d, 1H,
to afford 39 (240 mg, 99%).
Mp 100.0–101.0 ꢁC (recrystallized from hexane–EtOAc). [
a
]
J¼8.8 Hz, H-5a), 1.04 (s, 9H, tBu). 13C NMR:
d 161.6 and 161.5
D
ꢀ4.5 (c 1). Rf 0.34 (9:1 toluene–EtOAc). 1H NMR:
d
7.69 (d, 1H,
(Cl3CCONHꢂ2), 134.9 (–CH]CH2), 116.5 (–CH]CH2), 103.0, and
102.5 (C-1b and C-1d), 100.2 (C-1c), 94.8 (C-1a), 92.6, and 92.4
(–CCl3ꢂ2), 82.2 (C-3d), 71.5 (–CH2–CH]CH2), 59.3 (C-2a), 57.7 (C-
2c), 26.8 [–C(CH3)3], 19.1 [–C(CH3)3]. MALDI TOF MS: calcd for
C117H124N2O21Cl6Si (MþNa)þ m/z 2153.65. Found: 2153.97. Anal.
calcd for C117H124N2O21Cl6Si: C, 65.82; H, 5.85; N, 1.31. Found: C,
65.61; H, 5.88; N, 1.29.
J¼6.8 Hz, Ar), 7.69 (d, 1H, J¼7.8 Hz, Ar), 7.64 (d, 1H, J¼6.8 Hz, Ar),
7.64 (d, 1H, J¼8.3 Hz, Ar), 7.40–7.13 (m, 31H, Ar), 6.90 (d, 1H,
J¼7.8 Hz, –NH), 4.95 (d, 1H, J¼11.7 Hz, –CH2Ph), 4.93 (d, 1H,
J¼7.8 Hz, H-1a), 4.75 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.72 (d,
1H, J¼10.2 Hz, –CH2Ph), 4.60 (d, 1H, J¼11.7 Hz, –CH2Ph), 4.57 (d, 1H,
J¼11.7 Hz, –CH2Ph), 4.57 (d, 1H, J¼11.7 Hz, –CH2Ph), 4.56 (d, 1H,
J¼10.2 Hz, –CH2Ph), 4.44 (d, 1H, J¼7.8 Hz, H-1b), 4.40 (d,
1H, J¼12.2 Hz, –CH2Ph), 4.37 (d, 1H, J¼11.7 Hz, –CH2Ph), 4.37 (d, 1H,
J¼11.7 Hz, –CH2Ph), 4.26 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.26 (d,
1H, J¼12.2 Hz, –CH2Ph), 4.07 (br t, 1H, J¼8.5 Hz, H-4a), 3.89 (br t,
1H, J¼9.0 Hz, H-3a), 3.83 (d, 1H, J¼3.4 Hz, H-4b), 3.75 (dd, 1H, J¼7.8,
9.8 Hz, H-2a), 3.63 (dd, 1H, J¼2.9, 10.7 Hz, H-6a), 3.57–3.43 (m, 4H,
H-5b, H-6b, H-2b, H-3b), 3.36 (dd, 1H, J¼3.9, 8.3 Hz, H-6b), 3.34 (dd,
1H, J¼2.4, 10.7 Hz, H-6a), 3.08 (br d, 1H, J¼8.8 Hz, H-5a), 2.17 (d, 1H,
Byproduct 43: 1H NMR:
d
6.86 (d, 1H, J¼7.8 Hz, –NH), 6.66 (d, 1H,
J¼7.3 Hz, –NH), 6.60 (br d, 1H, J¼7.8 Hz, –NH), 5.94 (m, 1H,
–CH]CH2), 1.03 (s, 9H, tBu). MALDI TOF MS calcd for
C166H174N3O31Cl9Si (MþNa)þ: m/z 3077.31. Found: 3078.97.
Procedure B (glycosylation of 39 with 38). A mixture of 39
(1.36 g, 1.15 mmol), 38 (1.39 g, 1.21 mmol), and dried MS AW-300
powder (3.5 g) in anhydrous CH2Cl2 (35 ml) was cooled at ꢀ78 ꢁC
with stirring under Ar for 10 min. To the cold mixture was added
J¼8.8 Hz, –OH), 1.06 (s, 9H, tBu). 13C NMR:
d
161.5 (Cl3CCONH), 102.7
TMSOTf (10
m
l, 0.06 mmol). The mixture was stirred at ꢀ78 ꢁC for
(C-1b), 94.8 (C-1a), 92.6 (-CCl3), 80.4 (C-3b), 77.7 (C-3a), 76.1 (C-4a),
75.8 (C-4b), 75.2 (C-5a), 75.0 (–CH2Ph), 74.9 (–CH2Ph), 74.0 (C-5b),
73.9 (–CH2Ph), 73.3 (C-2b), 73.3 (–CH2Ph), 73.2 (–CH2Ph), 68.0 (C-
6b), 67.6 (C-6a), 59.4 (C-2a), 26.8 [–C(CH3)3],19.1 [–C(CH3)3]. MALDI
TOF MS: calcd for C65H70NO11Cl3Si (MþNa)þ m/z 1196.37. Found:
1196.55. Anal. calcd for C65H70NO11Cl3Si: C, 66.40; H, 6.00; N, 1.19.
Found: C, 66.16; H, 6.00; N, 1.19.
1 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 aq, water, and
brine, dried over MgSO4, and concentrated in vacuo. The crude
product was chromatographed on Bio-beads S-X3 with toluene–
EtOAc (3:1) and then on silica gel with toluene–EtOAc (14:1–9:1)
to give 40 (2.32 g, 94%).
4.1.32. tert-Butyldiphenylsilyl 3-O-allyl-2,4,6-tri-O-benzyl-
actopyranosyl-(1/4)-3, 6-di-O-benzyl-2-deoxy-2-tri-
chloroacetamido- -glucopyranosyl-(1/3)-2,4,6-tri-O-benzyl-
galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-tri-
chloroacetamido- -glucopyranoside 40. Procedure A (glycosyla-
tion of 39 with 37). A mixture of Cp2ZrCl2 (12 mg, 42 mol), AgClO4
(17 mg, 84 mol), and dried MS 4A powder (70 mg) in anhydrous
b
-
D
-gal-
4.1.33. 3-O-Allyl-2,4,6-tri-O-benzyl-
3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-
(1/3)-2,4,6-tri-O-benzyl- -galactopyranosyl-(1/4)-3,6-di-O-
benzyl-2-deoxy-2-trichloroacetamido- -glucopyranose
41. Compound 40 (978 mg, 0.46 mmol) was desilylated with 1 M
TBAF/THF (1.8 ml, 1.83 mmol) and AcOH (0.26 ml, 4.58 mmol) in
THF (5 ml) as described for 21. The crude product was chromato-
graphed on silica gel with toluene–EtOAc (5:1–4:1–2:1) to afford 41
(850 mg, 98%). Rf 0.40 and 0.22 (4:1 toluene–EtOAc). 1H NMR:
b
-
D
-galactopyranosyl-(1/4)-
b-D
-glucopyranosyl-
b
-
D
b
-
D
-
b-D
D
b-
D
m
m
CH2Cl2 (1 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 37 (41 mg, 42
m
mol) and 39 (41 mg, 42
m
mol)
d
7.37–7.08 (m, 50H, Ar), 6.82 (d, 1H, J¼8.8 Hz, –NH), 6.64 (d, 1H,
in anhydrous CH2Cl2 (1 ml). Then the temperature was raised to
ꢀ15 ꢁC during the period of 30 min and stirring was continued for
further 2.5 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 aq, water,
and brine, dried over MgSO4, and concentrated in vacuo. The
residue was purified by preparative TLC, which was eluted twice
with toluene–EtOAc (19:1) to give 40 (22 mg, 29%). Hex-
asaccharide 43 (8 mg, 16%) and fully protected disaccharide 35
(5 mg, 9%) were obtained from the more polar band and the less
J¼7.8 Hz, –NH), 5.93 (m, 1H, –CH]CH2), 5.36–5.30 (m, 2H,
–CH]CH2 and H-1a), 5.19 (dd, 1H, J¼1.5, 10.2 Hz, –CH]CH2), 5.09
(m, 1H, H-1c), 4.98 (d, 1H, J¼11.7 Hz, –CH2Ph), 4.96 (d, 1H,
J¼10.7 Hz, –CH2Ph), 4.94 (d, 2H, J¼11.2 Hz, –CH2Phꢂ2), 4.83 (d,
1H, J¼11.2 Hz, –CH2Ph), 4.80 (m, 1H, –CH2Ph), 4.78 (d, 1H,
J¼10.7 Hz, –CH2Ph), 4.71 (d, 1H, J¼11.7 Hz, –CH2Ph), 4.54 (d, 1H,
J¼11.2 Hz, –CH2Ph), 4.52 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.51 (d,
1H, J¼11.2 Hz, –CH2Ph), 4.48 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.47 (d, 1H,
J¼12.2 Hz, –CH2Ph), 4.45 (d, 1H, J¼10.2 Hz, –CH2Ph), 4.42 (d,
1H, J¼7.8 Hz, Gal H-1), 4.37 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.28 (d, 1H,
J¼11.7 Hz, –CH2Ph), 4.27 (d, 1H, J¼11.7 Hz, –CH2Ph), 4.26 (m, 1H, Gal
H-1), 4.25 (d, 1H, J¼10.2 Hz, –CH2Ph), 4.19 (d, 1H, J¼11.7 Hz,
–CH2Ph), 4.20–4.15 (m, 2H, –CH2CH]CH2), 4.14 (d, 1H, J¼11.7 Hz,
–CH2Ph), 4.10–3.99 (m, 2H, H-2a, H-4c), 3.93 (br t, 1H, J¼9.3 Hz, H-
4a), 3.90 (s, 1H, Gal H-4), 3.86 (d, 1H, J¼2.4 Hz, Gal H-4), 3.83–3.78
(m, 4H, H-5a, H-6c, H-3c, H-2c), 3.75–3.65 (m, 6H, H-6c, Gal H-2ꢂ2,
H-3a, Gal H-3, H-6a), 3.55 (m,1H, H-5c), 3.46–3.30 (m, 7H, H-6a, Gal
H-6, -OH, Gal H-5ꢂ2, Gal H-6, Gal H-3), 3.28–3.25 (m, 2H, Gal H-
polar band, respectively. Compound 40: [
a
]
ꢀ14.0 (c 1). Rf 0.41
D
(9:1 toluene–EtOAc). 1H NMR:
d
7.66 (d, 2H, J¼6.8 Hz, Ar), 7.62 (d,
2H, J¼6.8 Hz, Ar), 7.45–7.08 (m, 56H, Ar), 6.88 (d, 1H, J¼7.3 Hz,
–NH), 6.63 (d, 1H, J¼7.8 Hz, –NH), 5.93 (m, 1H, –CH]CH2), 5.33
(dd, 1H, J¼1.5, 17.1 Hz, –CH]CH2), 5.18 (dd, 1H, J¼1.5, 10.7 Hz,
–CH]CH2), 5.08 (d, 1H, J¼7.3 Hz, H-1c), 4.96 (d, 1H, J¼11.2
Hz, –CH2Ph), 4.94 (d, 2H, J¼9.8 Hz, –CH2Phꢂ2), 4.88 (d, 1H,
J¼10.2 Hz, –CH2Ph), 4.84 (d, 1H, J¼7.3 Hz, H-1a), 4.81 (d,
1H, J¼11.2 Hz, –CH2Ph), 4.77 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.66 (d,
1H, J¼12.2 Hz, –CH2Ph), 4.63 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.52
(d, 1H, J¼11.2 Hz, –CH2Ph), 4.50 (d, 1H, J¼10.2 Hz, –CH2Ph), 4.47 (d,
2H, J¼11.7 Hz, –CH2Phꢂ2), 4.45 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.42 (d,
1H, J¼7.8 Hz, Gal H-1), 4.38 (d, 1H, J¼7.3 Hz, Gal H-1), 4.37 (d, 1H,
J¼10.2 Hz, –CH2Ph), 4.36 (d, 1H, J¼11.7 Hz, –CH2Ph), 4.31 (d, 1H,
J¼11.7 Hz, –CH2Ph), 4.28 (d, 1H, J¼11.7 Hz, –CH2Ph), 4.21–4.14 (m,
5H, –CH2Phꢂ3, –CH2CH]CH2), 4.03 (br t, 1H, J¼8.0 Hz, H-4c), 4.01
(br t, 1H, J¼8.0 Hz, H-4a), 3.90 (d, 1H, J¼2.4 Hz, Gal H-4), 3.85 (d,
1H, J¼2.9 Hz, Gal H-4), 3.85–3.63 (m, 9H, H-6c, GlcNTCA H-3ꢂ2,
Gal H-3, GlcNTCA H-2ꢂ2, H-6c, Gal H-2ꢂ2), 3.52–3.41 (m, 5H, H-
6ꢂ2). 13C NMR:
161.7 and 161.6 (Cl3CCONHꢂ2), 134.9 (–CH]CH2),
d
116.5 (–CH]CH2), 103.0 and 102.8 (C-1b, C-1d), 100.3 (C-1c), 92.5
and 92.3 (–CCl3ꢂ2), 90.7 (C-1a), 82.2 (C-3d), 71.5 (–CH2–CH]CH2),
57.5 (C-2c), 54.4 (C-2a). MALDI TOF MS: calcd for C101H106N2O21Cl6
(MþNa)þ m/z 1915.53. Found: 1915.87. Anal. calcd for
C101H106N2O21Cl6: C, 63.96; H, 5.63; N, 1.48. Found: C, 64.00; H,
5.72; N, 1.53.
4.1.34. 3-O-Allyl-2,4,6-tri-O-benzyl-
3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-
(1/3)-2,4,6-tri-O-benzyl- -galactopyranosyl-(1/4)-3,6-di-O-
b
-
D
-galactopyranosyl-(1/4)-
b-D
-glucopyranosyl-
b-D

1756
A. Ueki et al. / Tetrahedron 66 (2010) 1742–1759
benzyl-2-deoxy-2-trichloroacetamido-
D
-glucopyranosyl (N-phenyl)-
2114.21. Anal. calcd for C114H120N2O21Cl6Si: C, 65.36; H, 5.77; N,1.34.
Found: C, 65.41; H, 5.84; N, 1.40.
2,2,2-trifluoroacetimidate 42. Compound 41 (739 mg, 0.39 mmol)
was reacted with (N-Phenyl)-2,2,2-trifluoroacetimidoyl chloride
(162 mg, 0.78 mmol) and K2CO3 (270 mg, 1.95 mmol) in acetone
(5 ml) as described for 38. The crude product was chromatographed
on Bio-beads S-X3 with toluene–EtOAc (3:1) to afford 42 (774 mg,
4.1.36. tert-Butyldiphenylsilyl 3-O-allyl-2,4,6-tri-O-benzyl-
actopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-tri-
chloroacetamido- -glucopyranosyl-(1/3)-2,4,6-tri-O-benzyl-
galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-tri-
chloroacetamido- -glucopyranosyl-(1/3)-2,4,6-tri-O-benzyl-
galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-tri-
chloroacetamido- -glucopyranosyl-(1/3)-2,4,6-tri-O-benzyl-
galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-tri-
chloroacetamido- -glucopyranoside 45. A mixture of 42 (702 mg,
b-D-gal-
b
-
D
b-D-
96%). Rf 0.41 (7:1 toluene–EtOAc). 1H NMR:
d 7.38–7.05 (m, 53H, Ar),
6.75–6.71 (m, 3H, ]NPhꢂ2, –NH), 6.59 (d, 1H, J¼7.8 Hz, –NH), 6.43
(br s, 1H, H-1a), 5.94 (m, 1H, –CH]CH2), 5.34 (dd, 1H, J¼1.5, 17.1 Hz,
–CH]CH2), 5.19 (dd, 1H, J¼1.5, 10.2 Hz, –CH]CH2), 5.12 (d, 1H,
J¼7.3 Hz, H-1c), 4.98 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.96–4.92 (m, 3H,
–CH2Phꢂ3), 4.84 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.80–4.75 (m, 2H,
–CH2Phꢂ2), 4.71 (d, 1H, J¼11.7 Hz, –CH2Ph), 4.58 (d, 1H, J¼11.2 Hz,
–CH2Ph), 4.54 (d, 1H, J¼11.7 Hz, –CH2Ph), 4.52 (d, 2H, J¼11.2 Hz,
–CH2Phꢂ2), 4.47 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.47 (d, 1H,
J¼10.2 Hz, –CH2Ph), 4.43 (d, 1H, J¼7.3 Hz, Gal H-1), 4.38 (d, 1H,
J¼10.2 Hz, –CH2Ph), 4.35 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.31 (d,
1H, J¼11.7 Hz, –CH2Ph), 4.30 (d, 1H, J¼7.3 Hz, Gal H-1), 4.29 (d, 1H,
J¼11.7 Hz, –CH2Ph), 4.20 (d, 2H, J¼11.7 Hz, –CH2Phꢂ2), 4.21–4.11
(m, 3H, –CH2CH]CH2, H-2a), 4.09 (br t,1H, J¼9.8 Hz, GlcNTCA H-4),
4.04 (br t, 1H, J¼8.6 Hz, GlcNTCA H-4), 3.93 (br s,1H, Gal H-4), 3.87–
3.83 (m, 3H, Gal H-4, GlcNTCA H-3, GlcNTCA H-6), 3.80–3.70 (m,
7H, H-2c, Gal H-2ꢂ2, Gal H-3, GlcNTCA H-3, GlcNTCA H-6ꢂ2), 3.62–
3.57 (m, 2H, GlcNTCA H-5ꢂ2), 3.48–3.32 (m, 7H, Gal H-6, GlcNTCA
H-6, Gal H-6ꢂ2, Gal H-5ꢂ2, Gal H-3), 3.27 (dd,1H, J¼4.9, 8.3 Hz, Gal
b
-
D
b-D-
b
-
D
b-D-
b-D
0.34 mmol), 44 (647 mg, 0.31 mmol), and dried MS AW-300 (1 g) in
anhydrous CH2Cl2 (10 ml) was cooled at ꢀ78 ꢁC with stirring under
Ar for 10 min. To the cold mixture was added TMSOTf (3 ml,
15
m
mol). Then the temperature was raised to ꢀ40 ꢁC over a period
of 30 min and stirring was continued for further 1.5 h, before the
reaction was quenched with satd NaHCO3 aq. The mixture was
diluted with EtOAc and filtered through Celite. The filtrate was
successively washed with satd NaHCO3 aq, water, and brine, dried
over MgSO4, and concentrated in vacuo. The crude product was
chromatographed on Bio-beads S-X1 with toluene–EtOAc (1:1) and
then by recycled HPLC [Mightysil Si 60 (20ꢂ250 mm, 5
mm, Kanto
Chemical Co.) with CHCl3–EtOAc (87:13)] to give 45 (1.14 g, 93%).
[a
]
D ꢀ20.7 (c 1). Rf 0.20 (9:1 toluene–EtOAc). 1H NMR:
d 7.65 (d, 1H,
H-6). 13C NMR:
d
161.8 (Cl3CCONH), 161.6 (Cl3CCONH), 143.0
J¼6.3 Hz, Ar), 7.65 (d, 1H, J¼7.8 Hz, Ar), 7.61 (d, 1H, J¼6.8 Hz, Ar),
7.61 (d, 1H, J¼7.8 Hz, Ar), 7.39–7.05 (m, 106H, Ar), 6.87 (d, 1H,
J¼7.8 Hz, –NH), 6.68 (d, 1H, J¼7.3 Hz, –NH), 6.64 (d, 1H, J¼7.8 Hz,
–NH), 6.60 (d, 1H, J¼7.8 Hz, –NH), 5.94 (m, 1H, –CH]CH2), 5.33 (dd,
1H, J¼1.5, 17.1 Hz, –CH]CH2), 5.19 (dd, 1H, J¼1.5, 10.7 Hz,
–CH]CH2), 5.11 (d, 1H, J¼7.3 Hz, GlcNTCA H-1), 5.04 (m, 1H,
GlcNTCA H-1), 5.01 (m, 1H, GlcNTCA H-1), 5.00–4.92 (m, 5H,
–CH2Phꢂ5), 4.89–4.69 (m, 10H, –CH2Phꢂ9, H-1a), 4.64 (d, 1H,
J¼11.7 Hz, –CH2Ph), 4.61 (d, 1H, J¼11.7 Hz, –CH2Ph), 4.53–4.45 (m,
10H, –CH2Phꢂ10), 4.43–4.25 (m, 13H, –CH2Phꢂ9, Gal H-1ꢂ4), 4.24–
4.12 (m, 7H, –CH2Phꢂ5, –CH2CH]CH2ꢂ2), 4.06–3.98 (m, 4H,
GlcNTCA H-4ꢂ4), 3.91 (d, 1H, J¼2.4 Hz, Gal H-4), 3.87–3.86 (m, 3H,
Gal H-4ꢂ3), 3.84–3.23 (m, 38H, GlcNTCA H-2ꢂ4, GlcNTCA H-3ꢂ4,
GlcNTCA H-5ꢂ3, GlcNTCA H-6ꢂ7, Gal H-2ꢂ4, Gal H-3ꢂ4, Gal H-
(>C]NPh), 134.9 (–CH]CH2), 116.5 (–CH]CH2), 103.1 and 102.6
(C-1b and C-1d), 100.3 (C-1c), 92.4 and 92.1 (–CCl3ꢂ2), 82.2 (C-3d),
71.6 (–CH2–CH]CH2), 57.7 (C-2c), 53.5 (C-2a). Anal. calcd for
C109H110N3O21Cl6F3: C, 63.31; H, 5.36; N, 2.03. Found: C, 63.50; H,
5.40; N, 2.04.
4.1.35. tert-Butyldiphenylsilyl
anosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-
glucopyranosyl-(1/3)-2,4,6-tri-O-benzyl- -galactopyranosyl-
(1/4)-3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido- -glucopyr-
2,4,6-tri-O-benzyl-b-D-galactopyr-
b-D-
b-D
b-D
anoside 44. Compound 40 (877 mg, 0.41 mmol) was deallylated by
Ir-catalyzed olefin isomerization and hydroiodination as described
for 39. The crude product was chromatographed on silica gel with
toluene–EtOAc (7:1) to afford 44 (837 mg, 97%). [
a
]D ꢀ14.3 (c 1). Rf
5ꢂ4, Gal H-6ꢂ8), 3.18 (dd, 1H, J¼2.0, 11.2 Hz, H-6a), 2.86 (br d, 1H,
0.50 (4:1 toluene–EtOAc). 1H NMR:
d
7.66 (d, 1H, J¼6.3 Hz, Ar), 7.66
J¼8.8 Hz, H-5a), 1.03 (s, 9H, Bu). 13C NMR:
d
161.6 (Cl3CCONHꢂ2),
t
(d, 1H, J¼7.8 Hz, Ar), 7.62 (d, 1H, J¼6.8 Hz, Ar), 7.62 (d, 1H, J¼8.3 Hz,
Ar), 7.39–7.09 (m, 56H, Ar), 6.88 (d, 1H, J¼7.8 Hz, –NH), 6.65 (d, 1H,
J¼8.3 Hz, –NH), 5.11 (d, 1H, J¼7.3 Hz, H-1c), 4.97 (d, 1H, J¼11.7 Hz,
–CH2Ph), 4.94 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.89 (d, 1H, J¼10.2 Hz,
–CH2Ph), 4.85 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.84 (d, 1H, J¼8.3 Hz, H-
1a), 4.75 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.70 (d, 1H, J¼11.2 Hz, –CH2Ph),
4.67 (d,1H, J¼11.7 Hz, –CH2Ph), 4.63 (d,1H, J¼11.7 Hz, –CH2Ph), 4.58
(d, 1H, J¼11.2 Hz, –CH2Ph), 4.53 (d, 1H, J¼10.7 Hz, –CH2Ph), 4.51 (d,
1H, J¼11.7 Hz, –CH2Ph), 4.50 (d, 1H, J¼10.2 Hz, –CH2Ph), 4.46 (d, 1H,
J¼11.2 Hz, –CH2Ph), 4.42 (d, 1H, J¼9.8 Hz, H-1d), 4.40 (d,
1H, J¼11.7 Hz, –CH2Ph), 4.39 (d, 1H, J¼8.3 Hz, H-1b), 4.37 (d, 1H,
J¼12.2 Hz, –CH2Ph), 4.33 (d, 1H, J¼11.7 Hz, –CH2Ph), 4.31 (d, 1H,
J¼11.7 Hz, –CH2Ph), 4.22 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.19 (d,
1H, J¼12.2 Hz, –CH2Ph), 4.16 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.07 (br t,
1H, J¼8.5 Hz, H-4c), 4.02 (br t, 1H, J¼8.5 Hz, H-4a), 3.91 (d, 1H,
J¼2.9 Hz, H-4b), 3.86–3.80 (m, 3H, H-4d, H-6c, H-3c), 3.78–3.70 (m,
5H, H-3a, H-3b, H-6c, H-2c, H-2a), 3.66 (dd, 1H, J¼7.8, 9.3 Hz, H-2b),
3.52–3.42 (m, 8H, H-5c, H-3d, H-2d, H-6a, H-6b, H-6d, H-5b, H-5d),
3.36–3.29 (m, 2H, H-6b, H-6d), 3.19 (dd, 1H, J¼2.0, 11.2 Hz, H-6a),
161.5, and 161.4 (Cl3CCONHꢂ2), 134.9 (–CH]CH2), 116.5
(–CH]CH2), 103.0, 102.9, 102.8, and 102.5 (Gal C-1ꢂ4), 100.2 (C-1),
100.1 and 100.1 (GlcNTCA C-1ꢂ3), 94.8 (C-1a), 92.6 (–CCl3), 92.3
(–CCl3ꢂ3), 82.2 (C-3h), 71.5 (–CH2–CH]CH2), 59.2 (C-2a), 57.6
(GlcNTCA C-2), 57.5 (GlcNTCA C-2ꢂ2), 26.8 [–C(CH3)3], 19.1
[–C(CH3)3]. MALDI TOF MS: calcd for C215H224N4O41Cl12Si (MþNa)þ
m/z 3996.15 (100%). Found: 3996.72. Anal. calcd for
C215H224N4O41Cl12Si: C, 64.99; H, 5.68; N, 1.41. Found: C, 64.89; H,
5.78; N, 1.43.
4.1.37. 3-O-Allyl-2,4,6-tri-O-benzyl-
3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-
(1/3)-2,4,6-tri-O-benzyl- -galactopyranosyl-(1/4)-3,6-di-O-
benzyl-2-deoxy-2-trichloroacetamido- -glucopyranosyl-(1/3)-
2,4,6-tri-O-benzyl- -galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-
deoxy-2-trichloroacetamido- -glucopyranosyl-(1/3)-2,4,6-tri-O-
benzyl- -galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-tri-
chloroacetamido- -glucopyranose 46. Compound 45 (908 mg,
b
-
D
-galactopyranosyl-(1/4)-
b-D
-glucopyranosyl-
b-D
b-D
b-D
b-D
b-D
D
0.23 mmol) was desilylated with 1 M TBAF/THF (0.9 ml, 0.91 mmol)
and AcOH (0.13 ml, 2.29 mmol) in freshly distilled THF (5 ml) as
described for 21. The crude product was chromatographed on Bio-
beads S-X1 with toluene–EtOAc (1:1) to quantitatively afford 46
2.87 (br d, 1H, J¼9.3 Hz, H-5a), 2.20 (d, 1H, J¼4.9 Hz, –OH), 1.04 (s,
t
9H, Bu). 13C NMR:
d
161.6 and 161.5 (Cl3CCONHꢂ2), 102.9 (1JCH
161.9 Hz, C-1d), 102.5 (1JCH 161.1 Hz, C-1b), 100.1 (1JCH 166.2 Hz, C-
1c), 94.8 (1JCH 164.5 Hz, C-1a), 92.6 and 92.4 (-CCl3ꢂ2), 75.2 (C-3d),
59.3 (C-2a), 57.7 (C-2c), 26.8 [–C(CH3)3], 19.1 [–C(CH3)3]. MALDI TOF
MS: calcd for C114H120N2O21Cl6Si (MþNa)þ m/z 2113.61. Found:
(854 mg). Rf 0.40 and 0.18 (4:1 toluene–EtOAc).1H NMR:
d 7.38–7.03
(m, 100H, Ar), 6.83 (d, 1H, J¼8.8 Hz, –NH), 6.70 (d, 1H, J¼7.8 Hz,
–NH), 6.68 (d, 1H, J¼9.8 Hz, –NH), 6.63 (d, 1H, J¼7.8 Hz, –NH), 5.94

A. Ueki et al. / Tetrahedron 66 (2010) 1742–1759
1757
(m, 1H, –CH]CH2), 5.34 (dq, 1H, J¼1.5, 17.1 Hz, –CH]CH2), 5.29 (br
t, 1H, J¼3.2 Hz, H-1a), 5.19 (br dd, 1H, J¼1.5, 10.2 Hz, –CH]CH2),
5.11 (br d, 1H, J¼6.8 Hz, GlcNTCA H-1), 5.04 (d, 1H, J¼7.3 Hz,
GlcNTCA H-1), 5.02 (d, 1H, J¼7.3 Hz, GlcNTCA H-1), 4.98 (d, 1H,
J¼12.2 Hz, –CH2Ph), 4.97–4.93 (m, 5H, –CH2Phꢂ5), 4.88 (d, 1H,
J¼10.2 Hz, –CH2Ph), 4.87 (d, 1H, J¼10.7 Hz, –CH2Ph), 4.83 (d, 1H,
J¼11.2 Hz, –CH2Ph), 4.82 (d, 1H, J¼11.7 Hz, –CH2Ph), 4.83–4.67 (m,
6H, –CH2Phꢂ6), 4.53–4.11 (m, 30H, –CH2Phꢂ24, Gal H-1ꢂ4,
–CH2CH]CH2ꢂ2), 4.06–3.26 (m, 49H, GlcNTCA H-2ꢂ4, GlcNTCA H-
3ꢂ4, GlcNTCA H-4ꢂ4, GlcNTCA H-5ꢂ4, GlcNTCA H-6ꢂ8, Gal H-
2ꢂ4, Gal H-3ꢂ4, Gal H-4ꢂ4, Gal H-5ꢂ4, Gal H-6ꢂ8, –OH). 13C NMR:
of 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 water and brine, dried over MgSO4, and concentrated
in vacuo. The crude product was chromatographed on Bio-beads S-
X1 with toluene–EtOAc (1:1) and then by recycled HPLC on JAIGEL-
2H with CH2Cl2 to give 48 (324 mg, 85%). [
a
]
D ꢀ2.1 (c 1). Rf 0.39 (4:1
toluene–EtOAc). 1H NMR:
d
7.73 (d, 2H, J¼7.8 Hz, Ar), 7.60 (d, 1H,
J¼6.8 Hz, Ar), 7.58 (d,1H, J¼6.8 Hz, Ar), 7.39–7.05 (m,124H, Ar), 6.99
(d, 1H, J¼7.8 Hz, TCANH), 6.69 (d, 1H, J¼7.8 Hz, TCANH), 6.68 (d, 1H,
J¼7.3 Hz, TCANH), 6.63 (d, 1H, J¼6.8 Hz, TCANH), 5.92 (m, 2H,
–CH]CH2ꢂ2), 5.67 (d, 1H, J¼9.3 Hz, FmocNH), 5.36–5.31 (m, 2H,
–OCH2CH]CH2 and –CO2CH2CH]CH2), 5.24–5.17 (m, 2H,
–CO2CH2CH]CH2 and –OCH2CH]CH2), 5.12 (br d, 1H, J¼6.8 Hz,
GlcNTCA H-1), 5.07–5.03 (m, 2H, GlcNTCA H-1ꢂ2), 5.02 (d, 1H,
J¼10.7 Hz, –CH2Ph), 4.98–4.85 (m, 11H, –CH2Phꢂ10, GlcNTCA H-1),
4.82–4.65 (m, 14H, –CH2Phꢂ11, GalN3 H-1, –CO2CH2CH]CH2ꢂ2),
d
161.6, 161.6, and 161.6 (Cl3CCONHꢂ4), 134.8 (–CH]CH2), 116.5
(–CH]CH2), 103.0 (Gal C-1), 102.8 (Gal C-1ꢂ2), 102.7 (Gal C-1),
100.2 (GlcNTCA C-1ꢂ2), 100.1 (GlcNTCA C-1), 92.5 (–CCl3), 92.3
(–CCl3ꢂ3), 90.7 (C-1a), 82.1 (C-3h), 71.5 (–CH2CH]CH2), 57.6, 57.4,
and 57.4 (GlcNTCA C-2ꢂ3), 54.4 (C-2a). MALDI TOF MS: calcd for
C199H206N4O41Cl12 (MþNa)þ m/z 3758.03 (100%). Found: 3758.85.
Anal. calcd for C199H206N4O41Cl12þH2O; C, 63.68; H, 5.59; N, 1.49.
Found: C, 63.57; H, 5.64; N, 1.61.
4.58–4.34 (m, 24H, –CH2Phꢂ17, Gal H-1ꢂ4, Thr-
bH, –CH2CHAr2,
H), 4.32–4.13 (m, 14H, –CH2Phꢂ9, Gal H-1, –CH2CHAr2,
Thr-
a
–CH2CHAr2, –OCH2CH]CH2ꢂ2), 4.11–3.26 (m, 60H, GalN3 H-2,
GalN3 H-3, GalN3 H-4, GalN3 H-5, GalN3 H-6ꢂ2, GlcNTCA H-2ꢂ4,
GlcNTCA H-3ꢂ4, GlcNTCA H-4ꢂ4, GlcNTCA H-5ꢂ4,, GlcNTCA H-
6ꢂ8, Gal H-2ꢂ5, Gal H-3ꢂ5, Gal H-4ꢂ5, Gal H-5ꢂ5, Gal H-6ꢂ10),
4.1.38. 3-O-Allyl-2,4,6-tri-O-benzyl-
3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-
(1/3)-2,4,6-tri-O-benzyl- -galactopyranosyl-(1/4)-3,6-di-O-
benzyl-2-deoxy-2-trichloroacetamido- -glucopyranosyl-(1/3)-
2,4,6-tri-O-benzyl- -galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-
deoxy-2-trichloroacetamido- -glucopyranosyl-(1/3)-2,4,6-tri-O-
benzyl- -galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-tri-
chloroacetamido- -glucopyranosyl (N-phenyl)-2,2,2-trifluoroacetim-
idate 47. Compound 46 (101 mg, 27 mol) was reacted with (N-
Phenyl)-2,2,2-trifluoroacetimidoyl chloride (11 mg, 54 mol) and
K2CO3 (19 mg, 135 mol) in acetone (0.5 ml) as described for 38.
b
-
D
-galactopyranosyl-(1/4)-
b-D
-glucopyranosyl-
b-D
b
-
D
3.05 (br s, 1H, –OH), 1.31 (d, 3H, J¼6.3 Hz, Thr-
g d 169.9
H). 13C NMR:
(–CO2All), 161.6 (Cl3CCONH), 161.5 (Cl3CCONHꢂ2), 161.5
b
-
D
b-D
(Cl3CCONH), 156.7 (–OCONH), 134.8 (–OCH2CH]CH2), 131.3
b
-
D
(–CO2CH2CH]CH2),
119.4
(–CO2CH2CH]CH2),
116.5
D
(–OCH2CH]CH2), 103.9 (C-1j), 103.0 and 102.8 (Gal C-1ꢂ2), 102.8
(Gal C-1ꢂ2), 100.2 (GlcNTCA C-1), 100.1 (GlcNTCA C-1ꢂ2), 99.8 (C-
1a), 99.7 (C-1b), 92.4 (–CCl3), 92.3 (–CCl3ꢂ3), 82.1 (C-3i), 75.8 (Thr
m
m
m
C-3),
71.5
(–OCH2CH]CH2),
67.4
(–CH2CHAr2),
66.4
The crude product was chromatographed on Bio-beads S-X1 with
toluene–EtOAc (1:1) to afford 47 (104 mg, 98%). Rf 0.19 (7:1 tolu-
(–CO2CH2CH]CH2), 59.0 (C-2b), 58.7 (Thr C-2), 57.5 and 57.4
(GlcNTCA C-2ꢂ3), 57.0 (C-2a), 47.0 (–CH2CHAr2), 18.7 (Thr C-4).
Anal. calcd for C261H270N8O54Cl12: C, 65.19; H, 5.66; N, 2.33. Found:
C, 65.24; H, 5.75; N, 2.33.
ene–EtOAc). 1H NMR:
d 7.38–7.03 (m, 103H, Ar), 6.75–6.68 (m, 5H,
]NPhꢂ2, –NHꢂ3), 6.61 (d, 1H, J¼7.3 Hz, –NH), 6.43 (br s, 1H, H-1a),
5.94 (m,1H, –CH]CH2), 5.34 (dd,1H, J¼1.5,17.1 Hz, –CH]CH2), 5.18
(br d, 1H, J¼10.2 Hz, –CH]CH2), 5.13 (d, 1H, J¼6.8 Hz, GlcNTCA H-
1), 5.07 (br d, 2H, J¼6.8 Hz, GlcNTCA H-1ꢂ2), 5.01–4.69 (m, 15H,
–CH2Phꢂ15), 4.59–4.25 (m, 25H, –CH2Phꢂ21, Gal H-1ꢂ4), 4.21–
3.99 (m, 11H, –CH2Phꢂ4, H-2a, –CH2CH]CH2ꢂ2, GlcNTCA H-4ꢂ4),
3.93–3.25 (m, 43H, GlcNTCA H-2ꢂ3, GlcNTCA H-3ꢂ4, GlcNTCA H-
5ꢂ4, GlcNTCA H-6ꢂ8, Gal H-2ꢂ4, Gal H-3ꢂ4, Gal H-4ꢂ4, Gal H-
4.1.40. N-(9-Fluorenylmethoxycarbonyl)-O-{3-O-allyl-2,4,6-tri-O-
benzyl-
2-deoxy-
b
-
b
D
-galactopyranosyl-(1/4)-2-acetamido-3,6-di-O-benzyl-
-glucopyranosyl-(1/3)-2,4,6-tri-O-benzyl- -gal-
-
D
b-D
actopyranosyl-(1/4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-
glucopyranosyl-(1/3)-2,4,6-tri-O-benzyl- -galactopyranosyl-
(1/4)-2-acetamido-3,6-di-O-benzyl-2-deoxy- -glucopyranosyl-
(1/3)-2,4,6-tri-O-benzyl- -galactopyranosyl-(1/4)-2-acet-
amido-3,6-di-O-benzyl-2-deoxy- -glucopyranosyl-(1/6)-[2,3,4,6-
tetra-O-benzyl- -galactopyranosyl-(1/3)]-2-acetamido-2-deoxy-
-galactopyranosyl}- -threonine allyl ester 49. Compound 48
(324 mg, 67 mol) was reduced with powdered Zn (1.32 g,
b-D-
b-D
b-D
5ꢂ4, Gal H-6ꢂ8). 13C NMR:
d
161.7, 161.5 (Cl3CCONHꢂ4), 142.9
b-D
(>C]NPh), 134.8 (–CH]CH2), 124.4, and 119.2 (]NPh), 116.4
(–CH]CH2), 103.0 (Gal C-1), 102.8 (Gal C-1ꢂ2), 102.4 (Gal C-1),
100.2 (GlcNTCA C-1ꢂ2), 100.1 (GlcNTCA C-1), 92.3 (–CCl3ꢂ3), 92.0
(–CCl3), 82.1 (C-3h), 71.5 (–CH2CH]CH2), 57.5 (GlcNTCA C-2ꢂ2),
b-D
b-D
a-D
L
m
57.4
(GlcNTCA
C-2),
53.4
(C-2a).
Anal.
calcd
for
20.22 mmol), and AcOH (1.35 ml, 23.6 mmol) in EtOAc (17 ml) un-
der microwave irradiation at 150 W for 1 h, and the crude product
was treated with Ac2O (0.7 ml) in a mixture of CH2Cl2 (5.6 ml) and
MeOH (1.4 ml) at room temperature for 2 h as described for 34. The
resulting product was chromatographed on Bio-beads S-X1 with
toluene–EtOAc (1:1) and then by recycled HPLC on JAIGEL-2H with
C207H210N5O41Cl12F3: C, 63.65; H, 5.42; N, 1.79. Found: C, 63.83; H,
5.50; N, 1.89.
4.1.39. N-(9-Fluorenylmethoxycarbonyl)-O-{3-O-allyl-2,4,6-tri-O-
benzyl-
chloroacetamido-
galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-tri-
chloroacetamido- -glucopyranosyl-(1/3)-2,4,6-tri-O-benzyl-
galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-tri-
chloroacetamido- -glucopyranosyl-(1/3)-2,4,6-tri-O-benzyl-
galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-tri-
chloroacetamido- -glucopyranosyl-(1/6)-[2,3,4,6-tetra-O-ben-
zyl- -galactopyranosyl-(1/3)]-2-azido-2-deoxy- -galactopyr-
anosyl}- -threonine allyl ester 48. A mixture of 4 (95 mg, 87 mol),
47 (309 mg, 79 mol), and dried MS AW-300 (400 mg) in anhy-
drous CH2Cl2 (4 ml) was cooled at –78 ꢁC with stirring under Ar for
b
-
D
-galactopyranosyl-(1/4)-3,6-di-O-benzyl-2-deoxy-2-tri-
b
-
D
-glucopyranosyl-(1/3)-2,4,6-tri-O-benzyl-
b
-
D
-
CHCl3 to afford 49 (261 mg, 88%). [
a
]
þ9.2 (c 1). Rf 0.30 (1:4 tol-
D
uene–EtOAc). 1H NMR:
d
7.74 (d, 2H, J¼7.3 Hz, Ar), 7.61 (d, 1H,
b
-
D
b
-
D
-
J¼7.3 Hz, Ar), 7.39–7.14 (m, 125H, Ar), 5.98–5.76 (m, 4H,
–CH]CH2ꢂ2, AcNHꢂ2), 5.66 (br d, 1H, J¼8.8 Hz, FmocNH), 5.33
(dd, 1H, J¼1.5, 17.1 Hz, –OCH2CH]CH2), 5.30 (br d, 1H, J¼17.1 Hz,
–CO2CH2CH]CH2), 5.24 (dd, 1H, J¼1.0, 10.2 Hz, –CO2CH2CH]CH2),
5.18 (dd, 1H, J¼1.5, 10.2 Hz, –OCH2CH]CH2), 5.17–5.15 (m, 3H,
AcNHꢂ3), 5.01–4.60 (m, 25H, –CH2Phꢂ19, GlcNAc H-1ꢂ4, GalNAc
H-1, –CH2CHAr2), 4.54–4.14 (m, 42H, –CH2Phꢂ29, Gal H-1ꢂ5,
b
-
D
b-D-
b-D
b
-
D
a-D
L
m
m
–CH2CHAr2, –CH2CHAr2, Thr-
3H, –COCH3), 1.44 (s, 9H, –COCH3ꢂ3), 1.29–1.23 (m, 6H, –COCH3,
Thr- 170.4, 170.0, 169.7, and 165.5 (–CO2All,
H). 13C NMR:
–NHCOCH3ꢂ5), 156.3 (–OCONH), 134.7 (–OCH2CH]CH2), 130.9
a
H, Thr-bH, CH2CH]CH2ꢂ4), 1.66 (s,
10 min. To the cold mixture was added 5% TMSOTf/CH2Cl2 (0.7
m
l,
g
d
4
m
mol). Then the temperature was raised to ꢀ40 ꢁC over a period

1758
A. Ueki et al. / Tetrahedron 66 (2010) 1742–1759
(–CO2CH2CH]CH2),
124.8,
119.7
(–CH2CHAr2),
119.3
to produce an octapeptide (SAPDTRPA)-resin by the Fastmoc
(–CO2CH2CH]CH2), 116.2 (–OCH2CH]CH2), 102.8, 102.8 (Gal
C-1ꢂ5), 101.6 (GlcNTCA C-1ꢂ4), 99.4 (GalNAc C-1), 75.6 (Thr C-3),
71.3 (–OCH2CH]CH2), 67.0 (–CO2CH2CH]CH2), 65.9 (–CH2CHAr2),
58.4 (Thr C-2), 55.9 (GlcNTCA C-2), 46.9 (–CH2CHAr2), 29.4 (Thr C-
4), 22.9 (–COCH3). MALDI TOF MS: calcd for C263H286N6O55 [aver-
age, (MþNa)þ] m/z 4434.08. Found: 4434.42. Anal. calcd for
C263H286N6O55$2H2O: C, 71.03; H, 6.57; N, 1.89. Found: C, 71.05; H,
6.55; N, 1.93.
program of the synthesizer, using 20% piperidine/NMP for
N-deprotection and HBTU/HOBt as the condensing agent. 2,2,4,6,7-
Pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) group was
employed for the protection of Arg, and t-Bu groups were used for
the protection of Thr, Ser and Asp, respectively. A part of the oc-
tapeptide resin (5
tube, to which a mixture of 50 (44 mg, 10
(40 l, 40 mol) and 0.1 M DCC/NMP (40
m
mol) was transferred into a polypropylene test
mol), 0.1 M HOBt/NMP
l, 40 mol) was added.
m
m
m
m
m
The mixture was heated for 5 h at 50 ꢁC in an oven with stirring by
a vortex mixer and then at room temperature for 12 h, and filtered.
The resin was washed with CH2Cl2–MeOH (1:1) and several times
with NMP. The N-terminal three amino acids were introduced
4.1.41. N-(9-Fluorenylmethoxycarbonyl)-O-{3-O-allyl-2,4,6-tri-O-
benzyl-
2-deoxy-
b
-
b
D
-galactopyranosyl-(1/4)-2-acetamido-3,6-di-O-benzyl-
-glucopyranosyl-(1/3)-2,4,6-tri-O-benzyl- -gal-
-
D
b-D
actopyranosyl-(1/4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-
glucopyranosyl-(1/3)-2,4,6-tri-O-benzyl- -galactopyranosyl-
(1/4)-2-acetamido-3,6-di-O-benzyl-2-deoxy- -glucopyranosyl-
(1/3)-2,4,6-tri-O-benzyl- -galactopyranosyl-(1/4)-2-acet-
amido-3,6-di-O-benzyl-2-deoxy- -glucopyranosyl-(1/6)-[2,3,4,6-
tetra-O-benzyl- -galactopyranosyl-(1/3)]-2-acetamido-2-deoxy-
-galactopyranosyl}- -threonine 50. A mixture of 49 (53 mg,
0.012 mmol), dimedone (34 mg, 0.24 mmol), and Pd(PPh3)4 (0.7 mg,
0.6 mol) in freshly distilled THF (0.5 ml) was stirred under Ar at
b
-
D
-
manually using Fmoc amino acid (20
m
mol), 0.1 M HOBt/NMP
b
-
D
(30 l, 30 mol) and 0.1 M DCC/NMP (30
m
m
m
l, 30 mol) to give
m
b
-
D
dodecaglycopeptide (HGVTSAPDTRPA)-resin (27 mg). 4-Methoxy-
trityl (Mmt) group was employed for the protection of His. To a part
of the resin (9 mg) was added an ice-cooled solution of reagent K
b-D
b-D
b
-
D
(TFA/phenol/water/thioanisole/ethanedithiol, 33:2:2:2:1, 150 ml),
a-D
L
and the mixture stirred at 0 ꢁC for 6 h. Then the resin was filtered
off, and the volatile materials in the mixture were evaporated in
a stream of N2. Diethyl ether was added to the residue to precipitate
the product, which was separated by centrifugation. The precipitate
was washed several times by suspending in diethyl ether and then
centrifuging to give a crude product, to which was added a mixture
m
room temperature for 30 min, before concentrated in vacuo. The
crude product was chromatographed on Bio-beads S-X1 with tolu-
ene–EtOAc (1:1) and then by recycled HPLC on JAIGEL-2H with CHCl3
to give 50 (43 mg, 83%). [
a
]D þ10.4 (c 2). Rf 0.39 (9:1 CHCl3–MeOH,1%
of TFA/DMS/m-cresol (5:3:1, 107 ml), and the mixture was cooled at
AcOH). 1H NMR (DMSO-d6):
d
12.88 (br s, 1H, –CO2H), 7.89–7.86 (m,
ꢀ15 ꢁC. A mixture of TFA/DMS/m-cresol/TfOH (5:3:1:1, 51
ml) was
7H, Arꢂ2, AcNHꢂ5), 7.73–7.70 (m, 2H, Ar, FmocNH), 7.38–7.12 (m,
125H, Ar), 5.96 (m, 1H, –CH]CH2), 5.35 (br d, 1H, J¼17.1 Hz,
–CH]CH2), 5.16 (br d, 1H, J¼9.8 Hz, –CH]CH2), 5.00–4.66 (m, 19H,
–CH2Phꢂ15, GlcNAc H-1ꢂ4), 4.66–4.08 (m, 46H, –CH2Phꢂ33, Gal H-
added to the mixture. The reaction mixture was stirred at ꢀ15 ꢁC
for 15 h, before the reaction was terminated by the addition of
diethyl ether preliminarily cooled at ꢀ80 ꢁC. The mixture was
centrifuged to separate the debenzylated product, which was
washed three times with diethyl ether and centrifuged as men-
tioned above to give a precipitate. The crude product was dissolved
in 50% CH3CN aq and purified by preparative HPLC on a column of
1ꢂ5, GalNAc H-1, –CH2CHAr2ꢂ2, –CH2CHAr2, Thr-
aH, Thr-bH,
CH2CH]CH2ꢂ2), 1.78 (s, 3H, –COCH3), 1.67 (s, 3H, –COCH3), 1.58 and
1.57 (sꢂ2, 9H, –COCH3ꢂ3), 1.08 (d, 3H, J¼5.9 Hz, Thr-
H). 13C NMR
(CDCl3):
193.1 (–CO2H), 169.9, 169.0, and 165.7 (, –NHCOCH3ꢂ5),
g
d
Mightysil (KANTO) RP-18 (5
m
m, 250ꢂ10 mm) with a gradient
156.5 (–OCONH), 135.5 (–CH]CH2), 125.3, 120.1 (–CH2CHAr2ꢂ2),
115.9 (–CH]CH2), 104.9, 102.7, 102.2, and 101.6 (Gal C-1ꢂ5, GlcNTCA
C-1ꢂ4), 98.8 (GalNAc C-1), 70.5 (–CH2CH]CH2), 65.7 (–CH2CHAr2),
46.8 (–CH2CHAr2), 29.4 (Thr C-4), 23.0 and 22.8 (–COCH3ꢂ5). MALDI
TOF MS: calcd for C260H282N6O55 [100% (MþNa)þ] m/z 4392.94.
Found: 4392.81. Anal. calcd for C260H282N6O55$4H2O: C, 70.28; H,
6.58; N, 1.89. Found: C, 70.28; H, 6.55; N, 1.92.
elution of aq CH3CN (23%–33%/20 min) containing 0.1% TFA (flow
rate: 2.5 ml/min). The major fraction was collected and lyophilized
to afford 54 (0.5 mg, 4.8% overall yield based on the value of Gly in
the amino-acid analysis). MALDI TOF MS: calcd for C135H207N23O69
(MþH)þ m/z 3255.35. Found: 3255.16. Amino-acid analysis: Asp0.92
Thr1.63 Ser0.92 Pro1.82 Gly1.00 Ala1.79 Val(þGlcNAc)1.98 His0.78 Arg0.93
.
Acknowledgements
4.1.42. N-(9-Fluorenylmethoxycarbonyl)-O-{
(1/4)-2-acetamido-2-deoxy- -glucopyranosyl-(1/3)-
actopyranosyl-(1/4)-2-acetamido-2-deoxy- -glucopyranosyl-
(1/3)- -galactopyranosyl-(1/4)-2-acetamido-2-deoxy- -glu-
copyranosyl-(1/3)- -galactopyranosyl-(1/4)-2-acetamido-2-
deoxy- -glucopyranosyl-(1/6)-[ -galactopyranosyl-(1/3)]-2-
acetamido-2-deoxy- -galactopyranosyl}- -threonine (De-
protection of decasaccharyl threonine 50). Decasaccharide 50
b
-
D
-galactopyranosyl-
b
-
D
b-D
-gal-
This work was supported by a grant-in-aid for creative scientific
research (17GS0420) from the Japan Society for the Promotion of
Science. We thank Dr. T. Chihara and his staff at Riken for com-
bustion analyses, and Tokyo Chemical Industry Co. Ltd. for the
generous gift of Phenyl 3-O-allyl-2,4,6-tri-O-benzyl-1-thio-b-D-
galactopyranoside. We also thank Tokai University for their support
by a grant-aid for high-technology research.
b-D
b
-
D
b-D
b-D
b
-
D
b-D
a
-
D
L
1
(10 mg, 2.4
(5:3:1, 270
mmol) was dissolved in a mixture of TFA/DMS/m-cresol
m
l), and the mixture was cooled at ꢀ15 ꢁC. TfOH (30
ml,
0.34 mmol) was added dropwise to the mixture. The reaction
mixture was stirred at ꢀ15 ꢁC for 4 h, before the reaction was ter-
minated by the addition of diethyl ether preliminarily cooled at
ꢀ80 ꢁC. The mixture was centrifuged to precipitate the product,
which was washed three times with diethyl ether and centrifuged
as mentioned above. The crude product was purified by preparative
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