Efficient and versatile synthesis of mucin-like glycoprotein mimics

Article abstract of DOI:10.1016/S0040-4020(02)01359-5

An efficient synthetic method of sequential glycopeptide-polymer by means of the combined chemical and enzymatic strategy was applied to the practical synthesis of some tandem repeating mucin-type glycoproteins. Versatility of the present synthetic strategy was demonstrated by elucidating some physicochemical and conformational analyses of these synthetic mucins.

Full text of DOI:10.1016/S0040-4020(02)01359-5

Tetrahedron 58 (2002) 10213–10224
Efficient and versatile synthesis of mucin-like glycoprotein mimics
Yuki Tachibana,a Naoki Matsubara,a Fumio Nakajima,b Tetsuro Tsuda,b Sakae Tsuda,c
Kenji Mondea and Shin-Ichiro Nishimuraa,d
,
*
aLaboratory for Bio-Macromolecular Chemistry, Division of Biological Sciences, Graduate School of Science, Hokkaido University,
Kita-ku, Sapporo 060-0810, Japan
bSapporo Laboratory for Glycocluster Project, Japan Bioindustry Association, Sapporo 060-0810, Japan
cStructural Biology Group, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo 062-8517, Japan
dGlycochemosynthesis Team, Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST),
Sapporo 062-8517, Japan
Received 4 September 2002; accepted 21 October 2002
Abstract—An efficient synthetic method of sequential glycopeptide-polymer by means of the combined chemical and enzymatic strategy
was applied to the practical synthesis of some tandem repeating mucin-type glycoproteins. Versatility of the present synthetic strategy
was demonstrated by elucidating some physicochemical and conformational analyses of these synthetic mucins. q 2002 Elsevier Science
Ltd. All rights reserved.
1. Introduction
synthetic glycoproteins have been expected to become nice
tools for the preparation of monoclonal antibody against
essential carbohydrate–peptide epitopes and novel class of
cell adhesive molecules in biomedical materials as well as
for the investigation of structure–activity relationship of
natural mucins. Here we would like to report a general
concept and feasibility of the combined chemical and
enzymatic synthesis of functional macromolecular mucins.
Carbohydrates of the glycoproteins are classified into two
major classes as N-glycans and O-glycans according to
chemical structures of the glycoside linkage between
oligosaccharides and peptide main chains.1 Proteoglycans
and mucins are typical O-glycans showing a variety of
specific functions as adhesive and signal molecules in
cell–cell interactions.2 These biological roles by O-glycans
might be deeply dependent on the multivalent structures of
carbohydrate branches bound to the peptide (protein) main
chains. In the course of the studies on structure–function
relationship of synthetic models for the glycoproteins,3 our
interest has been focused on a variety of tandem repeating
peptide motifs bearing sugar chains found in naturally
occurring mucins and proteoglycans.4 We considered that
the advent of the efficient synthetic strategy of these tandem
repeating glycoproteins will be one of the key technologies
to construct glycoclusters having both designated density
and desired orientation of carbohydrate side chains.5 These
Keywords: mucins; tandem-repeating glycoproteins; sialyl T antigenic
glycoproteins; multivalency; cluster effect.
Abbreviations: AFGP, antifreeze glycoprotein; CMP-Neu5Ac, cytidine
monophosphate-N-acetylneuramic acid; CD, circular dichroism; CSA,
(^)-camphor-10-sulfonic acid; DAST, (diethylamino)sulfur trifluoride;
DMF, N,N-dimethylformamide; DPPA, diphenylphosphorylazide;
GalNAc, N-acetyl galactosamine; GPC, gel permeation chromatography;
PPTS, pyridinium p-toluenesulfonate; TMSOTf, trimethylsiyl
trifluoromethansulfonate.
*
Corresponding author. Address: Laboratory for Bio-Macromolecular
Chemistry, Division of Biological Sciences, Graduate School of Science,
Hokkaido University, Kita-ku, Sapporo 060-0810, Japan. Tel.: þ81-11-
706-3807; fax: þ81-11-706-3435; e-mail: shin@glyco.sci.hokudai.ac.jp
Figure 1. Synthetic strategy of sequential glycopeptides in this study.
0040–4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)01359-5

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Y. Tachibana et al. / Tetrahedron 58 (2002) 10213–10224
Figure 2. Glycopeptides synthesized in this study.
2. Results and discussion
glycotripeptide unit and its direct polymerization under a
mild condition. In the present report, we have modified and
improved the synthetic method previously reported. To
8
Practical syntheses of mucin-type glycoprotein analogs
were basically performed by utilizing direct polymerization
of unprotected glycopeptide macromers in the presence of
diphenylphosphorylazide (DPPA)6,7 as a promotor (Fig. 1).8
Since DPPA selectively activates a peptide carboxyl group
even in the presence of unprotected hydroxyl groups on the
complex carbohydrate moiety, glycopeptide units can be
employed for the direct polymerization reactions without
any protective groups at sugar hydroxyl groups.9 Fig. 2
shows compounds synthesized in the present work and this
method provided glycoproteins with molecular mass from
6600 to 11,650 Da, corresponding to 8–15-mers of the
tripeptide repeating unit (Table 1). As shown in Schemes
1–3, a synthetic pathway of a typical mucin-like glyco-
protein (1), also as a model for antifreeze glycoprotein
(AFGP), involves an chemoselective synthesis10 of the key
prevent from racemization or b-elimination reaction in case
of O-deacylation of the intermediates under alkaline
condition, acetyl groups were converted to benzyl protec-
tions that can be readily removed by mild hydrogenation.
Moreover, a tripeptide unit (Ala-Ala-Thr) was changed to
(Ala-Thr-Ala) in order to avoid the effect of the steric factor
on the activity in the polymerization reactions at the
terminal carboxyl groups.
Schemes 1 and 2 indicate the synthetic route of the
disaccharide moiety of glycopeptide repeating unit. Here,
we employed glycosyl imidate 8 and glycosyl fluoride 7 as a
convenient set of starting materials for a chemoselective
glycosylation strategy. This greatly facilitated synthesis of a
key glycotripeptide intermediate 14. First, 3, 4, 6-tri-O-
acetyl-2-azide-2-deoxy-a,b-D-galactopyranosyl nitrate 4
was prepared from D-galactose in four steps,11 and the
anomeric nitrate group was subsequently replaced by
nucleophilic substitution with fluoride ion generated from
Et3N/3HF complex,12 giving rise to the glycosyl fluoride 5
in 76% yield (a/b¼2:3). At the same time, compound 6,
Table 1. Average molecular weights of synthetic glycopeptides estimated
from gel permeation chromatographical analysis
Compound
Mna
Mwb
1
2
3
3950
4100
6370
6600
5480
11,650
Experiments were carried out with TOSOH TSKgel G3000PWXL using
Pullulans (5.8, 12.2, 23.7, and 48.0 K) as standards.
a
Number average molecular weight.
Weight average molecular weight.
b
Scheme 2. Reagents: (a) NaOMe, MeOH; (b) PhCH(OMe)2, CSA, DMF,
˚
408C, 98% in two steps; (c) TMSOTf, MS 4 A, CH2Cl2, 2158C, 87%;
Scheme 1. Reagents: (a) Et3N/3HF, Et3N, CH3CN, 508C, 76% of 5
(a/b¼2:3), 11% of 6; (b) DAST, THF, 88% (a/b¼1:4).
(d) NaOMe, MeOH; (e) CSA, MeOH; (f) BnBr, NaH, DMF, 2158C to rt,
81% from 9.

Y. Tachibana et al. / Tetrahedron 58 (2002) 10213–10224
10215
˚
Scheme 3. Reagents: (a) Cp2ZrCl2, AgClO4, MS 4 A, CH2Cl2, 2208C to rt, 63% (a/b¼17:4); (b) AcSH, pyridine, 80%; (c) H2 gas, Pd/C, DMF, H2O, AcOH,
100%; (d) DPPA, Et3N, DMF, 08C to rt; (e) 25 mM NaOH, 100% in two steps.
which can be easily converted into 5 using DAST,13 was
obtained in 11% yield as a by-product. De-O-acetylation of
5 followed by benzylidenation furnished 7 having dual
functions in 98% yield. Then, glycosyl fluoride 7 as an
acceptor was coupled with galactosyl imidate 8 using
trimethylsilyl trifluoromethansulfonate (TMSOTf) as a
promoter,14 to provide desired disaccharide 9 in 82%
yield. Although both glycosyl imidate and glycosyl
Figure 3. 1H NMR spectra of compound 14, compound 1, and natural AFGP (AFGP-8).

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Y. Tachibana et al. / Tetrahedron 58 (2002) 10213–10224
monomer glycotripeptide 14. Finally, polymerization of 14
was carried out in the presence of DPPA as a promoter and
the mixture was further treated with 25 mM-NaOH.aq
solution to quench the active azide generated at the C
terminus of the polymer chains. After purification with gel
permeation chromatography the desired polymer fractions,
compound 1, was obtained in 100% yield. The present
method gave much improved polymerization yield in
comparison with the previous method,8 suggesting that
reactivity of the glycotripeptide macromonomer seems to be
enhanced by replacing the position of the threonin residue
having sterically hindered disaccharide from Ala-Ala-Thr to
Ala-Thr-Ala. It was clearly suggested from proton and 13C
NMR spectra that polymerization of the macromonomer
glycotripeptide 14 proceeded smoothly with no side
reaction to afford the macromolecular AFGP analog (1H
NMR spectra of compound 14, a tandem repeating
glycoprotein 1, and natural AFGP are shown in Fig. 3). It
was also demonstrated that the broad spectrum of the natural
AFGP involves some typical signals at 1.9–2.3 ppm due
to L-proline residues.20 Compound 1 showed broader
molecular weight distribution than that of natural AFGP
and the weight average molecular weight (Mw) was
estimated to be 6600 by gel permeation chromatography
(Fig. 4).21 In addition, intermediate 21 in which GalNAc
residue of 14 was replaced with Gal residue was also
synthesized by means of a conventional strategy and
polymerized as indicated in Schemes 4 and 5.
Figure 4. GPC elution profiles of a tandem repeating glycoprotein 1 and
natural AFGP (AFGP-8, m.w.¼2.6 K). TSKgel G3000PWXL was eluted
with 0.1 M ammonium bicarbonate buffer (pH 7.6, flow rate¼0.4 mL/min).
fluoride15 are known to be activated by TMSOTf, we have
succeeded in specifically activating glycosyl imidate by
running the reaction at low temperature (2208C) without
any side reactions. Both O-acetyl groups and benzylidene
group of the compound 9 were replaced with O-benzyl
protections. These procedures were necessary to solve two
serious problems: (a) the alkaline condition required for
the removal of O-acetyl groups might cause base-catalyzed
b-elimination of sugar moiety from peptide chain or base-
catalyzed epimerization of amino acids moiety, (b) the
benzylidene group became unstable in the presence of
electron donating benzyl groups.16 Thus, the intermediate 9
was converted into the per-O-benzylated glycosyl donor 10
in satisfactory yield (81% yield).
The versatility and importance of the present synthetic
strategy are evident because a variety of mucin-type
glycoproteins4 involving tandem-repeating peptide motifs
such as RNA polymerase II, Muc families, and proteo-
glycans have been reported to exhibit significant biological
functions as signals of cell adhesion. Moreover, enzymatic
modification study strongly contributes to the efficient
synthesis of further complicated mucin-type glycoproteins.
Actually, it was demonstrated that 3-O-sialylation reaction
by means of a commercially available sialyltransferase
(recombinant rat 2,3-sialyltransferase) greatly facilitated the
production of the sialylated glycoprotein (3) as shown in
Scheme 6 and Fig. 5. It was suggested that both periodate–
resorcinol method22 (98%) and integration data of 1H NMR
spectrum (100%) exhibited quantitative sialylation reaction
at O-3 position of the galactose residue by this enzyme. The
sialylated glycoprotein (3) as well as enzymatically
As shown in Scheme 3, disaccharidic donor 10 and a
tripeptide 11 were coupled in the presence of Cp2ZrCl2/
AgClO4 (1:2)17 in CH2Cl2 to give desired glycotripeptide 12
in 51% yield.18 At the same time, 12% of b-isomer (12b)
was yielded. Then, the azide group of 12 was converted into
N-acetyl group by treating with thioacetic acid–pyridine19
(80% yield) followed by quantitative hydrogenolytic
removal of all protecting groups to afford a key macro-
Scheme 4. Reagents: (a) CH3C(OCH3)3, TsOH, CH3CN; (b) 80% AcOH, 100% in two steps; (c) TMSOTf, CH2Cl2, 2208C, 84%; (d) NaOMe, MeOH;
(e) BnBr, NaH, DMF, 08C to rt, 80% in two steps; (f) PdCl2, NaOAc, 90% AcOH; (g) DAST, THF, 66% in two steps (a/b¼1:1).

Y. Tachibana et al. / Tetrahedron 58 (2002) 10213–10224
10217
˚
Scheme 5. Reagents: (a) Cp2ZrCl2, AgClO4, MS 4 A, CH2Cl2, 2208C to rt, 58%; (b) H2 gas, Pd/C, DMF, H2O, AcOH, 62%; (c) DPPA, Et3N, DMF, 08C to rt;
(d) 25 mM NaOH, 64% in two steps.
modified Tn-antigenic mucin mimics23,24 may be valuable
tools for investigating immunological and biological
significance of oligosaccharide sequences of O-linked type
glycoproteins.25,26
generate freezing point depression by adsorption to ice
surfaces and consequent inhibition of further growth of ice
crystals.28 The formation of the characteristic bipyramidal
ice crystals is one of the most important steps to inhibit the
growth of ice crystal. Fig. 6 clearly showed that over a
certain range of temperature (approximately from 0 to
20.58C) both 1 (a) and natural AFGP (d) induced the
formation of the typical hexagonal bipyramidal ice crystals.
On the other hand, compound 2 (b) exhibited no significant
Since a tandem repeating glycoprotein 1 may be a simple
model polymer of the naturally occurring AFGP,27 we
evaluated the inhibitory effect by 1 and analogs 2 and 3 on
ice crystal growth. It is known that the natural AFGPs
Scheme 6. Reagents: (a) CMP-Neu5Ac, a2,3-sialyltransferase, alkaline phosphatase, Triton CF-54, 50 mM sodium cacodyrate buffer (pH 7.0), 378C, 93%
(isolated yield).

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Y. Tachibana et al. / Tetrahedron 58 (2002) 10213–10224
Figure 8. Plausible structure of compound 1 generated by SYBYL/ad-
vanced computation module.
tains an ordered helix structure different from typical
a-helix or b-sheet such as a 3-fold left-handed helix
structure.29–32 As seen in the spectrum of the natural AFGP,
compound 1 has quite similar secondary structure to that of
natural AFGP. In addition, sialylated glycoprotein 3 which
showed no inhibitory effect on the ice growth still retains the
same secondary structure as that of natural AFGP. However,
Galb(1!3)Gal analog 2 exhibited typical CD spectrum
found in the polypeptides composed of random coil
32
structure.
It is likely that antifreeze activity by natural
1
Figure 5. H NMR spectrum of sialylated glycoprotein 3.
AFGP depends on the existence of this specific ordered
helix as well as key molecular motifs to adsorb efficiently
with ice lattice (Fig. 8). Further study of the antifreeze
activity by using a series of analogs is under examination
and the results will be reported in the nearest future.
In conclusion, a general synthetic strategy for the construc-
tion of tandem repeating polyglycopeptides was established
by means of direct polymerization of the repeating
glycopeptide unit as a macromonomer by DPPA without
any protective groups. The versatility of the combined
chemical and enzymatic synthesis of a biologically
important mucin was also demonstrated by employing a
recombinant rat a(2!3)sialyltransferase with synthetic
glycoprotein 1 in the presence of CMP-Neu5Ac as a
glycosyl donor substrate to give a sialylated glycoprotein 3
in a quantitative yield. The tandem repeating glycoproteins
synthesized here were found to act as valuable macro-
molecular models for antifreeze glycoprotein.
Figure 6. Ice crystal morphology in the presence of (a) a tandem repeating
glycoprotein 1, (b) Galb1!3Gal analog 2 (c) sialylated glycoprotein 3,
(d) natural AFGP (AFGP-8) in water (10 mg/mL). Photos were taken at
20.28C (a) and (d) or 0.08C (b) and (c). The melting points of all solutions
were 0.08C.
inhibitory effect and sialylated glycoprotein 3 (c) showed no
capacity to form the bipyramidal crystal. Moreover, the
glycomacromonomer 14, a repeating unit of the polymer,
did not show any inhibitory effect on the growth of the ice
(data not shown).
The CD spectra in the far-uv absorption region of synthetic
glycoproteins in aqueous buffer at neutral pH are shown in
Fig. 7. The CD patterns of 1 at 258C clearly show a positive
absorption at 218 nm in addition to a strong negative band at
195 nm, suggesting that this sequential glycoprotein con-
3. Experimental
3.1. General
Thin-layer chromatography (TLC) was performed on Merck
silica gel glass plates, 60F254; compounds were visualized
by treatment with a solution of (NH4)6Mo7O2·4H2O (20 g)
and Ce(SO4)2 (0.4 g) in 10% sulfuric acid (400 mL) and
heating at 1508C. Flash chromatography was performed
on Kanto Chemical silica gel N60 (40–50 mm).
NMR measurements were recorded at 278C on a JEOL
JNM-lambda-400 FT-NMR spectrometer (1H (400 MHz))
or a BRUKER AVANCE 600 (1H (600 MHz), 13C
(120 MHz)). FAB-mass spectra were obtained with a
JEOL JMS-HX 110 mass spectrometer, using m-nitro-
benzylalcohol (NBA) as matrix. All solvents were used as
commercially received. The molecular weights of synthe-
sized polyglycopeptides were estimated by gel permeation
chromatography with a TOSOH-TSKgel G3000PWXL
column (pullulans (5.8, 12.2, 23.7, and 48.0 K; Shodex
Standard) were used as standards). Recombinant rat a-2,3-
sialyltransferase and CMP-Neu5A were purchased from
Calbiochem CO. Ltd. Calf intestine alkaline phosphatase
were obtained from Sigma Co. Ltd. Natural AFGP was
Figure 7. CD spectra of natural AFGP and its analogs. All curves were
measured at 248C in water (0.1 mg/mL).

Y. Tachibana et al. / Tetrahedron 58 (2002) 10213–10224
10219
obtained from A/F protein Inc. Circular dichroism (CD)
spectra were measured in 1 mm path length quartz cells on a
JASCO J-720 spectropolarimeter. Molecular simulation
of AFGP was preliminarily performed by using SYBYL/
advanced computation module.33 Periodate-resorcinol
method was carried out for the quantitative analysis of
sialic acid content of 3 according to the method established
by G. W. Jourdian et al.22 The absorbance at 630 nm was
observed with HITACHI U-2010 spectrophotometer.
(606 mg, 2.61 mmol). The solution was stirred at 508C for
2 h, and neutralized with Et3N (1.8 mL, 13 mmol), then
concentrated. Flash column chromatography of the residue
(hexane/EtOAc¼4:1, then 2:1) yielded 1.51 g of 7 (98%) as
colorless syrup. The physical data were in agreement with
35
1
those reported by Ogawa et al.
Selected data of 7; H
NMR (400 MHz, CDCl3, d): 5.59 (s, 1H, Ph-CH–O), 5.06
(d, 1H, J1,2¼7.5 Hz, J1,F¼55.2 Hz, H-1), 4.40 (dd, 1H,
J5,6a¼1.6 Hz, J6a,6b¼12.8 Hz, H-6a), 4.10 (m, 1H, J5,6b
¼
1.8 Hz, H-6b), 3.75 (ddd, 1H, J2,F¼11.9 Hz, J2,3¼10.2 Hz,
1
The inhibitory effect on the ice crystal growth of synthetic
polyglycopeptides and natural AFGPs were evaluated by
observation of the ice crystal morphology using a Leica
DMLB 100 photomicroscope equipped with a Linkam LK
600 temperature controller as described previously.34 The
compounds were dissolved in water (10 mg/mL), momen-
tarily frozen (approximately 2228C), and warmed to 08C on
the sample stage of the optical microscope and create
several ice nucleolus in the solution. This solution was then
cooled at a rate of 0.078C/min, and the crystal morphologies
were monitored. The photos were taken at 20.28C in the
case the compound have ice crystal growth inhibition
activity, or at 0.08C for those did not have the activity.
H-2), 2.58 (d, 1H, J¼8.7 Hz, CHOH). (lit.35); H NMR
(CDCl3, d): 5.59 (s, 1H, Ph-CH–O), 5.06 (d, 1H, H-1,
J1,2¼7.6 Hz, J1,F¼52.2 Hz), 4.39 (dd, 1H, J5,6a¼1.5 Hz,
J6a,6b¼12.5 Hz, H-6a), 4.10 (m, 1H, J5,6b¼1.8 Hz, H-6b),
3.75 (ddd, 1H, J2,F¼11.9 Hz, J2,3¼10.4 Hz, H-2), 2.61 (d,
1H, J¼8.2 Hz, CHOH).
3.1.3. 2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl)-
(1!3)-2-azide-4,6-O-benzylidene-2-deoxy-b-D-galacto-
pyranosyl fluoride (9). A mixture of galactosyl imidate 8
(2.36 g, 4.8 mmol), glycosyl fluoride 7 (1.18 g, 4.0 mmol),
˚
and powdered molecular sieves 4 A (1.0 g) in dry CH2Cl2
(15 mL) was cooled to 2158C under nitrogen atmosphere.
Then, TMSOTf (77 mL, 0.40 mmol) in CH2Cl2 (0.5 mL)
was added dropwisely, and the mixture was stirred at 2158C
for 2 h, and then triethylamine (1.0 mL) was added to
quench TMSOTf. The solution was filtered, concentrated,
and purified by flash column chromatography (toluene/
EtOAc¼6:1 then 4:1) to give 9 (2.17 g, 87%). The physical
data were in agreement with those reported by Ogawa et al.36
3.1.1. 3,4,6-Tri-O-acetyl-2-azide-2-deoxy-D-galactopyra-
nosyl fluoride (5). To a solution of 4 (2.92 g, 7.8 mmol) in
CH3CN (15 mL) was added Et3N (0.75 mL) and Et3N/3HF
(3.1 mL) under nitrogen atmosphere and stirred at 508C for
18 h. Then, MeOH (5.0 mL) was added to quench
Et3N/3HF, and the solvent was concentrated. The residue
was diluted with CHCl3, washed with water, NaHCO3,
brine, dried (MgSO4), concentrated, and purified by flash
column chromatography (toluene/EtOAc¼4:1) to give 5
(1.97 g, 76%, a/b¼2:3) and 6 (0.28 g, 11%). Compound 6
was easily converted into 5 using DAST. To a solution of 6
(2.66 g, 8.0 mmol) in THF (30 mL) was added DAST
(1.3 mL, 8.2 mmol) at 2208C, and stirred at room
temperature for 1 h. Then, the solution was cooled to 08C,
and MeOH was added to quench DAST. The solution was
concentrated, and extracted with CHCl3, washed with water,
sat. NaHCO3, brine, dried (MgSO4), and concentrated. The
crude residue was purified by flash column chromatography
(toluene/EtOAc¼7:1, then 5:1) to give 5 (2.37 g, 89%,
a/b¼1:4). In this procedure, a-isomer (minor product)
could not be purified and b-isomer was used for the next
step. 5b: 1H NMR (400 MHz, CDCl3, d): 5.36 (s, 1H, H-4),
5.13 (d, 1H, H-1, J1,2¼7.5 Hz, J1,F¼51.7 Hz), 4.86 (dd, 1H,
Selected data of 9; 1H NMR (400 MHz, CDCl3, d): 5.50 (s,
0
0
0
1H, Ph-CH–O), 5.41 (d, 1H, J3 ,4 ¼3.4 Hz, H-4 ), 5.27 (dd,
0
0
0
0
0
1H, J1 ,2 ¼8.0 Hz, J3 ,4 ¼10.4 Hz, H-2 ), 5.07 (dd, 1H, J1,F
¼
0
52.6 Hz, J1,2¼7.5 Hz, H-1), 4.80 (d, 1H, H-1 ). (lit.36); H
1
NMR (CDCl3, d):0 5.54 (s, 1H, Ph-CH–O), 5.41 (d, 1H,
0
0
0
0
0
0
J3 ,4 ¼3.3 Hz, H-4 ), 5.28 (dd, 1H, J1 ,2 ¼7.9 Hz, J3 ,4
¼
10.3 Hz, H-20), 5.08 (dd, 1H, J1,F¼52.5 Hz, J1,2¼7.9 Hz,
H-1), 4.80 (d, 1H, H-10).
3.1.4. 2,3,4,6-Tetra-O-benzyl-b-D-galactopyranosyl)-
(1!3)-2-azide-4,6-di-O-benzyl-2-deoxy-b-D-galacto-
pyranosyl fluoride (10). To a solution of 9 (200 mg,
2.08 mmol) in MeOH (6 mL) was added NaOMe (2 mg,
0.032 mmol), and stirred at room temperature for 1 h. The
reaction mixture was then neutralized with Dowex 50W-X8
[Hþ] resin, filtered, and concentrated. To a solution of
residual amorphous in MeOH (6 mL) was added CSA
(38 mg, 0.16 mmol) and stirred at room temperature for 2 h.
Then, Et3N (0.18 mL, 1.28 mmol) was added to quench the
reaction, and concentrated. The residue was dissolved in
DMF (6 mL), and cooled to 2158C, at which time 60% NaH
(153 mg, 3.84 mmol) was added, and stirred at the same
temperature for 30 min. Benzyl bromide (0.69 mL,
5.76 mmol) was added dropwisely, and the mixture was
allowed to warm slowly to room temperature over 12 h.
Then, methanol was added and the solution was evaporated.
The residual syrup was extracted with CHCl3, and the
organic layer was washed with water, 1N HCl, sat.
NaHCO3, brine, dried (MgSO4), and concentrated. The
crude residue was purified by flash column chromatography
(hexane/EtOAc¼5:1, then 4:1) to give 10 (235 mg, 81%).
H-3, J2,3¼10.9 Hz, J3,4¼3.2 Hz), 4.18 (dd, 2H, J5,6a¼J5,6b
¼
6.4 Hz, J6a,6b¼2.0 Hz, H-6a,b), 4.00 (dd, 1H, H-5), 3.82 (m,
1H, J2,F¼18.4 Hz, H-2), 2.17–2.06 (s£3, 9H, CH3CvO£3);
HRMS-FAB (m/z): [MþH]þ calcd for C12H17FN3O7,
334.1051; found, 334.1076; elemental analysis calcd (%)
for C12H16FN3O7: C, 43.25; H, 4.84; N, 12.61; found: C,
43.22; H, 4.70; N, 12.61.
3.1.2. 2-Azide-4,6-O-benzylidene-2-deoxy-D-galacto-
pyranosyl fluoride (7). To a solution of 5b (1.74 g,
5.22 mmol) in MeOH (20 mL) was added NaOMe (27 mg,
0.52 mmol) and stirred at room temperature for 1 h. Then,
the solution was neutralized with Dowex 50W-X8 [Hþ]
resin, filtered, and concentrated. The concentrated mixture
was dissolved in DMF (20 mL), and was added benz-
aldehyde dimethylacetal (1.57 mL, 10.4 mmol) and CSA
1
10: H NMR (400 MHz, CDCl3, d): 7.37–7.18 (m, 30H,
aromatic), 5.02 (dd, 1H, J1,2¼7.4 Hz, J1,F¼52.6 Hz, H-1),

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Y. Tachibana et al. / Tetrahedron 58 (2002) 10213–10224
0
0
5.01–4.55 (m, 8H, Ph-CH–O), 4.63 (d, 1H, J1 ,2 ¼7.6 Hz,
with CHCl3, filtered thorough celite. The filtrate was
washed with sat. NaHCO3, brine, dried (MgSO4), and
concentrated. The crude residue was purified by flash
column chromatography (toluene/EtOAc¼5:1, then 4:1) to
give 12 (1.39 g, 51%) and its b-isomer 12b (337 mg, 12%).
H-10), 4.56–4.34 (m, 4H, Ph-CH–O), 3.95–3.87 (m, 1H,
H-2), 3.92 (dd, 1H, H-50), 3.90 (d, 1H, J3 ,4 ¼2.6 Hz, H-4 ),
0
0
0
0
0
0
3.85 (dd, 1H, J2 ,3 ¼9.6 Hz, H-2 ), 3.64–3.53 (m, 7H, H-4,
H-5, H-6a, H-6b, H-60a, H-60b), 3.52 (dd, 1H, J3 ,4 ¼2.6 Hz,
H-30), 3.44 (dd, 1H, J2,3¼8.1 Hz, J3,4¼4.6 Hz, H-3);
HRMS-FAB (m/z): [MþNa]þ calcd for C54H56FN3O9Na,
932.3899; found, 932.3907; elemental analysis calcd (%)
for C54H56FN3O9: C, 71.26; H, 6.21; N, 4.62; found: C,
71.02; 6.25; 4.52.
0
0
1
12: H NMR (400 MHz, CDCl3, d): 7.37–7.18 (m, 40H,
aromatic), 7.34 (d, 1H, J¼7.0 Hz, Ala-NH), 7.03 (d, 1H,
J¼6.7 Hz, Thr-NH), 5.26 (d, 1H, J¼6.2 Hz, Ala-NH), 5.23
(d, 1H, J1,2¼3.8 Hz, H-1), 5.19–4.94 (m, 6H, Ph-CH2–O),
4.77–4.29 (m, 13H, Ph-CH2–O, Ala-a-H, Thr-a-H, Thr-b-
0
0
0
H), 4.71 (d, 1H, J1 ,2 ¼7.6 Hz, H-1 ), 4.23 (m, 1H, Ala-a-H),
3.1.5. N-(Benzyloxycarbonyl)-L-alanyl-L-threonyl-L-ala-
nine benzyl ester (11). To a stirred solution of Boc-Thr-OH
(5.4 g, 24.6 mmol) and Ala-OBzl p-tosylate (9.9 g,
28.3 mmol) in DMF (50 mL) was added DPPA (6.1 mL,
28.3 mmol) in DMF (50 mL) at 08C, followed by the
dropwise addition of triethylamine (7.4 mL, 52.9 mmol) in
DMF (50 mL). The solution was stirred at 08C for 2 h, then
warmed to room temperature and stirred for 22 h. The
solution was concentrated, and extracted with EtOAc,
washed with 5% citric acid, sat. NaHCO3, brine, and dried
(MgSO4). Then the solution was evaporated, and the residue
was purified by flash column chromatography (hexane/
EtOAc¼1:1 then 2:3) to give Boc-Thr-Ala-OBzl (8.2 g,
88%).
4.17 (dd, 1H, J2,3¼10.6 Hz, J3,4¼2.8 Hz, H-3), 4.13 (dd,
1H, H-4), 4.00 (dd, 1H, J5 ,6 a¼J5 ,6 b¼6.1 Hz, H-50), 3.90 (d,
0
0
0
0
0
0
0
0
0
1H, J3 ,4 ¼3.0 Hz, H-4 ), 3.90–3.87 (m, 2H, J2 ,3 ¼9.8 Hz,
H-2, H-20), 3.66–3.56 (m, 3H, H-5,0H-6a, H-6b), 3.54–3.39
(m, 3H, J6 a,6 b¼9.6 Hz, H-30, H-6 a, H-60b), 1.40 (d, 3H,
J¼7.2 Hz, Ala-b-H), 1.37 (d, 3H, J¼7.2 Hz, Ala-b-H), 1.11
(d, 3H, J¼6.0 Hz, Thr-g-H); 13C NMR (120 MHz, CDCl3,
d): 172.7, 172.5, 166.6, 139.3, 139.1, 138.8, 138.5, 138.2,
136.5, 135.8, 129.0, 128.9, 128.8, 128.7, 128.6, 128.5,
128.4, 128.3, 128.2, 128.0, 127.9, 127.8, 105.6, 99.5, 82.8,
79.8, 76.7, 75.6, 75.0, 74.8, 74.3, 74.2, 74.0, 73.8, 73.7,
73.5, 70.7, 69.8, 69.3, 67.5, 67.3, 61.2, 60.8, 56.4, 51.4,
48.7, 19.1, 17.1, 14.6; HRMS-FAB (m/z): [MþNa]þ calcd
for C79H86N6O16Na, 1397.5998; found, 1397.6050; elemen-
tal analysis calcd (%) for C79H86N6O16: C, 68.98; H, 6.30;
N, 6.11; found: C, 68.88; H, 6.32; N, 5.94.
0
0
N-Protected dipeptide (Boc-Thr-Ala-OBzl, 3.3 g,
8.67 mmol) was dissolved in 4N HCl/dioxane (50 mL),
and stirred at room temperature for 1 h, then concentrated to
give colorless solid. To a stirred solution of the residual
colorless solid and Z-Ala-OH (3.5 g, 7.9 mmol) in DMF
(30 mL) was added DPPA (2.1 mL, 9.1 mmol) in DMF
(30 mL) at 08C, followed by the addition of triethylamine
(2.5 mL, 18.2 mmol) in DMF (30 mL). The solution was
stirred at 08C for 2 h, then warmed to room temperature and
stirred for 22 h. The solution was concentrated, and
extracted with EtOAc, washed successively with 5% citric
acid, sat. NaHCO3, brine, and dried (MgSO4), and
concentrated. Crystallization of the residue from EtOAc
giving 11 (3.6 g, 85%). 11: mp 1738C; 1H NMR (400 MHz,
CDCl3, d): 7.38–7.26 (m, 10H, aromatic), 7.12 (d, 1H,
J¼7.5 Hz, Thr-NH), 7.03 (d, 1H, J¼6.9 Hz, Ala-NH), 5.45
(d, 1H, J¼6.8 Hz, Ala-NH), 5.17 (dd, 2H, Ph-CH2–OH),
5.10 (s, 2H, Ph-CH2–OH), 4.55 (m, 1H, J¼7.3 Hz, Ala-a-
H), 4.42 (dd, 1H, J¼2.0 Hz, Thr-a-H), 4.31 (m, 2H, Thr-b-
H, Ala-a-H), 1.40 (d, 3H, Ala-b-H), 1.39 d, 3H, Ala-b-H),
1.12 (d, 3H, J¼6.2 Hz, Thr-g-H); HRMS-FAB (m/z):
[MþH]þ calcd for C25H31N3O7, 486.2241; found,
486.2201; elemental analysis calcd (%) for
C25H30N3O7·H2O: C, 59.63; H, 6.61; N, 8.34; found: C,
60.03; H, 6.42; N, 8.41.
1
12b: H NMR (400 MHz, CDCl3, d): 7.29–7.08 (m, 41H,
aromatic, Ala-NH), 6.92 (d, 1H, J¼6.6 Hz, Thr-NH), 5.24
(d, 1H, Ala-NH), 5.09–4.86 (m, 8H, Ph-CH2–O), 4.72–
0
0
4.63 (m, 4H, Thr-a-H, Ph-CH2–O), 4.57 (d, 1H, J1 ,2
¼
7.6 Hz, H-10), 4.52–4.48 (m, 3H, H-1, Ph-CH2–O), 4.35 (q,
1H, Ala-a-H), 4.32–4.28 (m, 3H, Ph-CH2–O), 4.20–4.10
(br, 2H, Thr-b-H, Ala-a-H), 3.86 (d, 1H, J3,4¼2.8 Hz, H-4),
0
0
0
0
0
3.83 (d, 1H, J3 ,4 ¼2.9 Hz, H-4 ), 3.76 (dd, 1H, J2 ,3 ¼9.5 Hz,
H-20), 3.75 (dd, 1H, H-2), 3.59 (dd, 1H, J5,6a¼6.2 Hz, J5,6b
¼
0
0
0
0
6.3 Hz, H-5), 3.54 (dd, 1H, J5 ,6 a¼4.9 Hz, J5 ,6 b¼5.4 Hz,
H-50), 3.51–3.46 (m, 4H, H-6a, H-6b, H-30, H-60a), 3.43 (dd,
1H, J2,3¼10.5 Hz, H-3), 3.38 (dd, 1H, H-6b), 1.25 (d, 3H,
J¼6.4 Hz, Ala-b-H), 1.18 (d, 3H, J¼7.1 Hz, Ala-b-H), 1.08
(d, 1H, J¼6.1 Hz, Thr-g-H); 13C NMR (120 MHz, CDCl3,
d): 172.3, 172.2, 168.2, 139.3, 139.0, 138.9, 138.3, 138.2,
136.8, 136.6, 136.0, 128.9, 128.8, 128.7, 128.6, 128.5,
128.3, 128.1, 128.0, 127.9, 127.8, 105.4, 103.0, 82.4, 80.1,
79.9, 75.6, 75.4, 75.0, 75.0, 74.4, 74.1, 74.0, 73.7, 73.7,
73.6, 69.2, 68.9, 67.4, 67.3, 63.7, 55.8, 51.2, 48.9, 19.2,
17.9, 16.0; HRMS-FAB (m/z): [MþH]þ calcd for
C79H87N6O16, 1375.6179; found, 1375.6143; elemental
analysis calcd (%) for C79H86N6O16·H2O: C, 68.18; H,
6.23; N, 6.04; found: C, 68.02; H, 6.24; N, 5.86.
3.1.6. N-(Benzyloxycarbonyl)-L-alanyl-O-[(2,3,4,6-tetra-
O-benzyl-b-D-galactopyranosyl)-(1!3)-(2-azide-4,6-di-
O-benzyl-2-deoxy-a-(12) and -b-D-galactopyranosyl)]-L-
threonyl-L-alanine benzyl ester (12b). A mixture of
Cp2ZrCl2 (1.13 g, 3.85 mmol), AgClO4 (1.60 g,
7.70 mmol), 11 (2.57 g, 4.62 mmol), and powdered mol-
3.1.7. N-(Benzyloxycarbonyl)-L-alanyl-O-[(2,3,4,6-tetra-
O-benzyl-b-D-galactopyranosyl)-(1!3)-(2-acetamido-
4,6-di-O-benzyl-2-deoxy-a-D-galactopyranosyl)]-L-
threonyl-L-alanine benzyl ester (13). A solution of 13
(90 mg, 65.4 mmol) in AcSH (1 mL) and pyridine (0.5 mL)
was stirred at room temperature for 20 h, and the residue
was purified by flash column chromatography (run 1; hexane/
EtOAc¼5:1, then EtOAc, run 2; hexane/EtOAc¼3:4) to
give 13 (73 mg, 80%). 13: 1H NMR (400 MHz, CDCl3, d):
7.31–7.18 (m, 40H, aromatic), 7.10 (d, 1H, J¼6.2 Hz,
Ala-NH), 7.03 (d, 1H, Thr-NH), 6.44 (d, 1H, J¼8.5 Hz,
˚
ecular sieves 4 A (2.5 g) in dry CH2Cl2 (40 mL) was stirred
at room temperature for 10 min under nitrogen atmosphere,
then cooled to 2208C. A solution of 10 (1.40 g, 1.54 mmol)
in dry CH2Cl2 (7 mL) was added, and the mixture was
stirred at 2208C to room temperature for 24 h, then diluted

Y. Tachibana et al. / Tetrahedron 58 (2002) 10213–10224
10221
CHNHCvO), 5.37 (d, 1H, J¼7.2 Hz, Ala-NH), 5.23–4.90
4.12 (m, 7H, Thr-a-H, Thr-b-H, H-10, Ala-a-H£2, H-2,
0
0
(m, 7H, Ph-CH2–O), 5.21 (d, 1H, J1,2¼3.0 Hz, H-1), 4.75–
H-4), 3.97–3.94 (m, 2H, H-5, H-3), 3.80 (d, 1H, J3 ,4
¼
0
4.63 (m, 6H, J1 ,2 ¼7.5 Hz, H-1 , H-2, Ph-CH2–O), 4.55
(m, 1H, Ala-a-H), 4.53–4.23 (m, 8H, Ala-a-H, Thr-a-H,
Thr-b-H, Ph-CH2–O), 4.07 (s, 1H, H-4), 4.00 (dd, 1H,
2.9 Hz, H-40), 3.66 0(m, 4H, H-6a, 6b, 6a0, 6b0), 3.97–3.53
0
0
(m, 2H, H-50, H-3 ), 3.42 (dd, 1H, J1 ,2 ¼7.8 Hz, J2 ,3
¼
0
0
0
0
9.8 Hz, H-20), 1.92 (s, 3H, NHCOCH3), 1.33 (m, 9H, Ala-b-
H£2, Thr-g-H); 13C NMR (120 MHz, D2O, d): 176.0, 174.5,
174.2, 171.2, 105.1, 99.4, 77.8, 76.6, 75.3, 72.9, 71.4, 71.0,
69.2, 69.0, 61.7, 61.4, 57.5, 49.5, 49.0, 48.7, 22.7, 18.9,
17.6, 17.2.
0
0
0
0
J3 ,4 ¼2.4 Hz, H-3 ), 3.98 (dd, 1H, H-5), 3.87 (d, 1H, H-4 ),
0
0
0
3.84 (dd, 1H, J2 ,3 ¼9.6 Hz, H-2 ), 3.61–3.39 (m, 6H, H-5,
H-6a, H-6b, H-30, H-60a, H-60b), 1.63 (s, 3H, NHCOCH3),
1.39 (d, 3H, J¼6.9 Hz, Ala-b-H), 1.34 (d, 3H, J¼7.0 Hz,
Ala-b-H), 1.11 (d, 3H, J¼6.4 Hz, Thr-g-H); 13C NMR
(120 MHz, D2O, d): 173.5, 172.7, 171.3, 169.3, 139.4,
139.3, 138.8, 138.7, 138.2, 136.5, 135.4, 129.1, 129.0,
128.9, 128.8, 128.7, 128.6, 128.5, 128.4, 128.2, 128.0,
127.9, 127.8, 106.1, 99.0, 82.6, 79.8, 78.3, 76.4, 75.3, 75.0,
74.5, 74.2, 74.0, 73.7, 73.3, 70.8, 70.3, 69.2, 68.0, 67.5,
56.6, 51.1, 51.0, 50.4, 48.9, 18.6, 17.5, 14.5; HRMS-FAB
(m/z): [MþNa]þ calcd for C81H90N4O17Na, 1413.6199;
found, 1413.6220; elemental analysis calcd (%) for
C81H90N4O17·H2O: C, 69.02; H, 6.58; N, 3.97; found: C,
69.40; H, 6.82; N, 3.78.
3.1.10. Allyl 4-O-acetyl-2,6-di-O-benzyl-a-D-galacto-
a
solution of 1537 (280 mg,
pyranoside (16). To
0.70 mmol) in CH3CN (10 mL) was added MeC(OMe)3
(270 mL) and a catalytic quantity of PPTS (10 mg).38,39
After 10 min, a solution of 80% AcOH (18 mL) was added
and stirred for 1.5 h. The solution was extracted with
CHCl3, washed with water, NaHCO3, brine, dried (MgSO4),
concentrated, and purified by flash column chromatography
(hexane/EtOAc¼6:1, then 4:1) to give 16 (306 mg, 100%)
1
as a white solid. 16: H NMR (600 MHz, CDCl3, d): 5.91
(m, 1H, CH2–CHvCH2), 5.45 (d, 1H, J1,2¼3.0 Hz, H-1),
5.31 (dd, 1H, CHvCH2), 5,21 (dd, 1H, CHvCH2), 4.92
(d, 1H, J3,4¼3.0 Hz, H-4), 4.55 (d, 1H, O–CH2-Ph), 4.45
(d, 1H, O–CH2-Ph), 4.20–4.14 (m, 2H, H-2, CH2–
CHvCH2), 4.12 (dd, 1H, J5,6a¼J5,6b¼6.2 Hz, H-5), 3.95
(dd, 1H, CH2–CHvCH2), 3.71 (dd, 1H, J2,3¼9.9 Hz, H-3),
3.71 (s, 1H, 3-OH), 3.49 (dd, 2H, H-6a, H-6b); HRMS-FAB
(m/z): [MþH]þ calcd for C25H31O7, 443.2070; found,
443,2085; elemental analysis calcd (%) for C25H30O7: C,
67.79; H, 6.89; found: C, 67.86; H, 6.83.
3.1.8. L-Alanyl-O-[b-D-galactopyranosyl-(1!3)-2-aceta-
mido-2-deoxy-a-D-galactopyranosyl]-L-threonyl-L-ala-
nine (14). To a solution of 14 (15 mg, 0.012 mmol) in DMF
(3 mL), acetic acid (1 mL), H2O (1 mL) was added 10%
Pd/C (100 mg), and stirred at room temperature for 48 h
under H2 gas atmosphere. Then, Pd/C was removed by
filtration, and the solution was evaporated. The residue was
purified by gel filtration chromatography (Sephadex G-10,
1
water as eluent) to give 14 (7.2 mg, 100%). 14: H NMR
(400 MHz, D2O, d): 4.88 (d, 1H, J1,2¼3.7 Hz, H-1), 4.43 (d,
1H, J¼2.1 Hz, Thr-a-H), 4.33 (dd, J¼2.1 Hz, Thr-b-H),
3.1.11. Allyl O-(2,3,4,6-tetra-O-acetyl-b-D-galactopyra-
nosyl)-(1!3)-4-O-acetyl-2,6-di-O- benzyl-a-D-galacto-
pyranoside (17). A mixture of galactosyl imidate 8
(240 mg, 0.49 mmol), 16 (1.18 g, 4.0 mmol), and powdered
0
0
0
4.36 (d, 1H, J1 ,2 ¼7.8 Hz, H-1 ), 4.20 (dd, 1H, J2,3
¼
11.1 Hz, H-2), 4.16 (q, 1H, Ala-a-H), 4.11 (d, 1H,
J3,4¼1.7 Hz, H-4), 4.05 (q, 1H, J¼7.1 Hz, Ala-a-H), 3.97
˚
0
0
(m, 1H, H-5), 3.94 (dd, 1H, H-3), 3.80 (d, 1H, J3 ,4 ¼3.2 Hz,
molecular sieves 4 A (500 mg) in dry CH2Cl2 (3 mL) was
H-40), 3.66 (m, 4H, H-6a, 6b, 6a0, 6b0), 3.97 (m, 1H, H-50),
cooled to 2158C under nitrogen atmosphere. Then,
TMSOTf (7.9 mL, 0.041 mmol) in CH2Cl2 (72 mL) was
added dropwisely, and the mixture was stirred at 2158C
for 2 h, and then triethylamine (1.0 mL) was added to
quench TMSOTf. The solution was filtered, concentrated,
and purified by flash column chromatography (toluene/
EtOAc¼6:1) to give 17 (305 mg, 96%). 17: 1H NMR
(600 MHz, CDCl3, d): 7.36–7.26 (m, 10H, aromatic), 5.91
3.53 (dd, 1H, H-30), 3.42 (dd, 1H, J2 ,3 ¼9.8 Hz, H-2 ), 1.91
(s, 3H, NHCOCH3), 1.50 (d, 3H, J¼7.0 Hz, Ala-b-H), 1.22
(d, 3H, Ala-b-H), 1.20 (d, 3H, Thr-g-H); 13C NMR
(120 MHz, D2O, d): 179.2, 174.8, 171.8, 170.3, 105.2,
98.7, 77.6, 75.5, 75.0, 73.0, 71.6, 71.1, 69.3, 69.1, 61.7,
61.5, 57.9, 51.1, 49.4, 48.9, 22.8, 18.3, 18.3, 17.4; HRMS-
FAB (m/z): [MþH]þ calcd for C24H43N4O15, 627.2726;
found, 627.2747.
0
0
0
(ddd, 1H, CH2–CHvCH2), 5.44 (d, 1H, J3,4¼3.4 Hz, H-4),
0
0
0
5.34 (d, 1H, J3 ,4 ¼3.4 Hz, H-4 ), 5.31–5.20 (m, 2H,
0
0
0
0
3.1.9. Poly-[L-alanyl-O-(b-D-galactopyranosyl-(1!3)-2-
acetamido-2-deoxy-a-D-galactopyranosyl)-L-threonyl-L-
alanine] (1). To a stirred solution of 14 (20 mg,
0.032 mmol) in DMF (450 mL) was added 10% DPPA in
DMF solution (90 mL, 0.042 mmol) at 08C, followed by the
addition of 10% triethylamine in DMF solution (100 mL,
0.073 mmol). The solution was stirred at 08C for 2 h, then
warmed to room temperature and stirred for 2 days. Then,
the product in DMF was precipitated by addition of diethyl
ether, and centrifuged. The crude product was then dis
solved in 25 mM-NaOH aq. (1.0 mL) at 08C, and stirred for
1h. Then, 0.1 M CH3COOH aq. (25 mL) was added, and the
mixture was subjected to the purification by gel filtration
(Sephadex G-25, water as eluent) to give 1 (20 mg, 100%).
The weight average molecular weight of 1 was estimated as
6600 by gel permeation chromatography. 1: 1H NMR
(400 MHz, D2O, d): 4.88 (d, 1H, J1,2¼3.7 Hz, H-1), 4.37–
CHvCH2), 5.17 (dd, 1H, J1 ,2 ¼7.9 Hz, J2 ,3 ¼10.5 Hz,
H-20), 4.97 (dd, 1H, H-30), 4.83 (d, 1H, H-10), 4.83 (d, 1H,
J1,2¼3.9 Hz, H-1), 4.71 (d, 1H, O–CH2-Ph), 4.52–4.45 (m,
3H, O–CH2-Ph), 4.18 (dd, 1H, J3,4¼10.0 Hz, H-3), 4.15
(dd, 1H, O–CH2–CH), 4.11–4.08 (m, 3H, H-60a, H-60b,
0
0
H-5), 3.99 (dd, 1H, O–CH2–CH), 3.84 (dd, 1H, J5 ,6
¼
6.7 Hz, H-50), 3.81 (dd, 1H, H-2), 3.50 (dd, 1H, H-6a), 3.44
(dd, 1H, H-6b), 2.16, 2.08, 2.06, 1.97, 1.94 (s£5, 3H£5,
OAc); HRMS-FAB (m/z): [MþH]þ calcd for C39H49O16,
773.3020; found, 773.3043; elemental analysis calcd (%)
for C86H93N3O17·1/2H2O: C, 59.92; H 6.31; found: C,
59.83; H,6.17.
3.1.12. Allyl O-(2,3,4,6-tetra-O-benzyl-b-D-galacto-
pyranosyl)-(1!3)-2,4,6-tri-O-benzyl-a-D-galactopyrano-
side (18). To a solution of 17 (305 mg, 0.40 mmol) in
MeOH (10 mL) was added NaOMe (10 mg, 0.16 mmol),

10222
Y. Tachibana et al. / Tetrahedron 58 (2002) 10213–10224
and stirred at room temperature for 1 h. The reaction
mixture was then neutralized with Dowex 50W-X8 [Hþ]
resin, filtered, and concentrated. To the residue was added
DMF (10 mL), and cooled to 2158C, at which time 60%
NaH in oil (104 mg, 2.6 mmol) was added, and stirred at the
same temperature for 30 min. Benzyl bromide (362 mL,
3.0 mmol) was added dropwisely, and the mixture was
allowed to warm slowly to room temperature over 12 h.
Then, methanol was added and the solution was evaporated.
The residual syrup was extracted with CHCl3, and the
organic layer was washed with water, brine, dried (MgSO4),
and concentrated. The crude residue was purified by flash
column chromatography (hexane/EtOAc¼8:1) to give 18
(276 mg, 67 %). 18: 1H NMR (600 MHz, CDCl3, d): 7.33–
7.18 (m, 35H, aromatic), 5.88 (ddd, 1H, CH2–CHvCH2),
aromatic), 6.90 (d, 1H, J¼4.3 Hz, Thr-NH), 5.31 (d, 1H,
J¼5.9 Hz, Ala-NH), 5.19 (d, 1H, J1,2¼3.7 Hz, H-1), 5.12–
0
0
0
4.89 (m, 8H, Ph-CH2–O), 4.79 (d, 1H, J1 ,2 ¼7.7 Hz, H-1 ),
4.75–4.57 (m, 5H, Ph-CH2–O), 4.50–4.31 (m, 6H,
Ph-CH2–O, Thr-a-H), 4.30 (m, 1H, Ala-a-H), 4.23–4.20
(m, 2H, J2,3¼9.8 Hz, H-3, Ala-a-H), 4.07 (dd, 1H, H-2),
4.05–4.02 (m, 3H, H-4, Thr-b-H, H-50), 3.94 (d, 1H, J3 ,4
¼
0
0
2.5 Hz, H-40), 3.88 (dd, 1H, J2 ,3 ¼9.7 Hz, H-2 ), 3.68 (dd,
1H, J5,6¼8.5 Hz, H-5), 3.57 (dd, 1H, H-6a), 3.52–3.44 (m,
4H, H-6b, H-50, H-60a, H-60b), 1.37 (d, 3H, J¼7.0 Hz,
Ala-b-H), 0.95 (d, 3H, J¼5.6 Hz, Thr-g-H), 0.87 (d, 3H,
J¼7.3 Hz, Ala-b-H); 13C NMR (120 MHz, CDCl3, d):
172.3, 171.5, 167.7, 138.9, 138.9, 138.7, 138.4, 138.2,
137.9, 137.4, 136.3, 135.5, 128.5–127.5, 82.6, 79.8, 77.5,
77.3, 77.2, 75.3, 74.8, 74.7, 74.6, 73.7, 73.6, 73.4, 73.3,
73.2, 72.8, 70.0, 69.2, 68.5, 66.7, 48.2, 19.1, 17.1, 15.1;
HRMS-FAB (m/z): [MþH]þ calcd for C86H94N3O17,
1440.6583; found, 1440.6620; elemental analysis calcd
(%) for C86H93N3O17: C, 71.69; H 6.51; N, 2.69; found: C,
71.70; H, 6.51; N, 2.92.
0
0
0
5.29–5.14 (m, 2H, CHvCH2), 5.04–5.96 (m, 3H,
0
0
0
Ph-CH2–O), 4.86 (d, 1H, J1 ,2 ¼7.7 Hz, H-1 ), 4.83–4.60
(m, 7H, H-1, Ph-CH2–O), 4.45–4.33 (m, 5H, Ph-CH2–O),
4.23 (dd, 1H, J2,3¼10.1 Hz, J3,4¼3.0 Hz, H-3), 4.09
(dd, 1H, H-6a), 3.99 (dd, 1H, J1,2¼3.7 Hz, H-2), 3.99–
3.93 (m, 4H, H-6b,0O–CH2–CHvCH2, H-40), 3.83 (dd, 1H,
0
0
0
0
0
J2 ,3 ¼9.4 Hz, H-2 ), 3.66 (dd, 1H, J5 ,6 ¼7.7 Hz, H-5 ),
3.58–3.43 (m, 4H, H-60a, H-30, H-5, H-60b); HRMS-FAB
(m/z): [MþNa]þ calcd for C64H68O11Na, 1035.4660; found,
1035.4670.
3.1.15. L-Alanyl-O-[b-D-galactopyranosyl-(1!3)-a-D-
galactopyranosyl]-L-threonyl-L-alanine (21). To a solu-
tion of 20 (50 mg, 0.035 mmol) in DMF (4 mL), acetic acid
(300 mL), H2O (1 mL) was added 10% Pd/C (300 mg),
and stirred at room temperature for 72 h under H2 gas
atmosphere. Then, Pd/C was removed by filtration, and the
solution was evaporated. The residue was purified by gel
filtration chromatography (Sephadex G-10, water as eluent)
3.1.13. 2,3,4,6-Tetra-O-benzyl-b-D-galactopyranosyl-
(1!3)-2,4,6-tri-O-benzyl-a,b-D-galactopyranosyl fluor-
ide (19). To a solution of 18 (250 mg, 0.25 mmol) in 90%
AcOH was added PdCl2 (88 mg, 0.49 mmol) and NaOAc
(243 mg, 2.96 mmol), and stirred at room temperature for
18 h. The solution was diluted with CHCl3, and the organic
layer was washed with sat. NaHCO3, brine, dried (MgSO4),
and concentrated. To a solution of the residue in THF
(10 mL) was added DAST (39 mL, 0.30 mmol) at 2208C,
and further stirred at room temperature for 1 h. Then, MeOH
was added to quench DAST, and the solution was
concentrated. The residue was extracted with CHCl3,
washed with sat. NaHCO3, brine, dried (MgSO4), concen-
trated, and purified by flash column chromatography
(hexane/EtOAc¼8:1) to give the anomeric mixture of 19
(160 mg, 66 %, a/b¼1:1). This syrupy crude material was
used for the next glycosidation reaction without further
purification and characterization.
1
to give 21 (12.6 mg, 62%). 21: H NMR (600 MHz, D2O,
0
0
d): 5.04 (d, 1H, J1,2¼4.0 Hz, H-1), 4.55 (d, 1H, J1 ,2
¼
7.7 Hz, H-10), 4.48 (d, 1H, J¼3.2 Hz, Thr-a-H), 4.31 (dd,
1H, Thr-b-H), 4.17 (d, 1H, J3,4¼2.9 Hz, H-4), 4.12 (q, 1H,
J¼7.1 Hz, Ala-a-H), 4.10 (q, 1H, J¼7.2 Hz, Ala-a-H), 3.99
(dd, 1H, J5,6a¼7.2 Hz, J5,6b¼5.0 Hz, H-5), 3.96 (dd, 1H,
0
0
J2,3¼10.3 Hz, H-3), 3.90 (dd, 1H, H-2), 3.86 (d, 1H, J3 ,4
¼
3.3 Hz, H-40), 3.72–3.59 (m, 6H, H-6a, H-6b, H-6a0, H-6b0,
H-50, H-30), 3.54 (dd, 1H, H-20), 1.55 (d, 3H, Ala-b-H), 1.29
(d, 3H, Ala-b-H), 1.24 (d, 3H, J¼6.4 Hz, Thr-g-H); 13C
NMR (120 MHz, D2O, d): 179.5, 171.5, 169.7, 104.4, 99.2,
79.1, 75.1, 74.3, 72.6, 71.1, 71.1, 69.3, 68.7, 67.4, 61.3,
61.1, 57.5, 51.2, 49.2, 17.7, 17.7, 16.8; HRMS-FAB (m/z):
[MþH]þ calcd for C22H40N3O15, 586.2454; found,
586.2458.
3.1.14. N-(Benzyloxycarbonyl)-L-alanyl-O-[(2,3,4,6-
tetra-O-benzyl-b-D-galactopyranosyl)-(1!3)-(2,4,6-tri-
O-benzyl-a-D-galactopyranosyl)]-L-threonyl-L-alanine
benzyl ester (20). A mixture of Cp2ZrCl2 (42 mg,
1.44 mmol), AgClO4 (60 mg, 2.88 mmol), 11 (87 mg,
3.1.16. Poly-[L-alanyl-O-(b-D-galactopyranosyl-(1!3)-
a-D-galactopyranosyl)-L-threonyl-L-alanine] (2). To a
stirred solution of 21 (9 mg, 0.015 mmol) in DMF
(150 mL) was added 10% DPPA in DMF solution (50 mL,
0.023 mmol) at 08C, followed by the addition of 10%
triethylamine in DMF solution (54 mL, 0.039 mmol). The
solution was stirred at 08C for 2 h, then warmed to room
temperature and stirred for 2 days. Then, the product in
DMF was precipitated by addition of diethyl ether, and
centrifuged. The crude product was then dissolved in
25 mM NaOH aq. (2.0 mL) at 08C, and stirred for 2 h.
Then, 0.1 M CH3COOH aq. (50 mL) was added, and the
mixture was subjected to the purification by gel filtration
(Sephadex G-25, water as eluent) to give 2 (5.8 mg, 64%).
The weight average molecular weight of 2 was estimated
˚
1.80 mmol), and powdered molecular sieves 4 A (1.0 g) in
dry CH2Cl2 (8 mL) was stirred at room temperature for 2 h
under nitrogen atmosphere, then cooled to 2208C. A
solution of 19 (70 mg, 0.72 mmol) in dry CH2Cl2 (2 mL)
was added, and the mixture was stirred at 2208C to room
temperature for 72 h, then diluted with CHCl3, filtered
thorough celite. The filtrate was washed with sat. NaHCO3,
brine, dried (MgSO4), and concentrated. The crude residue
was purified by flash column chromatography (toluene/
EtOAc¼7:1, 6:1 then 4:1) to give 20 (60 mg, 58%). The
trace amount of b-anomer was found in TLC analysis, but
1
as 5480 by gel permeation chromatography. 2: H NMR
(600 MHz, D2O, 0 d): 5.07 (br, 1H, H-1), 4.54 (d, 1H,
1
could not be isolated. 20: H NMR (600 MHz, CDCl3, d):
7.61 (d, 1H, J¼7.3 Hz, Ala-NH), 7.35–7.12 (m, 45H,
0
0
J1 ,2 ¼7.3 Hz, H-1 ), 4.46 (br, 1H, Thr-a-H), 4.36–4.30 (br,

Y. Tachibana et al. / Tetrahedron 58 (2002) 10213–10224
10223
3. Nishimura, S.-I.; Lee, Y. C. Synthetic Glycopolymers: New
Tools for Glycobiology. In Polysaccharides, Structural
Diversity and Functional Versatility; Dumitriu, S., Ed.;
Marcel Dekker: New York, 1998; pp 523–537.
4. For example, see: Hanisch, F. G.; Muller, S. Glycobiology
2000, 10, 439–449.
2H, Ala-a-H£2), 4.23 (br, 1H, Thr-b-H), 4.19 (br, 1H, H-4),
4.00–3.89 (m, 3H, H-5, H-3, H-2), 3.87 (br, 1H, H-400),
3.71–3.53 (m, 6H, 0H-6a, H-6b, H-6a0, H-6b0, H-50, H-3 ),
3.55 (dd, 1H, H-2 ), 1.37 (d, 3H, J¼7.0 Hz, Ala-b-H),
1.34 (d, 3H, J¼6.90 Hz, Ala-b-H), 1.22 (d, 3H, J¼4.3 Hz,
Thr-g-H).
5. Matsuura, K.; Hibino, M.; Yamada, Y.; Kobayashi, K. J. Am.
Chem. Soc. 2001, 123, 357–358.
3.1.17. Poly-[L-alanyl-O-(5-acetamido-3,5-dideoxy-a-D-
glycero-D-galacto-2-nonulopyranulosonic acid)-(2!3)-
b-D-galactopyranosyl-(1!3)-(2-acetamido-2-deoxy-a-D-
galactopyranosyl)]-L-threonyl-L-alanine] (sialylated glyco-
protein, 3). To a solution of 1 (20 mg) in 50 mM sodium
cacodyrate buffer (1.0 mL, pH 7.0, 0.1% Triton CF54) was
added CMP-Neu5Ac (15.4 mg, 41 mmol), a2,3-sialyl-
transferase (50 mU), alkaline phosphatase (20 mU), and
the mixture was incubated at 378C overnight. Then the
reaction mixture was taken in boiled water for 5 min,
filtered with syringe filter (0.45 mm) and separated by gel
filtration chromatography (Sephadex G-25, water as eluent),
giving 3 (27.6 mg, 93% (98% from periodate–resorcinol
method)). The weight average molecular weight of 3 was
estimated as 11,650 by gel permeation chromatography. 3:
6. Shioiri, T.; Ninomiya, K.; Yamada, S. J. Am. Chem. Soc. 1972,
94, 6203–6205.
7. Nishi, N. T.; Naruse, K.; Hagiwara, B.; Nakajima, S. Tokura.
Makromol. Chem. 1991, 192, 1799–1809.
8. Tsuda, T.; Nishimura, S.-I. Chem. Commun. 1996,
2779–2780.
9. Recently, it was also suggested that DPPA can be applied for
the blockwise synthesis of the glycopeptides with potent
biological activity. See: Matsuda, M.; Nishimura, S.-I.;
Nakajima, F.; Nishimura, T. J. Med. Chem. 2001, 44,
715–724.
10. Fraser-Reid, B.; Udodong, U. E.; Wu, Z.; Ottoson, H.; Merritt,
J. R.; Rao, C. S.; Roberts, C.; Madsen, R. Synlett 1992,
927–942.
1H NMR (600 MHz, D2O, d): 4.82 (br, 1H, H-1), 4.43 (d,
11. Lemieux, R. U.; Ratcliffe, R. M. Can. J. Chem. 1979, 57,
1244–1251.
0
0
0
1H, J1 ,2 ¼7.7 Hz, H-1 ), 4.41 (br, 1H, Thr-a-H), 4.34–4.31
(m, 2H, Ala-a-H£2), 4.25 (br, 1H, Thr-b-H), 4.17 (br, 1H,
H-2), 4.11 (br, 1H, H-4), 3.98–3.92 (m, 2H, H-3, H-30), 3.84
(br,01H, H-40), 3.81–3.48 (m,0013H, 0H0 -5, H-6a, H-6b, H-50,
H-6 a, H-60b, H-400, H-500, H-6 , H-7 , H-800, H-900a, H-900b),
12. McClinton, M. A. Aldarichim. Acta 1995, 28, 31–35.
13. Posner, G. H.; Haines, S. R. Tetrahedron Lett. 1985, 26, 5–8.
14. Schmidt, R. R.; Kinzy, W. Adv. Carbohydr. Chem. Biochem.
1994, 50, 21.
3.42 (dd, 1H, H-20), 2.66 (dd, 1H, J3 eq,3 ax¼12.0 Hz,
00
00
15. Hashimoto, S.; Hayashi, M.; Noyori, R. Tetrahedron Lett.
1984, 25, 1379–1382.
00
00
00
J3 eq,4 ¼3.7 Hz, H-3 eq), 1.92 0(0 s, 6H, NHCOCH3£2), 1.69
00
00
(dd, 1H, J3 ax,4 ¼12.0 Hz, H-3 ax), 1.39 (d, 3H, J¼7.1 Hz,
Ala-b-H), 1.29 (d, 3H, J¼6.9 Hz, Ala-b-H), 1.21 (d, 3H,
Thr-g-H), 1.33 (m, 9H, Ala-b-H£2, Thr-g-H).
16. For example, it was preliminarily suggested that glycosidation
reaction using a disaccharidic donor having both O-benzyl and
benzylidene protections reduces the yield of the coupling
reaction with peptide due to the significant degree of de-O-
benzylidenation. Interconversion from azide group into
acetamido group at C-2 position in the presence of thioacetic
acid also induced de-O-benzylidenation reaction.
17. Suzuki, K.; Maeta, H.; Matsumoto, T. Tetrahedron Lett. 1989,
30, 4853–4856.
Acknowledgements
This work was supported by a grant from NEDO for
“Research and Development on Glycocluster Controlling
Biomolecules” and by a grant-in-aid for scientific research
from the Ministry of Education, Science, and Culture
(Grants 11480157 and 12555256). We also thank Ms
A. Maeda, Ms H. Matsumoto, and Ms S. Oka of the
Center of Instrumental Analysis, Hokkaido University,
for measuring elemental analyses and mass spectroscopy.
We appreciate valuable comments and suggestions by
Dr R. W. Woody of the Department of Biochemistry,
Colorado State University and Dr Y. C. Lee of the Biology
Department, Johns Hopkins University.
18. Although the yield of this coupling reaction seems to be
similar to that by the previous method, per-O-benzyl
protection of carbohydrate moiety is apparently useful for
further simple deprotection and polymerization steps.
19. Elofsson, M.; Salvador, L. A.; Kihlberg, J. Tetrahedron 1997,
53, 369–390.
20. Burcham, T. S.; Osuga, D. T.; Rao, B. N. N.; Bush, C. A.;
Feeney, R. E. J. Biol. Chem. 1986, 261, 6384–6389.
21. Possibility of the epimerization in the DPPA-promoted
polymerization of some amino acids or peptides was
previously discussed by Nishi et al., see Ref. 7 and papers
cited therein. It has been widely accepted that this method
provides stereochemically pure polypeptides even in the
presence of free hydroxyl groups due to L-serine or L-threo-
nine residue.
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