Solid-phase synthesis of core 2 O-linked glycopeptide and its enzymatic sialylation

Article abstract of DOI:10.1016/j.tet.2003.08.047

The core 2-type tetrasaccharide building blocks (1a/1b) for solid-phase synthesis of glycopeptide were synthesized via stereoselective glycosylation of the disaccharyl Ser/Thr (3a/3b) with a glycosyl fluoride (2) carrying the 2-trichloroacetamido group that was readily converted into a 2-acetamido group by reduction. A segment of glycoprotein leukosialin (215-224) was synthesized by the solid-phase protocol, the building block (1b) being utilized. Cleavage of the synthetic glycopeptide from resin was effected with reagent K and subsequent treatment of the product with a cocktail for the 'low-acidity TfOH' facilitated complete removal of the benzyl groups with minimum loss of glycosidic linkages. To the deprotected glycopeptide (21), were enzymatically introduced N-acetylneuraminic acid (sialic acid) residues in remarkably high efficiency by using the specific sialyltransferases.

Full text of DOI:10.1016/j.tet.2003.08.047

Tetrahedron 59 (2003) 8415–8427
Solid-phase synthesis of core 2 O-linked glycopeptide
and its enzymatic sialylation
*
Yutaka Takano, Naoya Kojima, Yuko Nakahara, Hironobu Hojo and Yoshiaki Nakahara
Department of Applied Biochemistry, Institute of Glycotechnology, Tokai University, 1117 Kitakaname, Hiratsuka-shi,
Kanagawa 259-1292, Japan
Received 25 June 2003; accepted 19 August 2003
Abstract—The core 2-type tetrasaccharide building blocks (1a/1b) for solid-phase synthesis of glycopeptide were synthesized via
stereoselective glycosylation of the disaccharyl Ser/Thr (3a/3b) with a glycosyl fluoride (2) carrying the 2-trichloroacetamido group that was
readily converted into a 2-acetamido group by reduction. A segment of glycoprotein leukosialin (215–224) was synthesized by the solid-
phase protocol, the building block (1b) being utilized. Cleavage of the synthetic glycopeptide from resin was effected with reagent K and
subsequent treatment of the product with a cocktail for the ‘low-acidity TfOH’ facilitated complete removal of the benzyl groups with
minimum loss of glycosidic linkages. To the deprotected glycopeptide (21), were enzymatically introduced N-acetylneuraminic acid (sialic
acid) residues in remarkably high efficiency by using the specific sialyltransferases.
Published by Elsevier Ltd.
1. Introduction
carrying a biologically significant core 2 O-linked glycan.
The glycans are aberrantly expressed on the T-cell surface
glycoproteins due to altered O-glycosylation both when the
T-cells are activated by IL-2 and anti-CD 3 antibodies,6 and
when the T-cells are derived from patients with immuno-
deficiency syndromes such as Wiskott–Aldrich syndrome7
and AIDS.8 The typical structure of core 2 oligosaccharide,
disialylated glycohexaosyl threonine, has recently been
synthesized in a benzyl-protected form based on coupling
of the strategically designed trisaccharide donor and
acceptors.9 However, the quantity of hexasaccharide
obtained was inadequate to use further in the studies of
solid-phase synthesis of a glycopeptide. Therefore, we
decided to design an alternative route to the core 2
hexasaccharide by taking advantage of enzymatic sialyl-
ation. Enzymatic technologies are now readily applicable by
using commercial enzymes and have often been utilized for
the syntheses of sialoglycopeptides not only in solution but
also on resin.10,11 In this paper, we describe the stereo-
controlled synthesis of core 2 tetrasaccharide building
blocks, solid-phase synthesis of a glycopeptide with the
building block, and sialylation of the glycopeptide with the
commercial a2-3 sialyltransfases. An outline of this study is
depicted in Figure 1. The early part of this work has
preliminarily been reported.12
There is increasing demand for a variety of glycopeptide
samples to be used for studying glycoprotein-mediated
biological phenomena. Over the past 10 years, we have been
engaged in research directed toward the establishment of
facile synthetic methods for glycopeptides with unambigu-
ous glycan structure. The usefulness of the synthesized
glycopeptides in biological studies has recently been
demonstrated by a chitobiose-linked henhexacontapeptide,
the Ig domain I of extracellular matrix metalloproteinase
inducer (Emmprin) which is a metastasis-related glyco-
protein produced by tumor cells and stimulates fibroblasts to
release proteolytic enzymes. The synthesized glycopeptide
was proven to present remarkable matrix metalloproteinase
2 (MMP 2)-inducing activity, while little activity was
observed for the non-glycosylated peptide.1 More recently,
we have accomplished synthesis of the pentasaccharide-
linked Ig domain fully based on the Fmoc solid-phase
protocol.2 In addition, we have achieved the solid-phase
synthesis of O-linked (core 1) glycopeptide such as the
B-chain of a2HS glycoprotein,3 the N-terminal region of
glycophorin A,4 and the C-terminal of hCG (human
chorionic gonadotropin) b-subunit.5 In these studies either
benzyl-protected or non-protected oligosaccharide-linked
Fmoc amino acids were utilized as the key building blocks.
We also became interested in the synthesis of glycopeptide
2. Synthesis of the building block 1a and 1b
Keywords: glycosylation; glycopeptide; solid-phase synthesis;
sialyltransferase.
Synthetic studies were started for the key building blocks 1a
and 1b. Based on consideration of convergent synthesis
strategy, the tetrasaccharide was retrosynthesized into two
*
Corresponding author. Tel./fax: þ81-463-50-2075;
e-mail: yonak@keyaki.cc.u-tokai.ac.jp
0040–4020/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.tet.2003.08.047

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Y. Takano et al. / Tetrahedron 59 (2003) 8415–8427
Figure 1. Synthetic strategy for the core 2 hexasaccharide-linked glycopeptide.
disaccharide precursors (2 and 3). Since disaccharyl diol 3a
and 3b were readily produced by debenzylidenation of 4a
and 4b which had already been prepared in this group,13
another disaccharide 2 was synthesized as the glycosyl donor
in which an N-trichloroacetyl protecting group was employed
in order to facilitate b-selective glycosylation (Fig. 2).14 As
shown in Scheme 1, known t-butyldiphenylsilyl 2-azido-
3,6-di-O-benzyl-2-deoxy-b-D-glucopyranoside 69b was gly-
cosylated with 2,3,4,6-tetra-O-acetyl-a-D-galactopyranosyl
bromide 5 in the presence of AgOTf to give lactosamine
derivative 8. Compound 8 was deacetylated with NaOMe/
MeOH and then benzylidenated with benzaldehyde
dimethyl acetal and p-TsOH to afford 10. Protection of
the resulting hydroxyl groups by benzylation and sub-
sequent reduction of the azide group with Zn/AcOH gave
amine 14. Alternatively, compound 14 was also synthesized
from known N-phthaloylated derivative 9.15 After deacety-
lation of 9, benzylidenation was followed by benzylation to
afford 13, which was dephthaloylated with ethylenediamine
to give 14. Compound 14 was acylated with trichloroacetyl
chloride in pyridine, and then the silyl group was removed
under the conditions of fluoridolysis. The resulting hemi-
acetal (16) was converted into glycosyl fluoride 2. The
a-fluoride was produced in high stereoselectivity. Glyco-
sylation of 3a and 3b with 2 was promoted by Suzuki’s
condition16 to exclusively produce the desired tetrasacchar-
ide 17a and 17b in 72 and 70%, respectively. Transform-
ation of trichloroacetamide into acetamide was realized by
reduction with Zn–AcOH, with accompanying reduction of
the azide group. The products were acetylated with Ac2O in
MeOH. Despite the sluggish dechlorination, compounds
18a and 18b were obtained in high yields. Allyl ester was
then cleaved by catalysis of Pd(0) in the presence of
dimedone as a nucleophile in THF to furnish 1a and 1b.
3. Solid-phase synthesis of glycopeptide and deprotection
With the suitably protected building block (1b) in hand,
solid-phase synthesis of a glycopeptide carrying core 2
tetrasaccharide was next investigated. Human leukosialin
(CD 43) is a T-lymphocyte transmembrane glycoprotein
in which an extracellular domain is enriched with serine
and threonine residues, and is heavily O-glycosylated. As
described above, activated T-cells carry a leukosialin
expressing the core 2 O-hexasaccharide. A segment of
leukosialin (215–224) was synthesized as a model of core 2
glycopeptide, where 1b was incorporated as the building
block for Thr216. Solid-phase synthesis was performed on
the commercial Fmoc Sieber amide resin (0.25 mmol/g)
according to the Fmoc procedure.17 The resin can release
thesynthesized peptidecarboxamidesonexposuretoaweakly
acidic medium (e.g. 1–2% TFA in CH2Cl2). All the solid-
phase reactions were manually operated in a polypropylene
tube. Peptide condensation was facilitated by using excess
Fmoc amino acid (4 equiv.) activated with dicyclohexylcar-
bodiimide (DCC), 1-hydroxybenzotriazole (HOBt), and
diisopropylethylamine (DIEA) in N-methylpyrrolidone
Figure 2. Retrosynthetic scheme of the core 2 tetrasaccharide buildimg blocks 1a and 1b.

Y. Takano et al. / Tetrahedron 59 (2003) 8415–8427
8417
Scheme 1. Synthesis of the tetrasaccharide building blocks 1a and 1b.
(NMP), whereas N-deprotection was effected with 20%
piperidine in NMP. Side-chain functional groups of the
serine/threonine and asparagine residues were protected
with t-butyl and trityl groups, respectively. The unreacted
amino groups on the resin were capped with Ac2O at the end
of each peptide-coupling procedure until introduction of
the hydroxyl group-containing tetrasaccharide (1b). The
octapeptide (217–224) assembled on the resin was
N-deprotected and allowed to react with 2 equiv. of 1b in
the presence of O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetra-
methyluronium hexafluorophosphate (HATU) in NMP. The
crucial coupling step was run overnight. The coupling
efficiency seemed comparable with that of the reaction run
under activation with the standard DCC–HOBt combi-
nation. The resulting resin was N-deprotected and con-
densed with Fmoc-Pro–OH to complete the desired
Scheme 2. Solid-phase synthesis of the core 2 tetrasaccharide-linked peptide 21.

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Y. Takano et al. / Tetrahedron 59 (2003) 8415–8427
sequence. Conditions for deprotection of the carbohydrate
moiety were next explored with tetrasaccharide 1b, before
the synthesized glycopeptide was released from the resin.
Among the procedures tested, the ‘low-acidity TfOH’
method developed for debenzylation of the synthetic
peptides was the most promising.18 Compound 1b was
treated with TfOH in a mixture of dimethylsulfide (DMS),
m-cresol, and trifluoroacetic acid (TFA) at 2158C for 1 h.
The reaction was quenched with ethereal pyridine and the
products were analyzed by HPLC and MALDI TOF MS.
Figure 3(a) shows the chromatographic profile of the
products. The largest peak (1) represents the desired
tetrasaccharide (19), which comprises about 88% of the
carbohydrate-derived components. The small peaks 2 and 3
coincided in the mass spectra with the compounds lacking
one of the galactose residues, while peak 4 corresponded to
the disaccharide-linked threonine derived from scission of
the b-GlcNAc glycosidic linkage. Mass spectra of the peaks
Figure 3. HPLC of the debenzylation products of 1b (a), cleavage of the glycopeptide from the resin under the low-acidity TfOH conditions (b), the
debenzylation products obtained through reagent K treatment (c), and the fully deprotected glycopeptide 21 (d). Column: Mightysil RP-18 (150£4.6 mm).
Eluent A: distilled water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA. Flow rate: 1 ml/min.

Y. Takano et al. / Tetrahedron 59 (2003) 8415–8427
8419
in the less mobile fraction (5–6) agreed with those of the
compounds carrying one benzyl group. The deprotection
reaction performed at higher temperature (08C) gave rise to
apparent increases in the scission products. Based on these
experiments, we then moved to the deprotection studies
with the synthesized glycopeptide. The resin was treated
with the cocktail for the low-acidity TfOH for 1 h at 2158C
and the products precipitated by ethereal pyridine were
analyzed in the same manner. An HPLC profile of the
products is shown in Figure 3(b). Three major products were
separated and characterized by mass spectrometry. The
desired glycopeptide [20: (MþNa)þ: 1951.45] was eluted in
the fraction at the retention time of 22 min. The mass
spectrum of the second product showed the molecular ion
for the glycothreonine-missing product (octapeptide) with a
Trt group unremoved [(MþNa)þ: 1042.97]. The third
product exhibited the molecular ion (MþNa)þ of 2193.57
corresponding to the glycopeptide carrying a Trt group.
Therefore, we concluded that the procedure was insufficient
to cleave the N-Trt linkage. To achieve complete removal of
the Trt group, separate procedures for cleavage from resin
and for debenzylation were employed. The resin was treated
with reagent K (aq. CF3CO2H, thioanisole, 1,2-ethane-
dithiol, phenol) for 1 h at ambient temperature. The product
and the resin were precipitated from ether and subjected to
the debenzylation under the conditions of low-acidity TfOH
for 1 h at 2158C. The products were analyzed in detail,
and it was found that the desired glycopeptide (20) was
accompanied with the incompletely debenzylated glycopep-
tides (5–7%). Consequently, the debenzylation procedure
was performed for 2 h. Figure 3(c) exhibits an HPLC profile
of the mixture of products. In the chromatogram, peaks 1–6
are derived from (glyco)peptides. Peak 2 corresponds to
the tetrasaccharyl decapeptide (20) [(MþNa)þ: 1951.31],
while the highly hydrophilic component (peak 1) is the
unreacted octapeptide [(MþH)þ: 778.62]. Peaks 3
[(MþNa)þ: 1789.73] and 4 [(MþNa)þ: 1789.68] are the
glycopeptides losing a Gal residue. Peak 5 [(MþNa)þ:
1586.77] is attributable to the glycopeptide lacking a
Gal·GlcNAc residue. The peptide skipping the glycothreo-
nine unit appears as peak 6 [(MþNa)þ: 1120.83]. In a
similar manner, the fully deprotected glycopeptide (21) was
produced as follows. The tetrasaccharide-linked glycopep-
tide on resin was N-deprotected with 20% piperidine,
cleaved with reagent K, and debenzylated with the mixture
of diluted TfOH. An HPLC profile of the products is shown
in Figure 3(d). Three peaks are observed, in which the
largest one [(MþH)þ: 1707.89, (MþNa)þ: 1729.65]
represents 21. Peak 1 corresponds to the unreacted
octapeptide, whereas the glycopeptide lacking a Gal residue
is included in the fraction of peak 3. The isolation of
glycopeptide 21 was performed by preparative HPLC and
the overall yield was 27% (Scheme 2).
4. Enzymatic sialylation of the glycopeptide
Enzymatic sialylation of the synthesized glycopeptide was
next investigated. The neccessary enzymes, b-Gal-b1,3/
4-GlcNAc-a2,3-sialyltransferase and b-Gal-b1,3-GalNAc-
a2,3-sialyltransferase, are commercially available. The
former sialyltransferase promotes formation of an
a-Neu5Ac-(2!3)-b-Gal-(1!4)-GlcNAc oligosaccharide,
and the latter specifically gives an a-Neu5Ac-(2!3)-b-
Gal-(1!3)-GalNAc motif. In order to examine the effi-
ciency of the enzymatic reactions with these sialyl-
transferases, tetrasaccharide 19 was enzymatically
sialylated (Fig. 4). A mixture of substrate 19 and CMP–
sialic acid (5 equiv.) was incubated with the recombinant rat
b-Gal-b1,3/4-GlcNAc-a2,3-sialyltransferase, BSA and
Figure 4. Structures of the enzymatic sialylation products.

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Y. Takano et al. / Tetrahedron 59 (2003) 8415–8427
MOPS buffer (pH 7.4) at 378C. The products were analyzed
by HPLC and MALDI TOF MS. Figure 5(a) shows a
chromatogram of the reaction mixture of 24 h incubation,
where three new compounds are observed as peaks 1–3, and
unconverted 19 is detected as peak 4. Peak 1 (15%) was
identified as disialylated product 24 [(MþNa)þ: 1676.86],
whereas the mass spectra of peak 2 (8%) and 3 (57%)
demonstrated the molecular ions for monosialocompounds,
23 (1385.86) and 22 (1385.83), respectively. When the
incubation was extended to 48 h, reversed reaction took
place to afford a mixture of 24 (9%), 23 (13%), 22 (41%),
and 19 (37%). Further elongation of the incubation time to
72 h led to an increase in regenerated 19 (49%). On the other
hand, the recombinant rat b-Gal-b1,3-GalNAc-a2,3-sialyl-
transferase displayed high specificity in the sialylation of
19. The enzymatic reaction was carried out at pH 6.0 for
16 h. HPLC analysis of the product is shown in Figure 5(b),
where the specifically sialylated product (23: 86%) is
observed. The longer incubation again resulted in the
reversed reaction. Subsequently, we carried out the
concurrent sialy transfer reaction using two enzymes and
excess CMP–sialic acid (7.5 equiv. for each sialylation) at
pH 6.8. The results are shown in Figure 5(c). The desired
disialylation was predominant (24: 90%) in the mixture of
12 h incubation. The monosialo derivatives (23: 4% and 22:
6%) were also detected.
12 h under the same conditions as tested for concurrent
sialylation of 19. As shown in Figure 6, the disialylation was
remarkably successful to exclusively afford hexasaccharide-
linked peptide 25 (MþNa: 2311.35). In the upper half of the
figure is given a reference chromatogram derived from a
purposely prepared mixture of 21 (peak 1), the individually
prepared monosialoglycopeptides 26, 27 (peaks 2 and 3),
and 25 (peak 4). A 10 mM ammonium acetate buffer (pH
5.8) used as the eluent with acetonitrile was effective to
separate the glycopeptides on the C-18 reversed phase
column, while a combination of 0.1% TFA-containing
water and acetonitrile eluted all the glycopeptides, 19, 25,
26 and 27, as the indistinguishable peaks on the same
column. The disialylated product 25 was the least mobile in
the weakly acidic eluent. It has not been known whether this
unusual chromatographic behavior depends upon the
property of the ionized sialic acid residues or the altered
conformation of glycopeptide in this medium. As this
experiment demonstrated that the concurrent sialylation
proceeded in a highly efficient manner, a variety of core 2
sialoglycopeptides will be synthesized by applying the
simple procedure even in a larger scale.
In summary, the highly stereoselective formation of
b-GlcNAc-(1!6)-GalNAc linkage of the core 2 O-linked
glycan was accomplished by employing 2-trichloroacet-
amide as the stereocontrolling group. The tetrasaccharide-
linked threonine thus prepared in a benzyl-protected form
was used for solid-phase synthesis of the mucin type domain
of leukosialin (215–224). Deprotection was efficiently
On the basis of these results, we next examined the
sialylation of glycopeptide 21. The glycopeptide (100 nmol)
was incubated with two enzymes and CMP–sialic acid for
Figure 5. HPLC of the sialylation products of 19 with b-Gal-b1,3/4-GlcNAc-a2,3-sialyltransferase (a) the sialylation products of 19 with b-Gal-b1,3-
GalNAc-a2,3-sialyltransferase (b) and the sialylation products of 19 with b-Gal-b1,3/4-GlcNAc-a2,3-sialyltransferase and b-Gal-b1,3-GalNAc-a2,3-
sialyltransferase (c) Column: Mightysil RP-18 (150£4.6 mm). Eluent A: distilled water containing 0.1% TFA, B: acetonitrile containing 0.1% TFA. Flow rate:
1 ml/min.

Y. Takano et al. / Tetrahedron 59 (2003) 8415–8427
8421
(2,5-dihydroxybenzoic acid was used as a matrix). High
resolution Fab mass spectra were measured with JEOL JMS
HX-110 spectrometer (2,5-dihydroxybenzoic acid was used
as a matrix). All solid-phase reactions were performed at
room temperature in the capped polypropylene test tubes
with stirring on a vortex tube-mixer. HPLC was performed
using Mightysil RP-8, RP-18 (150£4.6 mm for analysis and
250£10 mm for preparation, Kanto Chemical Co.). Amino
acids were analyzed on a Hitachi L-8500 amino acid
analyzer. Fmoc Sieber amide MBHA resin was purchased
from NOVA Biochem. Sialyltransferases [a2,3-(N)-sialyl-
transferase, rat, recombinant, and a2,3-(O)-sialyltransfer-
ase, rat, recombinant] and CMP–sialic acid were purchased
from CALBIOCHEM.
5.1.1. N-(9-Fluorenylmethoxycarbonyl)-O-[2,3,4,6-tetra-
O-benzyl-b-D-galactopyranosyl-(1!3)-2-azido-2-deoxy-
a-D-galactopyranosyl]-L-serine allyl ester 3a. A mixture
of 4a (494 mg, 0.43 mmol) in CH2Cl2 (10 ml) and 80% aq.
TFA (4 ml) was stirred at 2158C for 1 h, then neutralized
with sat. NaHCO3, and extracted with CHCl3. The extract
was successively washed with water, sat. NaHCO3, and
brine, dried over Na2SO4, and concentrated in vacuo. The
residue was chromatographed on silica gel with toluene–
EtOAc (1:1) to afford 3a (340 mg, 74%). [a]D¼þ67.28 (c 1).
1
Rf 0.37 (1:1 toluene–EtOAc). H NMR: d 7.75 (d, 2H, J¼
7.3 Hz, Ar), 7.63–7.56 (m, 2H, Ar), 7.53–7.31 (m, 4H, Ar),
7.30–7.10 (m, 20H, Ar), 6.04 (d, 1H, J¼8.3 Hz, NH),
5.90 (m, 1H, –CH2CHvCH2), 5.32 (brd, 1H, J¼17.1 Hz,
–CH2vCH2), 5.24 (brd, 1H, J¼10.5 Hz, –CH2vCH2),
4.94 (d, 1H, J¼3.2 Hz, H-1a), 4.52 (d, 1H, J¼7.8 Hz, H-1b).
MALDI TOF MS: calcd for C61H64N4O14·Na m/z 1099.43.
Found 1099.04. Anal. calcd for C61H64N4O14: C, 68.02; H,
5.99; N, 5.20. Found: C, 67.75; H, 6.02; N, 5.09.
Figure 6. HPLC of the reaction mixture of the enzymatic sialylation of
21 (a) The upper chromatogram (b) shows a reference mixture of 21,
monosialoglycopeptides, and 25. Column: Mightysil RP-18 (150£4.6 mm).
Eluent A: 10 mM ammonium acetate buffer (pH 5.8), B: acetonitrile
containing 10% eluent A. Flow rate: 1 ml/min.
5.1.2. N-(9-Fluorenylmethoxycarbonyl)-O-[2,3,4,6-tetra-
O-benzyl-b-D-galactopyranosyl-(1!3)-2-azido-2-deoxy-
a-D-galactopyranosyl]-L-threonine allyl ester 3b. Com-
pound 4b (178 mg, 0.15 mmol) was debenzylidenated as
described above. Chromatography of the crude product on
silica gel with toluene–EtOAc (4:1) afforded 3b (138 mg,
84%). [a]D¼þ60.88 (c 1). Rf 0.15 (4:1 toluene–EtOAc). 1H
NMR: d 7.75 (d, 2H, J¼7.3 Hz, Ar), 7.60 (m, 2H, Ar),
7.41–7.24 (m, 24H, Ar), 5.92 (m, 1H, –CH2CHvCH2),
5.71 (d, 1H, J¼9.5 Hz, NH), 5.34 (brd, 1H, J¼17.1 Hz,
–CH2vCH2), 5.24 (brd, 1H, J¼10.5 Hz, –CH2vCH2),
5.05 (d, 1H, J¼3.7 Hz, H-1a), 4.52 (d, 1H, J¼7.8 Hz, H-1b),
1.32 (d, 1H, J¼6.3 Hz, Thr gH). MALDI TOF MS: calcd for
C62H66N4O14·Na m/z 1113.45. Found 1113.46.
achieved by utilizing the low-acidity TfOH conditions. The
resulting glycopeptide was subjected to enzymatic glyco-
sylation, being used as the substrate for the sialyltrans-
ferases. The desired hexasaccharide-linked glycopeptide
was successfully produced in high efficiency. Further efforts
to extend these results to synthesize glycopeptides with the
clustered oligosaccharides are currently underway in this
laboratory.
5. Experimental
5.1. General
Anal. calcd for C62H66N4O14: C, 68.24; H, 6.10. Found: C,
68.13; H, 6.16.
5.1.3. tert-Butyldiphenylsilyl 2,3,4,6-tetra-O-acetyl-b-D-
galactopyranosyl-(1!4)-2-azido-3,6-di-O-benzyl-2-
deoxy-b-D-glucopyranoside 8. To a stirred mixture of 6
(2.13 g, 3.42 mmol), AgOTf (3.52 g, 13.7 mmol), and
Optical rotations were determined with a Jasco DIP-370
polarimeter for solutions in CHCl3, unless noted otherwise.
Column chromatography was performed on silica gel PSQ
100B (Fuji Silysia). TLC and HPTLC were performed on
˚
1
Silica Gel 60 F254 (E. Merck). H and 13C NMR spectra
dried molecular sieves 4 A powder (20 g) in anhydrous
were recorded with a Jeol AL400 [1H (400 MHz), 13C
(100 MHz)] spectrometer. Chemical shifts are expressed in
ppm downfield from the signal for internal Me4Si for
solutions in CDCl3. MALDI TOF mass spectra were
obtained with a PerSeptive Voyager-DE PRO spectrometer
ClCH2CH2Cl (40 ml) was added a solution of 5 (1.69 g,
4.1 mmol) in ClCH2CH2Cl (20 ml) at 2158C under Ar. The
mixture was stirred at the temperature for 3.5 h before
the reaction was quenched with pyridine and water. The
mixture was filtered through Celite, and the filtrate was

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Y. Takano et al. / Tetrahedron 59 (2003) 8415–8427
extracted with CHCl3. The extract was successively washed
with water, sat. NaHCO3, and brine, dried over Na2SO4, and
concentrated in vacuo. The crude product was chromato-
graphed on silica gel with hexane–EtOAc (2:1) to give 8
(2.59 g, 79%). [a]D¼222.18 (c 1). Rf 0.47 (1:1 hexane–
EtOAc). 1H NMR: d 7.74–7.69 (m, 20H, Ar), 5.26 (brd, 1H,
H-4b), 5.08 (dd, 1H, J¼8.1, 10.3 Hz, H-2b), 4.93 (d, 1H,
J¼11.0 Hz, –CH2Ph), 4.84 (dd, 1H, J¼3.4, 10.3 Hz, H-3b),
4.72 (d, 1H, J¼10.5 Hz, –CH2Ph), 4.61 (d, 1H, J¼8.1 Hz,
H-1b), 4.55 (d, 1H, J¼12.0 Hz, –CH2Ph), 4.33 (d, 1H, J¼
7.8 Hz, H-1a), 4.27 (d, 1H, J¼12.0 Hz, –CH2Ph), 4.03–
3.97 (m, 2H, H-4a, H-6b), 3.84 (dd, 1H, J¼5.9, 11.2 Hz,
H-6b), 3.61 (m, 1H, H-5b), 3.54–3.45 (m, 2H, H-2a, H-6a),
3.29–3.24 (m, 2H, H-3a, H-6a), 2.85 (m, 1H, H-5a), 2.09,
1.98, 1.95, and 1.90 (4s, 12H, 4 Ac), 1.11 (s, 9H, t-Bu).
MALDI TOF MS: calcd for C50H59N3O14Si·Na m/z 976.17.
Found 975.96. Anal. calcd for C50H59N3O14Si: C, 62.94; H,
6.23; N, 4.40. Found: C, 62.91; H, 6.27; N, 4.19.
H-1b), 4.52 (d, 1H, J¼12.4 Hz, –CH2Ph), 4.46 (d, 1H,
J¼12.4 Hz, –CH2Ph), 4.08 (d, 1H, J¼3.6 Hz, H-4b), 3.66
(brt, 1H, H-2b), 1.62 (s, 9H, t-Bu). Anal. calcd for
C57H59NO12Si: C, 69.99; H, 6.08; N, 1.43. Found: C,
69.80; H, 6.10; N, 1.37.
5.1.6. tert-Butyldiphenylsilyl 2,3-di-O-benzyl-4,6-O-ben-
zylidene-b-D-galactopyranosyl-(1!4)-2-azido-3,6-di-O-
benzyl-2-deoxy-b-D-glucopyranoside 12. A mixture of 10
(370 mg, 0.42 mmol) and 60% NaH (68 mg, 1.70 mmol) in
anhydrous THF (70 ml) was heated at 608C with stirring for
1 h. To the mixture was added benzyl bromide (0.29 ml,
1.69 mmol). The resulting mixture was heated overnight at
the temperature. After cooling, a piece of ice was carefully
added to the mixture. The mixture was concentrated in
vacuo to the residue, which was extracted with ether–
EtOAc (1:1). The extract was successively washed with
water and brine, dried over Na2SO4, and concentrated in
vacuo. The crude product was chromatographed on silica
gel with hexane–EtOAc (4:1) to give 12 (405 mg, 91%).
[a]D¼26.48 (c 1). Rf 0.10 (4:1 hexane–EtOAc). 1H NMR:
d 7.72–7.70 and 7.53–7.08 (m, 35H, Ar), 5.46 [s, 1H,
PhCH(O–)2], 5.18 (d, 1H, J¼10.5 Hz, –CH2Ph), 4.76
(d, 1H, J¼10.9 Hz, –CH2Ph), 4.71 (brs, 2H, –CH2Ph), 4.69
(d, 1H, J¼11.2 Hz, –CH2Ph), 4.62 (d, 1H, J¼10.9 Hz,
–CH2Ph), 4.45 (d, 1H, J¼7.8 Hz, H-1b), 4.37 (d, 1H, J¼
11.9 Hz, –CH2Ph), 4.31 (d, 1H, J¼8.1 Hz, H-1b), 4.21 (brd,
1H, J¼11.2 Hz, H-6b), 4.16 (d, 1H, J¼11.9 Hz, –CH2Ph),
4.04 (d, 1H, J¼3.8 Hz, H-4b), 4.00 (t, 1H, J¼8.1 Hz, H-4a),
3.88 (dd, 1H, J¼1.7, 12.2 Hz, H-6b), 3.74–3.69 (m, 2H,
H-2b, H-6a), 3.46 (dd, 1H, J¼7.8, 9.8 Hz, H-2a), 3.41 (dd,
1H, J¼3.7, 7.8 Hz, H-3b), 3.27 (t, 1H, J¼8.8 Hz, H-3a),
3.18 (brd, 1H, J¼10.0 Hz, H-6a), 3.05 (brs, 1H, H-5b), 2.78
(brd, 1H, H-5a), 1.10 (s, 9H, t-Bu). MALDI TOF MS: calcd
for C63H67N3O10Si·Na m/z 1076.45. Found 1076.43. Anal.
calcd for C63H67N3O11Si: C, 71.77; H, 6.41; N, 3.99. Found:
C, 71.61; H, 6.38; N, 3.80.
5.1.4. tert-Butyldiphenylsilyl 4,6-O-benzylidene-b-D-
galactopyranosyl-(1!4)-2-azido-3,6-di-O-benzyl-2-
deoxy-b-D-glucopyranoside 10. A solution of 8 (1.06 g,
1.11 mmol) in MeOH (25 ml) was stirred with 1 M NaOMe/
MeOH (0.56 ml, 0.56 mmol) at room temperature for 1 h.
The mixture was neutralized with Amberlist 15 and filtered.
The filtrate was concentrated in vacuo to the residue, which
was dissolved in anhydrous CH3CN (30 ml) and stirred with
benzaldehyde dimethyl acetal (0.48 ml, 3.32 mmol) and a
catalytic amount of p-TsOH at room temperature for 1 h.
The reaction was quenched by an addition of sat. NaHCO3,
and the mixture was concentrated in vacuo. The residue
was extracted with ether. The extract was successively
washed with sat. NaHCO3, water, and brine, dried over
Na2SO4, and concentrated in vacuo. The crude product was
chromatographed on silica gel with hexane–EtOAc (1:1) to
afford 10 (0.87 g, 88%). [a]D¼223.78 (c 1). Rf 0.49
1
(EtOAc). H NMR: d 7.72–7.68 and 7.44–7.16 (m, 25H,
Ar), 5.49 [s, 1H, PhCH(O–)2], 5.00 (d, 1H, J¼11.0 Hz,
–CH2Ph), 4.86 (d, 1H, J¼11.0 Hz, –CH2Ph), 4.52 (d, 1H,
J¼7.8 Hz, H-1b), 4.51 (d, 1H, J¼12.2 Hz, –CH2Ph),
4.35 (d, 1H, J¼ 7.8 Hz, H-1a), 4.32 (d, 1H, J¼12.2 Hz,
–CH2Ph), 4.12–4.00 (m, 3H, H-4a, H-4b, H-6a), 3.80–3.76
(m, 2H, H-6a, H-6b), 3.73 (t, 1H, J¼7.8 Hz, H-2b), 3.52–
3.46 (m, 2H, H-2a, H-3b), 3.35 (t, 1H, J¼9.7 Hz, H-3a),
3.27 (br, 1H, H-6b), 2.95–2.93 (m, 2H, H-5a, H-5b), 1.12 (s,
9H, t-Bu). MALDI TOF MS: calcd for C49H55N3O10Si·Na
m/z 896.36. Found 896.52. Anal. calcd for C49H55N3O10Si:
C, 67.33; H, 6.34; N, 4.81. Found: C, 67.41; H, 6.38; N,
4.77.
5.1.7. tert-Butyldiphenylsilyl 2,3-di-O-benzyl-4,6-O-ben-
zylidene-b-D-galactopyranosyl-(1!4)-3,6-di-O-benzyl-
2-deoxy-2-phthalimido-b-D-glucopyranoside 13. A mix-
ture of 11 (694 mg, 0.70 mmol) and 60% NaH (85 mg,
2.1 mmol) in anhydrous DMF (15 ml) was stirred at 08C
under Ar for 15 min. Then benzyl bromide (0.25 ml,
2.1 mmol) was added to the mixture, which was stirred
overnight at room temperature. The reaction was quenched
by addition of ice–NH4Cl aq. and the mixture was
concentrated in vacuo. The product was extracted with
ether–EtOAc (1:1), washed successively with water and
brine, and dried over Na2SO4. Concentration of the extract
in vacuo gave the crude product, which was chromato-
graphed on silica gel with toluene–EtOAc (9:1) to give 13
(545 mg, 66%). [a]D¼þ12.38 (c 1). Rf 0.18 (9:1 toluene–
EtOAc). 1H NMR: d 7.39–7.17, 7.13–6.97, and 6.80–6.73
(m, 35H, Ar), 5.42 [s, 1H, PhCH(O–)2], 5.19 (d, 1H, J¼
7.8 Hz, H-1a), 5.02 (d, 1H, J¼12.7 Hz, –CH2Ph), 4.81
(d, 1H, J¼11.0 Hz, –CH2Ph), 4.75–4.68 (m, 3H, J¼3 Hz,
–CH2Ph), 4.60 (d, 1H, J¼12.5 Hz, –CH2Ph), 4.53 (d, 1H,
J¼7.8 Hz, H-1b), 4.48 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.32–
4.18 (m, 4H, H-2a, H-3a, H-6b, –CH2Ph), 4.11 (brt, 1H,
J¼8.9 Hz, H-4a), 4.06 (brd, 1H, J¼3.4 Hz, H-4b), 3.90 (brd,
J¼11.0 Hz, H-6b), 3.82–3.72 (m, 2H, H-6a, H-2b), 3.46
(dd, J¼3.4, 9.4 Hz, H-3b), 3.36 (brd, 1H, J¼11.2 Hz, H-6a),
5.1.5. tert-Butyldiphenylsilyl 4,6-O-benzylidene-b-D-
galactopyranosyl-(1!4)-3,6-di-O-benzyl-2-deoxy-2-
phthalimido-b-D-glucopyranoside 11. Compound
9
(6.33 g, 5.98 mmol) was deacetylated with NaOMe in
MeOH, and benzylidenated with benzaldehyde dimethyl
acetal (2.6 ml, 18.0 mmol) and p-TsOH in anhydrous
CH3CN 150 ml) as described for 10. The product was
chromatographed on silica gel with toluene–EtOAc (1:1) to
give 11 (4.70 g, 97%). [a]D¼þ12.88 (c 1). Rf 0.41 (1:1
1
toluene–EtOAc). H NMR: d 7.69–7.59, 7.41–7.16, and
7.07–6.84 (m, 25H, Ar), 5.45 [s, 1H, PhCH(O–)2], 5.19 (d,
1H, J¼7.6 Hz, H-1a), 4.92 (d, 1H, J¼12.4 Hz, –CH2Ph),
4.64 (d, 1H, J¼12.4 Hz, –CH2Ph), 4.60 (d, 1H, J¼7.6 Hz,

Y. Takano et al. / Tetrahedron 59 (2003) 8415–8427
8423
3.16 (brs, 1H, H-5b), 3.12 (m, 1H, H-5a), 0.88 (s, 9H, t-Bu).
Anal. calcd for C71H71NO12Si: C, 73.61; H, 6.18; N, 1.21.
Found: C, 73.61; H, 6.17; N, 1.21.
Si·0.5H2O: C, 66.01; H, 5.88; N, 1.18. Found: C, 66.06; H,
5.88; N, 1.06.
5.1.10. 2,3-Di-O-benzyl-4,6-O-benzylidene-b-D-galacto-
pyranosyl-(1!4)-3,6-di-O-benzyl-2-deoxy-2-trichloro-
acetamido-D-glucopyranose 16. To a mixture of 15
(236 mg, 0.20 mmol) and AcOH (127 ml, 2.01 mmol) in
freshly distilled THF (6 ml) was added 1 M n-Bu4NF/THF
(0.8 ml, 0.80 mmol). The mixture was stirred at room
temperature overnight and then concentrated in vacuo. The
residue was extracted with EtOAc, successively washed
with water and brine, dried over Na2SO4, and concentrated
in vacuo. The crude product was chromatographed on silica
gel with toluene–EtOAc (9:1) to give 16 (169 mg, 90%) as
crystals. Mp 189–189.58C. Rf 0.34 (7:3 toluene–EtOAc).
1H NMR: d 7.45–7.18 (m, 25H, Ar), 6.79 (brd, 1H, NH),
5.44 [s, 1H, PhCH(O–)2], 5.40 (t, 1H, J¼3.7 Hz, H-1a),
5.21 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.86 (d, 1H, J¼11.0 Hz,
–CH2Ph), 4.77 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.75–4.72
(m, 3H, J¼3 Hz, –CH2Ph), 4.54 (d, 1H, J¼12.0 Hz,
–CH2Ph), 4.18 (m, 1H, H-6a), 4.12–4.04 (m, 3H, H-2a,
H-4b, H-6b), 3.92 (dd, 1H, J¼3.7, 10.5 Hz, H-6a), 3.98–
3.82 (m, 3H, H-3a, H-4a, H-6b), 3.78 (brt, 1H, H-2b), 3.57
(m, 1H, H-5a), 3.22 (dd, J¼3.4, 9.5 Hz, H-3b), 3.05 (m, 1H,
H-5a), 3.00 (brs, 1H, OH). MALDI TOF MS: calcd for
C49H50Cl3NO11·Na m/z 955.23. Found 955.74. Anal. calcd
for C49H50Cl3NO11: C, 62.92; H, 5.39; N, 1.50. Found: C,
62.69; H, 5.39; N, 1.55.
5.1.8. tert-Butyldiphenylsilyl 2,3-di-O-benzyl-4,6-O-ben-
zylidene-b-D-galactopyranosyl-(1!4)-2-amino-3,6-di-O-
benzyl-2-deoxy-b-D-glucopyranoside 14. Procedure A
(reduction of 12). A mixture of 12 (324 mg, 0.31 mmol),
powdered Zn (7 g) and AcOH (0.26 ml) in CH2Cl2 (50 ml)
was stirred at room temperature for 3 h and then filtered
through Celite. The filtrate was concentrated in vacuo and
remaining AcOH was coevaporated with toluene. The
residue was chromatographed on silica gel with CHCl3 to
give 14 (300 mg, 95%).
[a]D¼þ4.98 (c 1). Rf 0.06 (CHCl3). 1H NMR: d 7.71–7.68
and 7.46–7.19 (m, 35H, Ar), 7.11–7.10 (m, 2H, NH2), 5.45
[s, 1H, PhCH(O–)2], 5.36 (d, 1H, J¼10.7 Hz, –CH2Ph),
4.77 (d, 1H, J¼11.2 Hz, –CH2Ph), 4.67 (brs, 2H, –CH2Ph),
4.56 (d, 1H, J¼10.7 Hz, –CH2Ph), 4.50 (d, 1H, J¼7.8 Hz,
H-1b), 4.40 (d, 1H, J¼12.0 Hz, –CH2Ph), 4.37 (d, 1H,
J¼7.6 Hz, H-1a), 4.25 (d, 1H, J¼12.2 Hz, –CH2Ph), 4.07–
4.03 (m, 2H, H-4a, H-6a), 3.90 (brd 1H, H-6b), 3.77–3.72
(m, 2H, H-6a, H-2b), 3.43 (dd, 1H, J¼3.7, 9.5 Hz, H-3b),
3.30 (t, 1H, J¼9.3 Hz, H-3a), 3.21 (br, 1H, H-4b), 3.09 (brs,
1H, H-5b), 3.00 (dd, 1H, J¼7.6, 9.5 Hz, H-2a), 2.89 (br, 1H,
H-5a), 1.08 (s, 9H, t-Bu). MALDI TOF MS: calcd for
C63H69NO10Si·Na m/z 1050.46. Found 1049.64. Anal. calcd
for C63H69NO10Si: C, 73.58; H, 6.76; N, 1.36. Found: C,
73.37; H, 6.80; N, 1.31.
5.1.11. 2,3-Di-O-benzyl-4,6-O-benzylidene-b-D-galacto-
pyranosyl-(1!4)-3,6-di-O-benzyl-2-deoxy-2-trichloro-
acetamido-a-D-glucopyranosyl fluoride 2. To a stirred
solution of 16 (114 mg, 0.12 mmol) in freshly distilled THF
(3 ml) was added Et2NSF3 (28 ml, 0.17 mmol) at 08C. The
mixture was stirred for 30 min before the reaction was
quenched with MeOH, and concentrated in vacuo. The
residue was extracted with ether–EtOAc (1:1), successively
washed with water and brine, dried over Na2SO4, and
concentrated in vacuo. The crude product was chromato-
graphed on silica gel with CHCl3–EtOAc (9:1) to give 2
(113 mg, 93%). Mp 141.5–1428C (recrystallized from
hexane–EtOAc). [a]D¼þ38.38 (c 1.4). Rf 0.68 (9:1
CHCl3–EtOAc). 1H NMR: d 7.45–7.22 (m, 25H, Ar),
6.60 (d, 1H, J¼7.3 Hz, NH), 5.77 (dd, 1H, J¼2.5, 53.7 Hz,
H-1a), 5.45 [s, 1H, PhCH(O–)2], 5.21 (d, 1H, J¼11.7 Hz,
–CH2Ph), 4.79 (d, 1H, J¼11.5 Hz, –CH2Ph), 4.78–4.74
(m, 3H, J¼3 Hz, –CH2Ph), 4.58 (d, 1H, J¼12.0 Hz,
–CH2Ph), 4.41 (d, 1H, J¼7.8 Hz, H-1b), 4.33 (d, 1H, J¼
12.0 Hz, –CH2Ph). MALDI TOF MS: calcd for C49H49Cl3
NO10F·Na m/z 958.22. Found 958.03. Anal. calcd for
C49H49Cl3NO10F: C, 62.79; H, 5.27; N, 1.49; Cl, 11.35.
Found: C, 62.76; H, 5.24; N, 1.45; Cl, 11.60.
Procedure B (dephthaloylation of 13). A mixture of 13
(3.43 g, 2.97 mmol) and ethylenediamine (2.98 ml,
44.5 mmol) in n-BuOH (40 ml) was heated at 958C for
18 h and then concentrated in vacuo. The residue was
extracted with CHCl3. The extract was successively washed
with water and brine, dried over Na2SO4, and concentrated
in vacuo. The crude product was chromatographed on silica
gel with CHCl3 to give 14 (2.99 g, 98%).
5.1.9. tert-Butyldiphenylsilyl 2,3-di-O-benzyl-4,6-O-ben-
zylidene-b-D-galactopyranosyl-(1!4)-3,6-di-O-benzyl-
2-deoxy-2-trichloroacetamido b-D-glucopyranoside 15.
To a solution of 14 (95 mg, 0.09 mmol) in anhydrous
pyridine (1.5 ml) was added trichloroacetyl chloride (80 ml,
0.28 mmol) at 08C. The mixture was stirred at the
temperature overnight and then diluted with ether. The
ethereal extract was successively washed with water and
brine, dried over Na2SO4, and concentrated in vacuo. The
residue was chromatographed on silica gel with hexane–
EtOAc (3:1) to give 15 (94 mg, 87%). [a]D¼þ4.68 (c 1). Rf
1
0.58 (1:1 hexane–EtOAc). H NMR: d 7.69–7.62, 7.46–
7.22, and 7.19–7.10 (m, 35H, Ar), 6.86 (d, 1H, J¼7.3 Hz,
NH), 5.44 [s, 1H, PhCH(O–)2], 5.15 (d, 1H, J¼10.7 Hz,
–CH2Ph), 4.86 (d, 1H, J¼6.9 Hz, H-1a), 4.87–4.64 (m, 5H,
J¼5 Hz, –CH2Ph), 4.50 (d, 1H, J¼7.1 Hz, H-1b), 4.36
(d, 1H, J¼12.0 Hz, –CH2Ph), 4.20–4.08 (m, 3H, H-6a,
H-6b, –CH2Ph), 4.04 (br, 1H, H-4b), 3.89–3.81 (m, 3H,
H-2a, H-6a, H-6b), 3.75–3.71 (m, 2H, H-3a, H-2b), 3.42
(dd, 1H, J¼3.2, 9.5 Hz, H-3b), 3.20 (m, 1H, H-4a), 3.08
(brs, 1H, H-5b), 2.96 (m, 1H, H-5a), 1,05 (s, 9H, t-Bu).
MALDI TOF MS: calcd for C64H68Cl3NO11Si·Na m/z
1194.36. Found 1194.27. Anal. calcd for C64H68Cl3NO11
5.1.12. N-(9-Fluorenylmethoxycarbonyl)-O-{2,3-di-O-
benzyl-4,6-O-benzylidene-b-D-galactopyranosyl-(1!4)-
3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-b-D-glu-
copyranosyl-(1!6)-[2,3,4,6-tetra-O-benzyl-b-D-galacto-
pyranosyl-(1!3)]-2-azido-2-deoxy-a-D-galactopyrano-
syl}-L-serine allyl ester 17a. A mixture of 3a (144 mg,
0.13 mmol), Cp2ZrCl2 (71 mg, 0.24 mmol), AgClO4
(100 mg, 0.48 mmol), and dried molecular sieves 4 A
(1.35 g) in anhydrous CH2Cl2 (8 ml) was stirred at 2158C
under Ar for 1 h. Then a solution of 2 (113 mg, 0.12 mmol)
˚

8424
Y. Takano et al. / Tetrahedron 59 (2003) 8415–8427
in anhydrous CH2Cl2 (3 ml) was added to the stirred
mixture. The mixture was stirred at the temperature for
2.5 h, before the reaction was quenched with aq. NaHCO3.
The mixture was diluted with CHCl3 and filtered through
Celite. The filtrate was successively washed with sat.
NaHCO3, water, and brine, dried over Na2SO4, and
concentrated in vacuo. The product was chromatographed
on Bio-beads S X3 with toluene–EtOAc (1:1) to afford 17a
(184 mg, 76%). [a]D¼þ34.48 (c 1). Rf 0.23 (4:1 toluene–
–CH2CHvCH2), 5.56 (d, 1H, J¼9.2 Hz, NH), 5.43 [s, 1H,
PhCH(O)2], 5.30 (brd, 1H, J¼17.1 Hz, –CHvCH2), 5.20
(brd, 1H, J¼10.5 Hz, –CHvCH2), 1.80 (s, 3H, Ac), 1.55 (s,
3H, Ac). 13C NMR: d 98.0 (1JCH¼172.5 Hz, GalNAc C-1),
100.3 (1JCH¼165.9 Hz, GlcNAc C-1), 101.2 [PhCH(O)2],
102.8 (1JCH¼163.4 Hz, Gal C-1), 104.2 (1JCH¼161.7 Hz,
Gal C-1). MALDI TOF MS: calcd for C112H119N3O25·Na
m/z 1928.81. Found 1928.84. Anal. calcd for C112H119N3O25:
C, 70.53; H, 6.29; N, 2.20. Found: C, 70.55; H, 6.22; N,
2.39.
1
EtOAc). H NMR: d 5.88 (m, 1H, –CH2CHvCH2), 5.72
(d, 1H, J¼7.3 Hz, NH), 5.46 [s, 1H, PhCH(O)2], 5.30 (brd,
1H, J¼17.0 Hz, –CHvCH2), 5.22 (brd, 1H, J¼10.5 Hz,
–CHvCH2), 4.95 (d, 1H, J¼9.0 Hz, GlcNTCA H-1), 4.87
170.7 Hz, GalN3 C-1), 99.4 (1JCH¼157.5 Hz, GlcNTCA
C-1), 101.3 [PhCH(O)2], 102.7 (1JCH¼160.9 Hz, Gal C-1),
103.9 (1JCH¼155.9 Hz, Gal C-1). MALDI TOF MS: calcd
for C110H112Cl3N5O24·Na m/z 2014.67. Found 2014.42.
Anal. calcd for C110H112Cl3N5O24: C, 66.24; H, 5.66; N,
3.51; Cl, 5.33. Found: C, 66.20; H, 5.81; N, 3.29; Cl, 5.63.
5.1.15. N-(9-Fluorenylmethoxycarbonyl)-O-{2,3-di-O-
benzyl-4,6-O-benzylidene-b-D-galactopyranosyl-(1!4)-
2-acetamido-3,6-di-O-benzyl-2-deoxy-b-D-glucopyrano-
syl-(1!6)-[2,3,4,6-tetra-O-benzyl-b-D-galactopyranosyl-
(1!3)]-2-acetamido-2-deoxy-a-D-galactopyranosyl}-L-
threonine allyl ester 18b. Compound 17b (86 mg,
0.04 mmol) was treated with Zn and AcOH in CH2Cl2,
and then the reduced product was acetylated in CH2Cl2–
MeOH as described above for 18a. The crude product was
purified by gel-permeation chromatography on Sephadex
LH-60 to afford 18b (80 mg, 96%). [a]D¼þ36.78 (c 1). Rf
0.37 (1:4 toluene–EtOAc). 1H NMR: d 5.84 (m, 1H,
–CH2CHvCH2), 5.67 (d, 1H, J¼9.0 Hz, NH), 5.62 (d, 1H,
J¼8.5 Hz, NH), 5.58 (d, 1H, J¼8.1 Hz, NH), 5.43 [s, 1H,
PhCH(O)2], 5.30 (brd, 1H, J¼17.6 Hz, –CHvCH2), 5.24
(brd, 1H, J¼10.3 Hz, –CHvCH2), 1.81 (s, 3H, Ac), 1.65 (s,
3H, Ac), 1.29 (d, 1H, J¼6.0 Hz, Thr gH). 13C NMR: d 99.5
(1JCH¼178.3 Hz, GalNAc C-1), 100.7 (1JCH¼164.2 Hz,
GlcNAc C-1), 101.1 [PhCH(O)2], 103.5 (1JCH¼159.2 Hz,
Gal C-1), 104.3 (1JCH¼159.2 Hz, Gal C-1). MALDI TOF
MS: calcd for C113H121N3O25·Na m/z 1942.83. Found
1943.17. Anal. calcd for C113H121N3O25: C, 70.64; H,
6.35; N, 2.19. Found: C, 70.38; H, 6.67; N, 2.05.
(brd, 1H, J¼3.4 Hz, GalN3 H-1). 13C NMR: d 98.0 (1JCH
¼
5.1.13. N-(9-Fluorenylmethoxycarbonyl)-O-{2,3-di-O-
benzyl-4,6-O-benzylidene-b-D-galactopyranosyl-(1!4)-
3,6-di-O-benzyl-2-deoxy-2-trichloroacetamido-b-D-glu-
copyranosyl-(1!6)-[2,3,4,6-tetra-O-benzyl-b-D-galacto-
pyranosyl-(1!3)]-2-azido-2-deoxy-a-D-galactopyrano-
syl}-L-threonine allyl ester 16b. Condensation of 3b
(146 mg, 0.13 mmol) and 2 (115 mg, 0.12 mmol) was per-
formed in the same procedure as described for 16a.
Chromatography of the crude product on Bio-beads S X3
gave 16b (172 mg, 70%). [a]D¼þ33.88 (c 1). Rf 0.30
(4:1 toluene–EtOAc). 1H NMR:
d 5.91 (m, 1H,
–CH2CHvCH2), 5.67 (d, 1H, J¼9.3 Hz, NH), 5.44 [s,
1H, PhCH(O)2], 5.33 (brd, 1H, J¼17.0 Hz, –CHvCH2),
5.23 (brd, 1H, J¼10.3 Hz, –CHvCH2), 5.19 (d, 1H, J¼
10.2 Hz, NH), 4.95 (d, 1H, J¼3.2 Hz, GalN3 H-1), 4.87 (d,
1H, J¼7.9 Hz, GlcNTCA H-1), 1.31 (d, 1H, J¼6.1 Hz, Thr
gH). 13C NMR: d 99.6 (1JCH¼172.5 Hz, GalN3 C-1), 99.8
(1JCH¼159.2 Hz, GlcNTCA C-1), 101.2 [PhCH(O)2], 102.6
(1JCH¼160.0 Hz, Gal C-1), 103.8 (1JCH¼162.5 Hz, Gal
C-1). MALDI TOF MS: calcd for C111H114Cl3N5O24·Na
m/z 2028.69. Found 2028.41. Anal. calcd for
C111H114Cl3N5O24·H2O: C, 65.78; H, 5.77; N, 3.46.
Found: C, 65.89; H, 5.78; N, 3.28.
5.1.16. N-(9-Fluorenylmethoxycarbonyl)-O-{2,3-di-O-
benzyl-4,6-O-benzylidene-b-D-galactopyranosyl-(1!4)-
2-acetamido-3,6-di-O-benzyl-2-deoxy-b-D-glucopyrano-
syl-(1!6)-[2,3,4,6-tetra-O-benzyl-b-D-galactopyranosyl-
(1!3)]-2-acetamido-2-deoxy-a-D-galactopyranosyl}-L-
serine 1a. To a mixture of 18a (61 mg, 0.03 mmol),
5,5-dimethyl-1,3-cyclohexanedione (dimedone, 89 mg,
0.6 mmol), and Pd(PPh3)4 (8 mg, mmol) was added freshly
distilled THF (30 ml) under Ar. The mixture was stirred
overnight at room temperature and then concentrated in
vacuo. The residue was chromatographed on silica gel with
(CHCl3–EtOH (14:1, 1% AcOH) and then on Sephadex
LH-60 with CHCl3–MeOH (1:1) to give 1a (57 mg, 97%).
[a]D¼þ28.18 (c 1). Rf 0.15 (14:1 CHCl3–EtOH, 1%
5.1.14. N-(9-Fluorenylmethoxycarbonyl)-O-{2,3-di-O-
benzyl-4,6-O-benzylidene-b-D-galactopyranosyl-(1!4)-
2-acetamido-3,6-di-O-benzyl-2-deoxy-b-D-glucopyrano-
syl-(1!6)-[2,3,4,6-tetra-O-benzyl-b-D-galactopyranosyl-
(1!3)]-2-acetamido-2-deoxy-a-D-galactopyranosyl}-L-
serine allyl ester 18a. A mixture of 17a (142 mg,
0.07 mmol), AcOH (1.5 ml), and Zn powder (1.5 g) in
CH2Cl2 (35 ml) was stirred at room temperature for 72 h,
and then filtered through Celite. The filtrate was concen-
trated in vacuo to the residue, which was dissolved in
MeOH–CH2Cl2 (1:1, 1 ml) and stirred with Ac2O (36 ml,
0.36 mmol) for 40 min. The mixture was diluted with
CH2Cl2 and successively washed with sat. NaHCO3, water,
and brine, dried over Na2SO4, and concentrated in vacuo.
The residue was chromatographed on Sephadex LH-60
with CHCl3–MeOH (1:1) to give 18a (130 mg, 92%).
[a]D¼þ31.58 (c 1). Rf 0.23 (1:4 toluene–EtOAc). 1H NMR:
d 6.34 (d, 1H, J¼7.3 Hz, NH), 5.94–5.83 (m, 2H, NH,
1
AcOH). H NMR (DMSO-d6): d 5.63 [s, 1H, PhCH(O)2],
1.78 (s, 3H, Ac), 1.64 (s, 3H, Ac). HR Fab MS: calcd for
C109H115N3O25·Na m/z 1889.7751. Found 1889.7744.
5.1.17. N-(9-Fluorenylmethoxycarbonyl)-O-{2,3-di-O-
benzyl-4,6-O-benzylidene-b-D-galactopyranosyl-(1!4)-
2-acetamido-3,6-di-O-benzyl-2-deoxy-b-D-glucopyrano-
syl-(1!6)-[2,3,4,6-tetra-O-benzyl-b-D-galactopyranosyl-
(1!3)]-2-acetamido-2-deoxy-a-D-galactopyranosyl}-L-
threonine 1b. As described for 1a, compound 18b (114 mg,
0.06 mmol) was deallylated with dimedone (165 mg,
1.18 mmol) and Pd(PPh3)4 (14 mg, 0.01 mmol), and the
product was purified by chromatography to give 1b
(104 mg, 93%). [a]D¼þ34.48 (c 1). Rf 0.08 (14:1

Y. Takano et al. / Tetrahedron 59 (2003) 8415–8427
8425
1
CHCl3–EtOH, 1% AcOH). H NMR (DMSO-d6): d 5.63
Cleavage and debenzylation under the conditions of low
acidity TfOH. A mixture of the resin (1 mg), dimethylsulfide
(3 ml), m-cresol (0.8 ml), 1,2-ethanedithiol (0.2 ml) and TFA
(5 ml) in a polypropylen microcentrifuge tube was cooled at
2158C with stirring. TfOH (1 ml) was added to the mixture,
which was stirred for 1 h. The reaction was quenched with
ethereal pyridine and the work-up procedure was done as
described for 19. The product was analyzed by HPLC and
MALDI TOF MS (Fig. 3(b)).
[s, 1H, PhCH(O)2], 1.78 (s, 3H, Ac), 1.67 (s, 3H, Ac), 1.10
(d, 3H, J¼5.6 Hz, Thr gH). 13C NMR (CDCl3): d 98.9
(GalNAc C-1), 100.8 (GlcNAc C-1), 101.1[PhCH(O)2],
102.8 (Gal C-1), 104.3 (Gal C-1).
HR Fab MS: calcd for C110H117N3O25·Na m/z 1902.7874.
Found 1902.7854.
5.1.18. N-(9-Fluorenylmethoxycarbonyl)-O-{b-D-galac-
topyranosyl-(1!4)-2-acetamido-2-deoxy-b-D-glucopyr-
anosyl-(1!6)-[b-D-galactopyranosyl-(1!3)]-2-aceta-
mido-2-deoxy-a-D-galactopyranosyl}-L-threonine 19. A
mixture of 1b (1 mg), dimethylsulfide (3 ml), m-cresol
(1 ml) and TFA (5 ml) in a polypropylen microcentrifuge
tube was cooled at 2158C with stirring. TfOH (1 ml) was
added to the mixture, which was stirred for 1 h. Then a
precooled (2808C) solution of pyridine (200 ml) in ether
(1 ml) was added and the mixture was vigorously stirred for
1 min with a vortex mixer. The precipitate was separated by
centrifugation, the ethereal layer was decanted, and the
residual precipitate was washed two times with ether by
vortex-mixing, centrifugation and decantation. The product
thus obtained was dissolved in 20% aq. CH3CN and
analyzed by HPLC. (for chromatographic conditions see
Fig. 3(a))
Peak 1 (20): calcd for C82H124N14O39·Na m/z 1951.81.
Found 1951.45. Peak 2: C49H68N10O14·Na m/z 1043.48.
Found 1042.97. Peak 3: C101H138N14O39·Na m/z 2193.91.
Found 2193.57.
Cleavage and debenzylation under the conditions involving
reagent K. The resin (1 mg) was stirred with reagent K
[CF3CO2H–phenol–deionized water–thioanisole–ethane-
dithiol (82.5:5:5:5:2.5), 10 ml] for 1 h. The volatile com-
ponents in the mixture were removed by blowing nitrogen.
Ether was added to the residue, and the mixture was centri-
fuged. The ether layer was decanted, and the precipitate was
again washed with ether. The precipitated mixture of glyco-
peptide and resin was subjected to the procedure for the low
acidity TfOH as described above. The operation for the
debenzylation procedure was performed for either 1 or 2 h.
Figure 3(c) shows chromatogram of the products via the 2 h
operation. MALDI TOF MS: Peak 1: calcd for C30H55N10O14
m/z 779.39. Found 778.62. Peak 2 (20): found 1951.31. Peak
3: calcd for C76H114N14O34·Na m/z 1789.75. Found
1789.73. Peak 4: found 1789.68. Peak 5: calcd for
C68H101N13O29·Na m/z 1586.67. Found 586.77. Peak 6:
calcd for C50H71N11O17·Na m/z 1120.49. Found 1120.83.
5.1.19. N-(9-Fluorenylmethoxycarbonyl)-L-Prolyl-O-{b-
D-galactopyranosyl-(1!4)-2-acetamido-2-deoxy-b-D-
glucopyranosyl-(1!6)-[b-D-galactopyranosyl-(1!3)]-2-
acetamido-2-deoxy-a-D-galactopyranosyl}-L-threonyl-
L-threonyl-L-seryl-L-threonyl-L-asparaginyl-L-alanyl-L-
seryl-L-threonyl-L-valylamide 20. All the solid-phase
reactions were performed in a polylpropylene tube equipped
with a coarse filter and three-way stopcock by stirring with a
vortex mixer. Commercial Sieber amide resin (403 mg,
0.25 mmol) was stirred with 20% piperidine/NMP (5 ml)
for 5 min. After filtration the resin was again stirred
with 20% piperidine/NMP (5 ml) for 15 min to complete
N-deprotection. The resin was washed several times with
NMP (5 ml) and then stirred with Fmoc-Val-OH (339 mg,
1 mmol), 1 M DCC/NMP (1 ml, 1 mmol), 1 M HOBt/NMP
(1 ml, 1 mmol) in NMP (5 ml) for 1 h. The mixture was
filtered and the resultant resin was washed successively with
NMP and MeOH–CH2Cl2 (1:1). The unreacted amino
group on the resin was acetylated with 10% Ac2O–5%
DIEA/NMP (5 ml). After washing with NMP, the resin was
N-deprotected in the same manner as described above, and
used for further peptide assembling with Fmoc-Thr(But)-
OH, Fmoc-Ser(But)-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-
OH, Fmoc-Thr(But)-OH, Fmoc-Ser(But)-OH, Fmoc-
Thr(But)-OH. Fmoc amino acids (4 equiv.), DCC
(4 equiv.), and HOBt (4 equiv.) were used for each coupling
reaction. N-Deprotection and acetyl capping reaction were
performed as described. A part of the octapeptide-resin
thus prepared was subjected to the condensation with 1b.
The N-deprotected resin (50 mg, 20 mmol) was stirred with
1b (75 mg, 40 mmol), HATU (14 mg, 38 mmol), and DIEA
(8 ml, 45 mmol) in NMP (0.6 ml) overnight.
5.1.20. L-Prolyl-O-{b-D-galactopyranosyl-(1!4)-2-aceta-
mido-2-deoxy-b-D-glucopyranosyl-(1!6)-[b-D-galacto-
pyranosyl-(1!3)]-2-acetamido-2-deoxy-a-D-galactopyr-
anosyl}-L-threonyl-L-threonyl-L-seryl-L-threonyl-L-
asparaginyl-L-alanyl-L-seryl-L-threonyl-L-valylamide
21. The de-N-Fmoc resin was prepared from the above
glycopeptide-resin by the routine treatment with 20%
piperidine. The resulting resin (11 mg) was treated with
reagent K (110 ml), and the cleaved glycopeptide was
debenzylated at 2158C for 2 h with dimethylsulfide (12 ml),
m-cresol (3.2 ml), 12-ethanedithiol (0.8 ml), TFA (20 ml)
and TfOH (4 ml) as described for 20. The product was
purified by HPLC (Fig. 3(d)), the collected fractions were
concentrated and then lyophilized to give 21 (1.6 mg, 27%).
MALDI TOF MS: calcd for C67H115N14O37·Na m/z
1707.74. Found 1707.70; for C67H114N14O37·Na m/z
1729.74. Found 1729.65.
Amino acid analysis: Asp/Asn1.01Thr3.55Ser1.68Pro0.96
Ala1.00Val1.24
.
5.1.21. N-(9-Fluorenylmethoxycarbonyl)-O-{(5-aceta-
mido-3,5-dideoxy-D-glycero-a-D-galacto-2-nonulopyrano-
sylonic acid)-(2!3)-b-D-galactopyranosyl-(1!4)-2-aceta-
mido-2-deoxy-b-D-glucopyranosyl-(1!6)-[b-D-galacto-
pyranosyl-(1!3)]-2-acetamido-2-deoxy-a-D-galactopyr-
anosyl}-L-threonine 22. To a solution of 19 (100 nmol) in
deionized water (10 ml) were added 0.5 M 3-(N-morpho-
lino)propanesulfonic acid (MOPS) buffer (pH 6.8, 39 ml),
The resin was washed with NMP and MeOH–CH2Cl2, then
N-deprotected, and condensed with Fmoc-Pro-OH to
complete the sequence.

8426
Y. Takano et al. / Tetrahedron 59 (2003) 8415–8427
bovine serum albumin (BSA) in MOPS buffer (5 mg/ml;
5 ml), 0.1 M CMP–sialic acid (5 ml), and recombinant rat
b-Gal-b1,3/4-GlcNAc-a2,3-sialyltransferase (1 ml, 3.7 mU).
The mixture was incubated at 378C, and analyzed by
HPLC in combination with MALDI TOF MS. The
chromatogram for the mixture of 24 h incubation is depicted
in Figure 5(a).
C78H131N15O45(þHþ) m/z 1998.85. Found 1998.52; calcd
for C78H131N15O45·Na m/z 2020.83. Found 2020.67.
5.1.26. L-Prolyl-O-{b-D-galactopyranosyl-(1!4)-2-aceta-
mido-2-deoxy-b-D-glucopyranosyl-(1!6)-[(5-aceta-
mido-3,5-dideoxy-D-glycero-a-D-galacto-2-nonulopyra-
nosylonic acid)-(2!3)-b-D-galactopyranosyl-(1!3)]-2-
acetamido-2-deoxy-a-D-galactopyranosyl}-L-threonyl-
L-threonyl-L-seryl-L-threonyl-L-asparaginyl-L-alanyl-L-
seryl-L-threonyl-L-valylamide 27. Glycopeptide 21
(100 nmol) was sialylated and isolated in a similar manner
as described for 25. MALDI TOF MS: calcd for
C78H131N15O45(þHþ) m/z 1998.85. Found 1998.56; calcd
for C78H131N15O45·Na m/z 2020.83. Found 2020.60.
5.1.22. N-(9-Fluorenylmethoxycarbonyl)-O-{b-D-galacto-
pyranosyl-(1!4)-2-acetamido-2-deoxy-b-D-glucopyrano-
syl-(1!6)-[(5-acetamido-3,5-dideoxy-D-glycero-a-D-
galacto-2-nonulopyranosylonic acid)-(2!3)-b-D-galacto-
pyranosyl-(1!3)]-2-acetamido-2-deoxy-a-D-galactopyr-
anosyl}-L-threonine 23. A solution of 19 (100 nmol) in
deionized water (10 ml) was incubated with 0.1 M CMP–
sialic acid (5 ml), recombinant rat b-Gal-b1,3-GalNAc-
a2,3-sialyltransferase (1 ml, 0.9 mU), BSA solution (5 ml)
and 0.5 M MOPS buffer (pH 6.0, 29 ml) at 378C. The
chromatogram for the reaction mixture of 16 h incubation is
shown in Figure 5(b).
Acknowledgements
We are indebted to Dr T. Chihara and his staff (Riken) for
the combustion analyses. This work was supported by
Grant-in-Aid for Scientific Research from the Ministry
of Education, Culture, Sports, Science, and Technology of
Japan [(B) No.13460051] and in part by CREST program of
Japan Science and Technology Corporation. The authors
also acknowledge Tokai University for a grant-aid for high-
technology research.
5.1.23. N-(9-Fluorenylmethoxycarbonyl)-O-{(5-aceta-
mido-3,5-dideoxy-D-glycero-a-D-galacto-2-nonulopyra-
nosylonic acid)-(2!3)-b-D-galactopyranosyl-(1!4)-2-
acetamido-2-deoxy-b-D-glucopyranosyl-(1!6)-[(5-aceta-
mido-3,5-dideoxy-D-glycero-a-D-galacto-2-nonulopyra-
nosylonic acid)-(2!3)-b-D-galactopyranosyl-(1!3)]-2-
acetamido-2-deoxy-a-D-galactopyranosyl}-L-threonine
24. A solution of 19 (100 nmol) in deionized water (10 ml)
was incubated with 0.1 M CMP–sialic acid (15 ml),
recombinant rat b-Gal-b1,3/4-GlcNAc-a2,3-sialyltransfer-
ase (1 ml, 3.7 mU), recombinant rat b-Gal-b1,3-GalNAc-
a2,3-sialyltransferase (1 ml, 0.9 mU), BSA solution (5 ml)
and 0.5 M MOPS buffer (pH 6.8, 18 ml) at 378C. The
chromatogram for the reaction mixture of 12 h incubation is
shown in Figure 5(c).
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