Chemoenzymatic synthesis of NeuAcα-(2→3)-Galβ-(1→3)-[NeuAcα-(2→6)]- GalNAcα1-O-(Z)-Serine (N-protected MUC II oligosaccharide-serine)

Article abstract of DOI:10.1081/CAR-120003741

An efficient synthesis of NeuAcα-(2→3)-Galβ-(1→3)-[NeuAcα-(2→6)]- GalNAcα1-O-(Z)-Serine (N-protected MUC II oligosaccharide-serine, 14) by a chemoenzymatic strategy is described. The enzymatic reaction of GalNAcα1-O-(Z)-Ser-OAll 7 with pNP-β-Gal in the presence of recombinant β1,3-galactosidase from Bacillus circulans gave Galβ-(1→3)-GalNAcα1-O-(Z)-Ser-O All3 in 68%. The introduction of two sialic acids into 3 was accomplished by a stepwise method. The branched Galβ-(1→3)-[NeuAcα-(2→6)]-GalNAcα1-O-(Z)-Ser-OAll 11 was constructed by a chemical method. Sialylation at the C-3 position of the terminal Gal residue on Galβ-(1→3)-[NeuAcα-(2→6)]-GalNAcα1-O-(Z)-Serine 2 using α2,3-(O)-sialyltransferase from rat liver gave a target compound 14 in a practical yield.

Full text of DOI:10.1081/CAR-120003741

Journal of Carbohydrate Chemistry
ISSN: 0732-8303 (Print) 1532-2327 (Online) Journal homepage: http://www.tandfonline.com/loi/lcar20
CHEMOENZYMATIC SYNTHESIS OF NeuAcα-
(2→3)-Galβ-(1→3)-[NeuAcα-(2→6)]-
GalNAcα1- O-(Z)-Serine (N-PROTECTED MUC II
OLIGOSACCHARIDE–SERINE)
Katsuhiko Suzuki , Ichiro Matsuo , Megumi Isomura & Katsumi Ajisaka
To cite this article: Katsuhiko Suzuki , Ichiro Matsuo , Megumi Isomura & Katsumi Ajisaka (2002)
CHEMOENZYMATIC SYNTHESIS OF NeuAcα-(2→3)-Galβ-(1→3)-[NeuAcα-(2→6)]-GalNAcα1-
O-(Z)-Serine (N-PROTECTED MUC II OLIGOSACCHARIDE–SERINE), Journal of Carbohydrate
Chemistry, 21:1-2, 99-111, DOI: 10.1081/CAR-120003741
To link to this article: http://dx.doi.org/10.1081/CAR-120003741
Published online: 28 Oct 2011.
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J. CARBOHYDRATE CHEMISTRY, 21(1&2), 99–111 (2002)
CHEMOENZYMATIC SYNTHESIS OF
!
!
!
NeuAcA-(2 3)-GalB-(1 3)-[NeuAcA-(2 6)]-
GalNAcA1-O-(Z)-Serine (N-PROTECTED MUC II
OLIGOSACCHARIDE–SERINE)
2,y
Katsuhiko Suzuki,1, Ichiro Matsuo, Megumi Isomura,2
*
and Katsumi Ajisaka2
1Department of Enviromental Science, Faculty of Science and
Engineering, Iwaki Meisei University, 5-5-1 Iino, Chuohdai,
Iwaki, Fukushima, 970-8551 Japan
2Nutrition Research Institute, Meiji Milk Products Co., Ltd.,
540 Naruda, Odawara, Kanagawa, 250-0862 Japan
ABSTRACT
An efficient synthesis of NeuAca-(2 !3)-Galb-(1!3)-[NeuAca-(2!6)]-
GalNAca1-O-(Z)-Serine (N-protected MUC II oligosaccharide–serine, 14)
by a chemoenzymatic strategy is described. The enzymatic reaction of
GalNAca1-O-(Z)-Ser-OAll 7 with pNP-b-Gal in the presence of recombinant
b1,3-galactosidase from Bacillus circulans gave Galb-(1!3)-GalNAca1-O-
(Z)-Ser-OAll 3 in 68%. The introduction of two sialic acids into 3 was
accomplished by a stepwise method. The branched Galb-(1!3)-[NeuAca-
(2 ! 6)]-GalNAca1-O-(Z)-Ser-OAll 11 was constructed by a chemical
method. Sialylation at the C-3 position of the terminal Gal residue on
Galb-(1!3)-[NeuAca-(2 ! 6)]-GalNAca1-O-(Z)-Serine 2 using a2,3-(O)-
sialyltransferase from rat liver gave a target compound 14 in a practical yield.
Keywords: Mucin II oligosaccharide; Glycopeptide; Chemoenzymatic
synthesis
* Corresponding author. Fax: +81-246-29-0577; E-mail: ksuzuki@iwakimu.ac.jp
y Current address: The Institute of Physical and Chemical Resarch (RIKEN), 2-1 Hirosawa, Wako,
Saitama, 351-01, Japan.
99
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100
SUZUKI ET AL.
Figure 1.
INTRODUCTION
Natural type oligosaccharides are necessary for understanding various biological
phenomena originated by sugar chains in glycoconjugates. To use oligosaccharides as
tools for biological research, many chemical synthetic reactions of carbohydrates have
been reported,[1a,1b] but large-scale synthesis of oligosaccharides for glycoconjugates
requires many steps due to the protection of hydroxyl groups for unambiguous
glycosylations resulting in low yields. On the other hand, enzymatic synthesis using
glycosyltransferases[2a–2j] or glycosidases,[3a–3c] which catalyzes glycosylation without
protection of hydroxyl groups, can be an efficient method. For example, sialyl-T antigen
was synthesized using a one-pot multi-enzyme system.[4a–4c] Recently, various glyco-
sidases have become readily available, and various oligosaccharide units have been
synthesized by two types of reactions, reverse hydrolysis and transglycosylation.[5a–5h]
We have discovered a useful enzyme, a recombinant b-galactosidase, from Bacillus cir-
culans[6,7] for the synthesis of the Galb-(1 ! 3)-GalNAc linkage, which is a partial
structure of the mucin-type glycopeptide. Additionally, we have demonstrated the syn-
thesis of the Galb-(1!3)-GalNAca1-O-Ser derivative in excellent yield.[8] The appro-
priately protected Galb-(1!3)-GalNAca1-O-Ser derivative was expected to be a key
compound for the synthesis of various mucin type oligosaccharides.
MUC II 1 is disialylated tetrasaccharide-L-serine isolated from the urine of a patient
suffering from mucolipidosis I.[9a,9b] The MUC II oligosaccharide, linking two sialic
acids on Galb-(1!3)-GalNAca1-O-Serine, has already been synthesized by a pure
chemical approach.[10] In the present report, we would like to show an efficient
synthesis of MUC II as N-protected form by the combination of chemical and enzymatic
methods (Figure 1).
RESULTS AND DISCUSSION
Our synthetic plan for obtaining 1 is based on a stepwise introduction of two
sialic acids into Galb-(1!3)-GalNAca1-O-Ser derivative (Scheme 1). The route in
Scheme 1, including individual glycosylation at the C-3 position of Gal and the C-6
position of GalNAc, seems to be widely applicable for the synthesis of various mucin
type oligosaccharides which have Galb-(1!3)-GalNAca1-O-Ser/Thr at the reducing
end as a core structure. Sialylation at the terminal Gal residue on trisaccharide 2 might
be carried out using a2,3-(O)-sialyltransferase.[11] However, the enzymatic construction

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SYNTHESIS OF MUC II
101
Scheme 1.
of a branched structure at the GalNAc residue of Galb-(1 !3)-[NeuAca-(2 !6)]-
GalNAc]-Ser/Thr derivative is still challenging even at present. In this study, we
undertook construction of a branched NeuAca-(2 !6)-GalNAc structure by a chem-
ical method.
We first began synthesis of a Galb-(1!3)-GalNAca1-O-Ser derivative, which
was essential for the total synthesis of mucin-type oligosaccharides of various
structures. GalNAca1-O-Ser 6 was synthesized by a reverse hydrolysis reaction using
GalNAc and serine in the presence of N-acetyl-a-D-galactosaminidase from Aspergillus
niger [EC 3.2.1.49].[12a,12b] The transglycosylation of the Gal residue of pNP-b-D-
galactoside to GalNAca1-O-Ser 6 was performed by using recombinant b1,3-galacto-

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102
SUZUKI ET AL.
sidase from B. circulans [EC 3.2.0.23]. The mixture of GalNAca1-O-Ser 6 and pNP-b-
Gal in 20% DMF–80% potassium phosphate buffer (KPB, pH 6.0, 0.1 M) was
incubated at 37 °C in the presence of the b-galactosidase. The progress of the reaction
was monitored by a HPLC fitted with a UV detector (215 nm) and a NH2-column
(CAPCELL PAK). Two small peaks appeared after 3 hours but thereafter the reaction
proceeded no further. The yield of the transglycosylation product was very low. We
considered that the low reactivity and regioselectivity of this transglycosylation reaction
might be due to the carboxyl and/or amino group of the serine residue interacting with
the enzyme.
Therefore, we used a serine derivative as an acceptor whose amino and carboxyl
groups were protected. Treatment of GalNAca1-O-Ser 6 with benzyl chloroformate in
aqueous NaOH afforded GalNAca1-O-(Z)-Ser. Reaction of GalNAca1-O-(Z)-Ser with
allyl bromide in the presence of triethylamine gave GalNAca1-O-(Z)-Ser-OAll 7 in 77%
yield. Transglycosylation using GalNAca1-O-(Z)-Ser-OAll 7 and pNP-b-Gal in the
presence of b1,3-galactosidase from B. circulans gave Galb-(1!3)-GalNAca1-O-(Z)-
Ser-OAll 3 as a single product in 68% yield. The structure of the product was supported
1
by H and 13C NMR spectroscopy and the coupling constant J10,20 =7.8 Hz proved the
newly constructed b-structure. The Galb-(1!3)-linkage was also confirmed by an NOE
interaction between the H-10 and H-3 protons.[8]
For the chemical construction of the branched Galb-(1!3)-[NeuAca-(2 ! 6)]-
GalNAc-structure, protection of hydroxyl groups on 3 was performed. Isopropylidenation
of disaccharide-serine 3 by use of 2,2-dimethoxypropane in the presence of ( )-cam-
phorsulfonic acid gave a di-O-isopropylidene derivative 8 in good yield. After the
toluoylation of remaining hydroxyl groups, the 4,6-O-isopropylidene group on the
GalNAc residue was cleaved selectively to afford a diol derivative 10. Sialylation of
10 using the S-methyl donor 5[13] promoted by NIS-TfOH[14] at ꢀ40 °C gave mono-
sialoside 11 as an anomeric mixture at the NeuAc residue (a:b= 74:26). The a-(2 !6)-
linkage of 11 was determined by conversion into the acetylated derivative 12, which
showed a signal for H-4 at d 5.44 as a doublet with J3,4 =2.6 Hz in the 1H NMR spectrum.
The anomeric configuration of the NeuAc residue at C-2 of 11 was determined as a by
NMR (NeuAc H-3e d 2.57, NeuAc H-3a d 1.89, NeuAc C-3 d 37.33).[15] The 3,4-O-
Isopropylidene group of 11 was easily removed by 70% aq HOAc to give a triol 13.
Deallylation of 13 was cleanly achieved by use of Pd(PPh3)4 in EtOH–H2O,[16] and the
product was deacylated to give OH-free trisaccharide 2. Two methods (chemical and
enzymatic) were considered to introduce a second NeuAc residue into the triol 13, but we
chose to use a2,3-(O)-sialyltransferase [EC 2.4.99.4] from rat liver, which was known to
be highly regio- and stereo-selective for the synthesis of NeuAca-(2!3)-Galb-(1!3)-
GalNAc derivatives. A mixture of trisaccharide 2 and CMP-NeuAc 4 was incubated for
12 hours at 37 °C in the presence of a2,3-(O)-sialyltransferase in 0.1 M cacodylate buffer
(0.2 mL, pH 6.0) containing bovine serum albumin. The reaction was monitored with
HPLC using an ODS column (Mightysil) connected to a UV monitor (215 nm). The
target tetrasaccharide 14 was produced quantitatively. In the 1H NMR spectrum of 14, the
H-3a and H-3e signals of a-(2 !6)-GalNAc-linked sialic acid appeared at d 1.59 and d
2.59, respectively, and those of a-(2! 3)-Gal-linked sialic acid appeared at d 1.69 and d
2.64, respectively. From the empirical rule of the chemical shifts in sialic acid linkage,[17]
the structure of 14 was confirmed to be NeuAca-(2!3)-Galb-(1!3)-[NeuAca-(2 !6)]-
GalNAc (Scheme 2).

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SYNTHESIS OF MUC II
103
Scheme 2. a) 1. Benzyl chloroformate, 1M NaOH 2. Allyl bromide, Et3N, DMF; b) pNP-b-
galactoside, b(1,3)-galactosidase, DMF, 0.1M KPB; c) 2,2-Dimethoxypropane, ( )-Camphorsul-
fonic acid, acetone; d) Toluoyl chloride, pyridine; e) Me3SiCl, ethylene glycol, MeOH; f) 5, NIS,
TfOH, MS3A, MeCN, ꢀ40 °C; g) Ac2O, Pyridine; h) 70% HOAc; i) 1. Pd(PPh3)4, EtOH, H2O 2. 1
M NaOH, MeOH; j) 4, a(2,3)-O-Sialyltransferase, BSA, cacodylate buffer (0.1 M, pH 6.0).
In conclusion, the mucin type oligosaccharide NeuAca-(2 !3)-Galb-(1 !3)-
[NeuAca-(2! 6)]-GalNAca1-O-Ser was synthesized by the stepwise introduction of
two sialic acids into a Galb-(1!3)-GalNAca-1-O-(Z)-Ser-OAll. The branched Galb-
(1 !3)-NeuAca-(2 !6)-GalNAc derivative was constructed by use of a chemical
method. Sialylation on the terminal Gal residue was accomplished by use of sialyl-
transferase from rat liver. The present method, including individual glycosylation at the
C-3 position of Gal and the C-6 position of a GalNAc of Galb-(1!3)-GalNAca1-O-Ser/

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104
SUZUKI ET AL.
Thr derivative by a chemoenzymatic strategy, was shown to be effective for the synthesis
of various mucin type oligosaccharides.
EXPERIMENTAL
General Methods. Optical rotations were measured at 25 °C with a HORIBA
polarimeter SEPA-300. NMR spectra were measured on a UNITY INOVA 500
1
spectrometer (500 MHz) or JEOL JNM-ECP 400 spectrometer (400 MHz). H NMR
were recorded in CDCl3 or D2O using Me4Si (d 0.00) or DOH (d 4.65) as the internal
standard. 13C NMR spectra were recorded in CDCl3 or D2O using CDCl3 (d 77.0) or
1,4-dioxane (d 67.6) as the internal standard. Coupling constants were measured in Hz.
TOF–MS spectra were recorded on Finnigan mat LASERMAT 2000 with 2,5-dihy-
droxybenzoic acid as the matrix. High resolution mass spectra were obtained on JMS-
HX110 (Jeol) spectrometer with diethanolamine as the matrix. Silica gel column
chromatography was performed using Wakogel C-300 (Wako Pure Chemical Indus-
tries). Analytical TLC was performed on aluminium plates coated with silica gel 60
F254 (Merck). Preparative TLC was performed on glass plates coated with silica gel 60
˚
F254 (Merck). Molecular sieves (3 A) were purchased from Aldrich Chemical
Company, Inc., and activated at 180 °C under vacuum immediately prior to use.
a2,3-(O)-Sialyltransferase (EC 2.4.99.4) was purchased from Carbiochem–Novabio-
chem Corporation.
N-(Benzyloxycarbonyl)-O-(2-acetamido-2-deoxy-A-D-galactopyranosyl)-
!
(1 3)-L-serine Allyl Ester (7). To a 1 M NaOH solution (50 mL) of GalNAca1-O-Ser
6 (4.0 g, 13.0 mmol) was added slowly benzyl chloroformate (6.6 g, 39.0 mmol), and the
mixture was stirred for 10 hours at 0 °C. After the reaction mixture was washed with
CHCl3, an aqueous layer was neutralized with ion-exchange resin (Dowex 50W) and
concentrated in vacuo. To the DMF solution (130 mL) of the residue containing Et3N
(3.95 g, 39.0 mmol) was added allyl bromide (4.72 g, 39.0 mmol) at 0 °C. The mixture
was stirred for 20 hours at room temperature and concentrated in vacuo. The residue was
purified by silica gel column chromatography (EtOAc ꢁ EtOAc:MeOH = 7:3) to afford
GalNAca1-O-(Z)-Ser OAll 7 (4.83 g, 77%) as crystals: mp 185–187 °C (EtOH);
1
[a]D +115.3° (c 2.08, MeOH); H NMR (500 MHz, D2O) d 7.36–7.29 (m, 5H, Phenyl),
5.95–5.87 (m, 1H, CH2–CH == C H 2), 5.33 (d, 1H, CH2–CH == CH2, J = 16.9 Hz), 5.23
(d, 1H, CH2–CH ==CH2, J =10.3 Hz), 5.13 (d, 1H, benzyl, J =12.1 Hz), 5.09 (d, 1H,
benzyl), 4.78 (d, 1H, H-1, J = 3.3 Hz), 4.62 (m, 2H, CH2–CH ==CH2), 4.49 (br s, 1H, Ser-
a), 4.22 (dd, 1H, H-2, J =11.0 Hz), 3.94 (dd, 1H, Ser-b, J =3.7, 11.0 Hz), 3.89 (dd, 1H,
Ser-b, J =4.4 Hz), 3.86 (d, 1H, H-4, J =2.2 Hz), 3.79–3.65 (m, 4H, H-3,5,6,60), 1.95 (s,
3H, Ac); 13C NMR (125 MHz, CDCl3) d 173.76, 171.53, 158.53, 138.01, 133.08, 129.49,
129.10, 128.96, 119.06, 99.96, 72.83, 70.20, 69.48, 68.90, 67.87, 67.07, 62.73, 56.00,
51.34, 22.90.
Anal. Calcd for C22H30N2O10: C, 54.77; H, 6.27; N, 5.81. Found: C, 54.56; H, 6.31;
N, 5.82.
!
N-(Benzyloxycarbonyl)-O-(B-D-galactopyranosyl)-(1 3)-O-(2-acetamido-2-
!
deoxy-A-D-galactopyranosyl)-(1 3)-L-serine Allyl Ester (3). To the b1,3-galactosi-

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SYNTHESIS OF MUC II
105
dase solution (recombinant, from B. circulans, 0.1 mL, 130 mU)[6,7] was added 20%
DMF–80% KPB (pH 6.0, 0.1 M) buffer (0.6 mL) including pNP-b-Gal (10.7 mg,
35.3ꢂ10ꢀ3 mmol) and GalNAca1-O-(Z)-Ser-OAll 7 (51.1 mg, 106ꢂ10ꢀ3 mmol) and
the mixture was incubated for 4.5 hours at 37 °C. The reaction was stopped by heating at
100 °C for 5 min, then filtered and purified by HPLC [ODS (Waters AccQ TAG), eluent:
30% aq MeOH–50% aq MeOH] to afford Galb1-3GalNAca1-O-(Z)-Ser-OAll 3 (15.5 mg,
1
68%) as crystals: mp 196–198 °C (EtOH); [a]D +70.0° (c 1.10, H2O); H NMR (500
MHz, D2O) d 7.42–7.34 (m, 5H, Phenyl), 5.88–5.85 (m, 2H, CH2–CH == C H 2), 5.29 (d,
1H, CH2–CH ==CH2, J =17.1 Hz), 5.23 (d, 1H, CH2–CH == CH2, J =10.5 Hz), 5.14 (d,
1H, benzyl, J =12.5 Hz), 5.09 (d, 1H, benzyl), 4.81 (d, 1H, H-1, J =3.2 Hz), 4.51 (br s, 1H,
Ser-a), 4.40 (d, 1H, H-10, J =7.8 Hz), 4.23 (dd, 1H, H-2, J =11.0 Hz), 4.17 (d, 1H, H-4,
J = 2.9 Hz), 3.94 (dd, 1H, H-3, J =3.2 Hz), 3.85 (d, 1H, H-40, J =2.9 Hz), 3.59 (m, 1H, H-
50), 3.56 (dd, 1H, H-30, J = 10.0 Hz), 3.46 (dd, 1H, H-20), 1.93 (s, 3H, Ac); 13C NMR (125
MHz, CDCl3) d 103.57 (C-10), 97.03 (C-1), 75.87 (C-3), 73.96 (C-50), 71.49 (C-30), 69.88
(C-5), 69.58 (C-20), 67.55, 67.52 (C-4,40), 60.06, 59.94 (C-6,60), 47.51 (C-2).
Anal. Calcd for C28H40N2O15: C, 52.17; H, 6.25; N, 4.35. Found: C, 51.83; H, 6.27;
N, 4.37.
N-(Benzyloxycarbonyl)-O-(3,4-O-isopropylidene-B-D-galactopyranosyl)-
!
!
(1 3)-O-(2-acetamido-2-deoxy-4,6-O-isopropylidene-A-D-galactopyranosyl)-(1 3)-
L-serine Allyl Ester (8). To an acetone solution (46 mL) of Galb1-3GalNAca1-O-(Z)-
Ser-OAll 3 (1.16 g, 1.8 mmol) was added 2,2-dimethoxypropane (2.2 g, 21.6 mmol)
containing ( )-camphorsulfonic acid (230 mg, 0.99 mmol) and the mixture was stirred for
24 hours at room temperature. After the reaction was stopped by addition of Et3N (0.165
mL, 1.19 mmol), the solution was concentrated in vacuo. The residue was purified by
silica gel column chromatography (EtOAc:MeOH =98:2) to afford diacetonide 8 (910
1
mg, 70%): [a]D + 77.0° (c 0.96, CHCl3); H NMR (500 MHz, CDCl3) d 7.37–7.34 (m,
5H, Phenyl), 5.92–5.82 (m, 2H, CH2–CH == CH2 and NH), 5.70 (br d, 1H, NH, J =5.7
Hz), 5.34 (d, 1H, CH2–CH ==CH2, J = 17.3 Hz), 5.30 (d, 1H, CH2–CH ==CH2, J =10.5
Hz), 5.14 (s, 2H, benzyl), 4.87 (d, 1H, H-1, J = 3.7 Hz), 4.67 (d, 2H, CH2–CH ==CH2,
J = 5.1 Hz), 4.61 (ddd, 1H, H-2, J =9.3, 10.7 Hz), 4.58 (br s, 1H, Ser-a), 4.30 (d, 1H, H-4,
J = 2.9 Hz), 4.26 (d, 1H, H-10, J = 8.1 Hz), 4.15 (dd, 1H, H-40, J = 1.7, 5.5 Hz), 4.03 (dd,
1H, H-30, J =7.7 Hz), 4.01–3.65 (m, 9H, H-3, 5, 6a, 6b, 50, 60a, 60b, Ser-b), 3.59 (dd, 1H,
H-20), 1.98 (s, 3H, Ac), 1.52, 1.49, 1.46, 1.34 (each s, 12H, acetonide); 13C NMR (125
MHz, CDCl3) d 171.6, 170.2, 155.9, 135.9, 130.9, 128.6, 128.4, 128.3, 119.6, 110.4, 104.4,
99.1, 78.6, 77.2, 73.8, 73.4, 72.2, 69.0, 68.7, 67.3, 66.5, 63.3, 62.5, 54.5, 47.8, 29.3, 28.0,
26.3, 23.3, 18.4.
Anal. Calcd for C34H48N2O15: C, 56.35; H, 6.68; N, 3.87. Found: C, 56.00; H, 6.74;
N, 3.68.
N-(Benzyloxycarbonyl)-O-(3,4-O-isopropylidene-2,6-di-O-toluoyl-B-D-galacto-
!
pyranosyl)-(1 3)-O-(2-acetamido-2-deoxy-4,6-O-isopropylidene-A-D-galactopyra-
!
nosyl)-(1 3)-L-serine Allyl Ester (9). To a pyridine solution (5 mL) of diacetonide 8
(300 mg, 0.414 mmol) was added toluoyl chloride (320 mg, 2.07 mmol) and the mixture
was stirred for 2 h at room temperature. After the reaction was stopped by addition of
MeOH (5 mL), the solution was concentrated in vacuo. The residue was purified by silica
gel column chromatography (n-hexane:EtOAc =1:1 ꢁ 1:2 ꢁ EtOAc) to afford ditoluoyl

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106
SUZUKI ET AL.
derivative 9 (328 mg, 82%): [a]D + 69.7° (c 1.01, CHCl3); 1H NMR (500 MHz, CDCl3) d
7.94–7.91 (m, 4H, Phenyl), 7.36–7.20 (m, 13H, Phenyl), 5.89–5.78 (m, 2H, CH2–
CH == C H 2 and NH), 5.59 (br d, 1H, NH, J = 6.9 Hz), 5.30 (d, 1H, CH2–CH ==CH2, J = 16.9
Hz), 5.24 (d, 1H, CH2–CH ==CH2, J = 10.1 Hz), 5.13–5.10 (m, 3H, H-20, benzyl, H-1),
4.88 (d, 1H, H-10, J =6.4 Hz), 4.41 (ddd, 1H, H-2, J =11.1 Hz), 4.18 (m, 1H, H-50), 3.92 (dd,
1H, Ser-b, J =3.2, 11.3 Hz), 3.85 (dd, 1H, Ser-b, J = 2.7 Hz), 3.75 (dd, 1H, H-3, J =2.5 Hz),
2.41, 2.40 (each s, 6H, toluoyl), 1.62 (s, 3H, Ac), 1.47, 1.41, 1.36, 1.31 (12H, each s,
acetonide); 13C NMR (125 MHz, CDCl3) d 170.21, 169.83, 166.29, 165.45, 155.90, 144.36,
144.22, 135.99, 131.19, 129.92, 129.55, 129.22, 128.55, 128.29, 128.21, 126.62, 119.15,
110.93, 102.67, 99.30, 98.91, 76.22, 73.18, 72.94, 71.17, 69.28, 68.03, 67.18, 66.22, 63.78,
63.08, 62.54, 54.60, 48.27.
Anal. Calcd for C50H60N2O17: C, 62.49; H, 6.29; N, 2.91. Found: C, 62.08; H, 6.48;
N, 2.88.
N-(Benzyloxycarbonyl)-O-(3,4-O-isopropylidene-2,6-di-O-toluoyl-B-D-galacto-
!
!
pyranosyl)-(1 3)-O-(2-acetamido-2-deoxy-A-D-galactopyranosyl)-(1 3)-L-serine
Allyl Ester (10). To a MeOH solution (10 mL) of ditoluoyl derivative 9 (338 mg,
0.352 mmol) containing ethylene glycol (39ꢂ10ꢀ3 mL, 0.704 mmol) was added
Me3SiCl (22ꢂ10ꢀ3 mL, 0.176 mmol) at 0 °C. After being stirred for 30 min, the
reaction was stopped by addition of Et3N (0.102 mL, 0.704 mmol) and concentrated
in vacuo. The residue was purified by silica gel column chromatography (n-
hexane:EtOAc = 1:2 ꢁEtOAc) to afford monoacetonide 10 (243 mg, 75%):
[a]D + 203.7° (c 0.35, MeOH); 1H NMR (500 MHz, CDCl3) d 7.96–7.90 (m, 4H,
Phenyl), 7.35–7.26 (m, 13H, Phenyl), 5.84–5.77 (m, 1H, CH2–CH == C H 2), 5.73 (d, 1H,
NH, J = 8.4 Hz), 5.29 (d, 1H, NH, J = 7.6 Hz), 5.26 (d, 1H, CH2–CH ==CH2, J = 15.5
Hz), 5.21 (d, 1H, CH2–CH == CH2, J = 10.6 Hz), 5.17 (dd, 1H, H-20, J = 6.6 Hz), 5.11 (d,
1H, benzyl, J = 12.2 Hz), 5.07 (d, 1H, benzyl), 4.87 (d, 1H, H-1, J = 3.7 Hz), 4.74 (d, 1H,
H-10, J = 6.9 Hz), 3.89 (dd, 1H, Ser-b, J = 2.2, 10.8 Hz), 3.83 (dd, 1H, Ser-b, J = 3.2 Hz),
2.42, 2.40 (each s, 6H, toluoyl), 1.63 (s, 3H, Ac), 1.45, 1.36 (each s, 6H, acetonide); 13C
NMR (125 MHz, CDCl3) d 169.79, 169.68, 166.16, 164.89, 155.90, 144.14, 144.10,
135.90, 130.87, 129.66, 129.48, 129.05, 128.33, 128.08, 127.98, 126.52, 126.44, 119.01,
110.89, 100.60, 98.67, 78.83, 75.86, 72.79, 72.56, 70.88, 69.57, 68.85, 68.25, 66.99,
63.43, 62.31, 54.26.
Anal. Calcd for C47H56N2O17: C, 61.30; H, 6.13; N, 3.04. Found: C, 60.97; H, 6.19;
N, 3.08.
N-(Benzyloxycarbonyl)-O-(3,4-O-isopropylidene-2,6-di-O-toluoyl-B-D-galacto-
!
pyranosyl)-(1 3)-O-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-
!
glycero-A-D-galacto-2-nonulopyranosyl)onate-(2 6)]-O-(2-acetamido-2-deoxy-A-D-
!
galactopyranosyl)-(1 3)-L-serine Allyl Ester (11). The mixture of acceptor 10 (261
˚
mg, 0.284 mmol), donor 5 (296 mg, 0.568 mmol) and molecular sieves 3 A (600 mg) in
MeCN (0.5 mL) was stirred for 1 h at room temperature under an argon atmosphere. To
this mixture was added the MeCN solution (0.5 mL) of NIS (383 mg, 1.7 mmol)
containing TfOH (50ꢂ10ꢀ3 mL, 0.568 mmol) at ꢀ40 °C. After being stirred for 14 hours,
the solution was diluted with CH2Cl2, filtered and washed with saturated NaHSO3 and
brine, respectively. The organic layer was dried over MgSO4 and concentrated in vacuo.
The residue was purified by gel filtration (Sephadex LH-20, MeOH) to afford sialosides

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SYNTHESIS OF MUC II
107
as an anomeric mixture (a:b =76:24). These isomers were separated by preparative TLC
(CH2Cl2:MeOH = 19:1, 2 times) to give a isomer 11 (166 mg, 42%) and b isomer (50 mg,
1
13%) respectively. Compound 11: [a]D + 34.5° (c 0.35, MeOH); H NMR (500 MHz,
CDCl3) d 7.94–7.90 (m, 4H, Phenyl), 7.37–7.23 (m, 13H, Phenyl), 4.87 (d, 1H, H-10,
J = 6.1 Hz), 4.78 (d, 1H, H-1, J =3.7 Hz), 3.77 (s, 3H, OMe), 2.57 (dd, 1H, NeuAc H-3e,
J = 4.7, 12.3 Hz), 2.42, 2.40 (each s, 6H, toluoyl), 2.11, 2.02, 1.98, 1.87, 1.63, 1.53, 1.36
(each s, 24H, Ac and acetonide), 1.89 (dd, 1H, NeuAc H-3a, J =12.3 Hz); 13C NMR (125
MHz, CDCl3) d 170.63, 170.50, 169.97, 169.93, 167.75, 165.97, 164.99, 144.05, 143.86,
131.01, 129.71, 129.53, 129.05, 128.38, 128.11, 128.02, 126.81, 126.57, 119.16, 110.74,
101.04, 98.78, 98.70, 78.68, 75.40, 72.68, 72.49, 72.44, 70.22, 68.99, 68.92, 68.44, 67.50,
67.03, 66.00, 63.17, 63.04, 62.30, 52.59, 49.31, 47.77, 37.33, 29.48, 26.77, 25.55, 22.97,
22.40, 21.45, 20.79, 20.56, 20.51.
Anal. Calcd for C67H83N3O29: C, 57.71; H, 6.00; N, 3.01. Found: C, 57.27; H, 5.52;
N, 3.05.
N-(Benzyloxycarbonyl)-O-(3,4-O-isopropylidene-2,6-di-O-toluoyl-B-D-galacto-
!
pyranosyl)-(1 3)-O-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-
!
glycero-A-D-galacto-2-nonulopyranosyl)onate-(2 6)]-O-(2-acetamido-4-O-acetyl-2-
!
deoxy-A-D-galactopyranosyl)-(1 3)-L-serine Allyl Ester (12). To a pyridine sol-
ution (1 mL) of 11 (35 mg, 25.1ꢂ10ꢀ3 mmol) was added Ac2O (0.5 mL) and the
mixture was stirred for 30 min at 70 °C. The reaction was stopped by addition of
MeOH (5 mL), and the solution was concentrated in vacuo. The residue was
purified by silica gel column chromatography (CH2Cl2:MeOH = 15:1) to afford
acetate 12 (30 mg, 83%): 1H NMR (400 MHz, CDCl3) d 7.96–7.90 (m, 4H,
Phenyl), 7.36–7.16 (m, 13H, Phenyl), 5.82 (m, 1H, CH2–CH ==CH2), 5.76 (d, 1H,
NH, J = 8.1 Hz), 5.66 (d, 1H, NH, J =7.7 Hz), 5.44 (d, 1H, H-4, J = 2.6 Hz), 5.15
(dd, 1H, H-20, J = 6.1, 6.4 Hz), 4.92 (d, 1H, H-1, J = 3.3 Hz), 4.85 (m, 1H, NeuAc
H-4), 4.73 (d, 1H, H-10, J =6.4 Hz), 4.39 (dd, 1H, H-30, J =6.1 Hz), 3.73 (s, 3H,
OMe), 2.54 (dd, 1H, NeuAc H-3e, J =4.8, 12.8 Hz), 2.41, 2.38 (each s, 6H, toluoyl),
2.11, 2.09, 2.07, 2.02, 1.87, 1.64, 1.57, 1.33 (each s, 27H, Ac and acetonide); 13C
NMR (100 MHz, CDCl3) d 170.87, 170.64, 170.17, 170.12, 169.93, 169.68, 167.73,
166.29, 165.38, 155.83, 144.28, 143.91, 136.06, 131.21, 129.86, 129.69, 129.21,
129.14, 128.54, 128.28, 128.22, 119.28, 110.69, 100.62, 98.53, 98.49, 77.21, 76.21,
73.18, 72.81, 72.57, 71.00, 96.03, 68.44, 68.21, 67.30, 67.12, 66.17, 63.76, 63.01,
62.36, 54.48, 52.79, 49.28, 49.10, 37.57, 27.20, 25.92, 23.16, 21.64, 20.93, 20.81,
20.78, 20.70.
!
N-(Benzyloxycarbonyl)-O-(2,6-di-O-toluoyl-B-D-galactopyranosyl)-(1 3)-O-
[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-A-D-galacto-2-
!
nonulopyranosyl)onate-(2 6)]-O-(2-acetamido-2-deoxy-A-D-galactopyranosyl)-
!
(1 3)-L-serine Allyl Ester (13). The aq HOAc solution (70%, 5 mL) of sialoside 11
(129 mg, 93ꢂ10ꢀ3 mmol) was stirred for 48 hours at room temperature. The solution was
1
concentrated in vacuo to give a triol 13 (95 mg, 76%): [a]D +38.0° (c 0.90, MeOH); H
NMR (400 MHz, CDCl3) d 7.92–7.88 (m, 4H, Phenyl), 7.37–7.18 (m, 13H, Phenyl),
6.00 (d, 1H, NH, J = 9.9 Hz), 5.71 (d, 1H, NH, J =8.1 Hz), 5.56 (d, 1H, NH, J =9.2 Hz),
5.27 (d, 1H, CH2–CH == CH2, J = 17.2 Hz), 5.21 (d, 1H, CH2–CH ==CH2, J =10.3 Hz),
4.78 (d, 1H, H-10, J = 7.3 Hz), 4.61 (d, 1H, H-1, J =3.3 Hz), 3.84 (s, 3H, OMe), 2.56 (dd,

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108
SUZUKI ET AL.
1H, NeuAc H-3e, J =4.8, 12.8 Hz), 2.40, 2.36 (each s, 6H, toluoyl), 2.14, 2.12, 2.03, 1.97,
1.85, 1.19 (each s, 18H, Ac and acetonide).
Anal. Calcd for C64H79N3O29: C, 56.76; H, 5.88; N, 3.10. Found: C, 56.42; H, 5.83;
N, 3.16.
!
N-(Benzyloxycarbonyl)-O-(B-D-galactopyranosyl)-(1 3)-O-[(5-acetamido-3,5-
!
dideoxy-D-glycero-A-D-galacto-2-nonulopyranosylonic acid)-(2 6)]-O-(2-acet-
!
amido-2-deoxy-A-D-galactopyranosyl)-(1 3)-L-serine (2). To an EtOH–H2O solu-
tion (2 mL, 1:1) of triol 13 (14.7 mg, 10.9ꢂ10ꢀ3 mmol) was added Pd(PPh3)4 (12.5 mg,
10.9ꢂ10ꢀ3 mmol), and the mixture was stirred for 18 hours at room temperature. The
mixture was filtered through a celite pad and the filtrate was concentrated in vacuo. The
residue was purified by preparative TLC (EtOAc:MeOH = 7:3) to afford an acid. To the
MeOH solution (1 mL) of the acid was added 1 M NaOH (1 mL), and the mixture was
stirred for 10 hours at room temperature. The solution was neutralized with ion-exchange
resin (Dowex 50Wx8, H+ form) and concentrated in vacuo. The residue was purified by
gel filtration (Sephadex G-15, H2O) to give compound 2 (7.8 mg, 80%): [a]D +27.8° (c
0.44, H2O); 1H NMR (500 MHz, D2O) d 7.35–7.29 (m, 5H, Phenyl), 5.06 (s, 2H, benzyl),
4.33 (d, 1H, H-10, J = 7.9 Hz), 4.18 (dd, 1H, J =3.7, 11.0 Hz), 4.06 (d, 1H, H-1, J =3.0 Hz),
4.08 (m, 1H, Ser-a), 3.38 (dd, 1H, H-20, J =9.8 Hz), 2.61 (dd, 1H, NeuAc H-3e, J = 4.4,
12.3 Hz), 1.92, 1.87 (each s, 6H, Ac), 1.55 (dd, 1H, NeuAc H-3a, J =12.3 Hz); 13C NMR
(125 MHz, CDCl3) d 104.87, 100.54, 98.15. TOFMS m/z: 918.6 [M+Na]+. HRMS (FABꢀ)
Calcd for C36H52O23N3 [M–H]ꢀ: 894.2992; Found: 849.2957.
N-(Benzyloxycarbonyl)-O-(5-acetamido-3,5-dideoxy-D-glycero-A-D-galacto-2-
!
nonulopyranosylonic acid)-(2 3)-O-(B-D-galactopyranosyl)-(1 3)-O-[(5-acet-
!
!
amido-3,5-dideoxy-D-glycero-A-D-galacto-2-nonulopyranosylonic acid)-(2 6)]-O-
!
(2-acetamido-2-deoxy-A-D-galactopyranosyl)-(1 3)-L-serine (14). A mixture of
compound 2 (13.2 mg, 14.7ꢂ10ꢀ3 mmol), CMP-NeuAc 4 (19.4 mg, 29.4ꢂ10ꢀ3 mmol),
a2,3-(O)-sialyltransferase (EC 2.4.99.4) from rat liver (100 mU in 50 mM MES, pH 6.0,
250 mM NaCl, 0.5 mM b-mercaptoethanol, 50% glycerol) in 0.1 M cacodylate buffer
(0.7 mL, pH 6.0) containing bovine serum albumin (2 mg) was incubated for 12 hours at
37 °C. The solution was purified by HPLC (ODS, 10% MeCN containing 0.1% TFA) and
gel filtration (Sephadex G-15, H2O) to afford tetrasaccharide-serine 14 (16 mg, 83%):
[a]D +19.3° (c 0.29, H2O); 1H NMR (500 MHz, D2O) d 7.38–7.28 (m, 5H, Phenyl), 5.07
(s, 2H, benzyl), 4.40 (d, 1H, H-10, J = 7.8 Hz), 4.15 (dd, 1H, J = 3.7, 10.8 Hz), 4.12 (br s,
1H, H-1), 3.41 (dd, 1H, H-20, J =9.8 Hz), 2.64 (dd, 1H, NeuAc H-3e, J = 4.4, 12.3 Hz),
2.59 (dd, 1H, NeuAc H-3e, J =4.7, 12.5 Hz), 1.92, 1.86 (each s, 6H, Ac), 1.69 (dd, 1H,
NeuAc H-3a, J =12.3 Hz), 1.59 (dd, 1H, NeuAc H-3a, J =12.3 Hz); 13C NMR (125 MHz,
CDCl3) d 104.87, 100.54, 98.15. TOFMS m/z: 1211.9 [M +Na]+. HRMS (FABꢀ) Calcd
for C47H69O31N4 [M–H]ꢀ: 1185.3946; Found: 1185.3993.
ACKNOWLEDGMENTS
This work was performed as a part of the Research and Development Project of the
Industrial and Technology Program supported by the New Energy and Industrial
Technology Development Organization.

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SYNTHESIS OF MUC II
109
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SYNTHESIS OF MUC II
111
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Received June 1, 2001
Accepted January 2, 2002