Solid-phase synthesis of sialyl Tn antigen

Article abstract of DOI:10.1080/07328300600778785

Solid-phase synthesis of sialyl Tn [α-Neu5Ac-(2→6)-α- GalNAc-(1→ O)-Ser] antigen with Kenner's acylsulfonamide linker is described. The acylsulfonamide bond was found to be stable under glycosylation reactions using dimethyl(methylthio)sulfonium triflate

Full text of DOI:10.1080/07328300600778785

Journal of Carbohydrate Chemistry
ISSN: 0732-8303 (Print) 1532-2327 (Online) Journal homepage: http://www.tandfonline.com/loi/lcar20
Solid‐Phase Synthesis of Sialyl Tn Antigen
Takuya Kanemitsu , Shusaku Daikoku & Osamu Kanie
To cite this article: Takuya Kanemitsu , Shusaku Daikoku & Osamu Kanie (2006) Solid‐Phase
Synthesis of Sialyl Tn Antigen, Journal of Carbohydrate Chemistry, 25:5, 361-376, DOI:
10.1080/07328300600778785
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Date: 05 June 2016, At: 01:37

Journal of Carbohydrate Chemistry, 25:361–376, 2006
Copyright # Taylor & Francis Group, LLC
ISSN: 0732-8303 print 1532-2327 online
DOI: 10.1080/07328300600778785
Solid-Phase Synthesis of
Sialyl Tn Antigen
Takuya Kanemitsu, Shusaku Daikoku, and Osamu Kanie
Mitsubishi Kagaku Institute of Life Sciences (MITILS), Tokyo, Japan
Solid-phase synthesis of sialyl Tn [a-Neu5Ac-(2 ! 6)-a-GalNAc-(1 ! O)-Ser] antigen
with Kenner’s acylsulfonamide linker is described. The acylsulfonamide bond was
found to be stable under glycosylation reactions using dimethyl(methylthio)sulfonium
triflate (DMTST) as a promoter and basic conditions used for the removal of protecting
groups. The solid-phase reaction was monitored by the inverse gated decoupling 13C
NMR technique, which enabled quantitative analysis of the reaction progress. At the
end of the synthesis, the sulfamyl group of the linker was activated by treatment with
(trimethylsilyl)diazomethane to provide a N-methyl-N-acylsulfonamide. The acyl
group was displaced with hydroxide to give the corresponding precursors of sialyl Tn
antigen and its anomeric isomers, which were deprotected to afford the target molecules.
Keywords Oligosaccharide synthesis, Solid-phase synthesis, Thioglycosides,
Monitoring, Kenner’s linker
INTRODUCTION
A disaccharide, namely sialyl Tn, is known as a tumor-associated antigen present
in glycoproteins expressed on the surface of cancer cells[1] and is also found in the
envelope glycoprotein gp 120 of the human immunodeficiency virus (HIV).[2]
A
Received September 21, 2005; accepted November 12, 2005.
Address correspondence to Osamu Kanie, Mitsubishi Kagaku Institute of Life Sciences
(MITILS), Minamiooya 11, Machida-shi, Tokyo 194-8511, Japan. E-mail: kokanee@
libra.ls.m-kagaku.co.jp
361

T. Kanemitsu et al.
362
synthetic cancer vaccine[3–6] and a potential target for HIV immuno interven-
tion[7] based on sialyl Tn antigen has thus been a target of investigations.
Although several chemical or enzymatic syntheses of sialyl Tn epitope have
been reported,[8–12] there is no report based on solid-phase synthesis. One of
the reasons for this is the difficulty associated with the sialylation reaction.
As a part of our ongoing investigation of the solid-phase synthesis of oligo-
saccharides and a non-destructive monitoring method using inverse gated
decoupling13C NMR,[13,14] we selected an aNeuAc(2 ! 6)aGalNAc ! Ser
(sialyl Tn-Ser) epitope to demonstrate the applicability of our solid-phase syn-
thetic method in the practical synthesis of an oligosaccharide. Also, we decided
to synthesize all of its possible anomers. The primary aim of a current trend in
the chemical synthesis of oligosaccharides that includes solid-phase synthesis
is to synthesize a “desired” product, which has been isolated and structurally
defined, and found to play important biological roles. However, it may be
possible to find useful structures in nonnatural oligosaccharides. Merging
with the concept of combinatorial chemistry, the synthesis of an oligosacchar-
ide library should be recognized as a potential pool for finding lead compounds
in the development of pharmaceuticals, especially those for treating infectious
diseases.
Despite the importance of solid-phase synthesis of oligosaccharides,[15–26]
there is still room for improvement. One of the important factors is a linker
that is stable during glycosylation and deprotection reactions, which can be
cleaved easily as needed. Also, the quantitative nondestructive monitoring of
the reaction process remains to be solved. Regarding a suitable linker for our
solid-phase oligosaccharide synthesis, we have selected Kenner’s acylsulfona-
mide linker,[27–29] which is stable under basic and strongly nucleophilic
reaction conditions. Examination of the utility of the linker in oligosaccharide
synthesis revealed that the acylsulfonamide bond was stable in a glycosylation
reaction using DMTST[30,31] as a promoter and basic conditions used for the
removal of acetyl groups. Furthermore, the sulfamyl group of the resin could
be activated successfully by treatment with (trimethylsilyl)diazomethane to
provide an N-methyl-N-acylsulfonamide. The acyl group was displaced with
hydroxide to give the corresponding free acid. For the monitoring of solid-
phase oligosaccharide synthesis, we have been investigating a nondestructive
monitoring technique using an inverse gated decoupling 13C NMR.[32] The 13C-
enriched protecting groups were used for the glycosylating agents together
with13C-enriched linker as an internal integral marker.[13,14] Hence, the
reaction yields are given as relative to the internal standard, and the actual
chemical yields for the corresponding reactions are obtained.
We report herein the solid-phase synthesis of sialyl Tn antigen as a Boc-
protected form and all its stereoisomers using acylsulfonamide linker, the
reaction course of which is quantitatively monitored by an inverse gated
decoupling 13C NMR technique.

Solid-Phase Synthesis of Sialyl Tn Antigen
363
RESULTS AND DISCUSSION
Synthesis of L-Serine Attachment to
Acylsulfonamide Linker
N-(t-butoxycarbonyl)-L-serine (1) was treated with acetic-1-13C anhydride,
prepared from acetic-1-13C acid, in the presence of 4-dimethylaminopyridine
(DMAP) in CH2Cl2 to give compound 2, which was coupled with 4-sulfamoyl-
benzoic acid benzyl ester (4) using benzotriazole-1-yl-oxy-tris(pyrrolidino)
phosphonium hexafluorophosphate (PyBop) in CH2Cl2 to obtain a conjugate
of L-serine-linker with a 13C-enriched tag (5) in 72% yield. Compound 5 was
debenzylated by hydrogenolysis to give compound 6 (Scheme 1).
Synthesis of GalN Glycosyl Donor
The primary OH group of the phenylthioglycoside of 2-azide-2-deoxy-galac-
tose 7[10] was tritylated and the remaining hydroxy groups were subsequently
benzylated to give 9. Compound 9 was detritylated with TFA to give compound
10, which was converted to the GalN glycosyl donor (11) after acetylation with
acetic-1-13C acid for solid-phase synthesis (Scheme 2).
Solid-Phase Synthesis of Sialyl Tn Antigen
The 13C acetyl-protected L-serine attached sulfonamide linker 6 was first
incorporated into the TentaGel carrying a 1-13C-glycine (12) as an internal
Scheme 1: Synthesis of L-Ser attached to the acylsulfonamide linker. i. acetic-1-13
C
anhydride, DMAP/CH2Cl2, rt, 12 h, 92%; ii. BnOH, DIPC, HOBt / DMF, rt, 12 h, 85%; iii. PyBop,
iPr2EtN/CH2Cl2, 2208C, 10 h, 72%; iv. 10% Pd/C, H2/MeOH, rt, 12 h, 88%.

T. Kanemitsu et al.
364
Scheme 2: Synthesis of GalN donor. i. TrCl, pyridine, rt, 24 h, 90%; ii. BnBr, NaH/DMF, rt, 2 h, 90%;
iii. 10% TFA/CH2Cl2, rt, 1 h, 83%; iv. acetic-1-13C acid, DCC, DMAP/CH2Cl2, rt, 4 h, 94%.
integral standard. The coupling yield of the reaction was estimated by
gated decoupling 13C NMR (97%) after filtration. Subsequent deacetylation
of 13 afforded a glycosyl acceptor 14 quantitatively. The solid-bound L-Ser
14 was glycosylated with GalN donor 11 (2 equiv.) in the presence of
DMTST (8 equiv.) in CH2Cl2-Et2O at 08C. The reaction was carried out
twice to obtain a resin, 15, in 82% yield, which was confirmed by gated
decoupling 13C NMR. The remaining hydroxyl groups of resin-bound L-Ser
were capped using t-butyldimethylsilylchloride (TBDMS-Cl) to eliminate the
[33]
sequence deletion.
After deprotection of the acetyl group (16, quant.), a
second glycosylation reaction was performed using methyl (13C) 5-acetoamide-
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-methylthio-D-glycero-b-D-galacto-2-nonu-
lopyranosylonate.[14] The sialylation reaction was carried out in the presence
of DMTST at 2158C in CH3CN. Solvent selection was based on some
preliminary experiments to obtain all anomers where the desired distribution
of the mixture was equal. Cleavage of the solid-bound compounds was
achieved by a sequential treatment with TMS-diazomethane and NaOH to
give the glycopeptides 19a–d as a mixture of the combination of the
possible anomers. The yield for the cleavage reaction was 62% based on gravi-
metric analysis of the released products and on the reaction yields estimated
by the gated decoupling technique. The resin should be glycosylated in various
degrees of order since each glycosylation reaction was not quantitative, and
individual compounds are given as a mixture of anomers. In order to
determine the anomeric configurations in each glycosidic bond, compounds
19a–d released from the resin were purified by gel permeation chromato-
graphy and repeated silica gel column chromatography. The product was
found to consist of four isomers based on the anomeric configurations of the
Gal and Sia residues, and it was revealed that the desired anomeric
combination [a a(Sia/Gal)] was a major component among the anomers:
a/a (19a, 25%), a/b (19b, 19%), b/a (19c, 12%), and b/b(19d, 6%). Each
compound was analyzed by HPLC, the chromatogram of which is shown

Solid-Phase Synthesis of Sialyl Tn Antigen
365
in Figure 1. Assignment of the anomeric configurations for the sialic acid
residue was based on an empirical rule where the chemical shifts of the equa-
torial proton of H-3 appeared in the lower field for the a-glycosides.[34] The
observed chemical shifts of the proton for compounds 19a–d were d 2.85,
2.89, 2.41, and 2.47, respectively (Scheme 3).
Deprotection of Sialyl Tn Antigen
The isolated compounds 19a–d were first subjected to hydrogenolysis to
deprotect benzyl groups and at the same time to convert the azide into an
amine. Subsequent N-acetylation gave sialyl Tn antigen and its anomeric
isomers (20a–d) as a Boc-protected form, which could be used in peptide syn-
thesis (Scheme 4).
Conclusion
The synthesis of sialyl Tn-Ser antigen and its stereoisomers with a Boc-
protecting group was achieved on the solid support, where we have used two
13C enriched markers for the inverse gated decoupling 13C NMR technique.
The strategy should fit suitably into the small-scale solid-phase synthesis of oligo-
saccharides and perhaps other organic compounds, especially for optimization of
the reaction conditions. The advantage of this method is that actual chemical
yields are obtained since the yield is given directly from integrals in 13C NMR
without any manipulation, which sometimes results in inaccurate calculations
due to resin breakdown. For the cleavage of the synthesized molecule from
resin, Kenner’s acylsulfonamide linker was used. The acylsulfonamide bond
was found to be stable to the glycosylation reaction conditions using DMTST as
a promoter and basic conditions used for the removal of protecting groups.
Figure 1: Chromatograms of purified compounds 19a–d.

T. Kanemitsu et al.
366
Scheme 3: Solid-phase synthesis of sialyl Tn antigen. i. 6, DIPC-HOBt-iPr2EtN/DMF, rt, 12 h, 97%;
ii. 0.05 M NaOMe/MeOH-DMF (1:1), rt, 12 h, quant.; iii. 11, DMTST/CH2Cl2-Et2O (1 : 1), 08C, 12 h,
twice, 82%; iv. TBDMSCl-imidazole / CH2Cl2, rt, 12 h; v. 17, DMTST/CH3CN, 2158C, 12 h, twice,
80%; vi. a) TMS-diazomethane, iPr2EtN/THF, rt, 24 h; b) 0.05 N NaOH/H2O-THF (1 : 1), rt, 12 h, 62%
(in two steps).

Solid-Phase Synthesis of Sialyl Tn Antigen
367
Scheme 4: Deprotection reactions. i. (a) Pd(OH)2-H2/MeOHH2O-AcOH, rt, 12 h, (b) Ac2O,
pyridine, rt, 12 h, (c) 0.05M NaOMe / MeOH, r.t., 3 h.
EXPERIMENTAL
General Methods
TentaGelTM S-NH2 resin was purchased from Fluka. Dried solvents were
used for all reactions. Solvents were evaporated under reduced pressure
at a bath temperature not exceeding 508C. Analytical thin layer chromato-
graphy (TLC) was performed on Merck Art. 5715, Kieselgel 60 F254/0.25-mm
thickness plates. Visualization was accomplished with UV light, phosphomo-
lybdic acid, or sulfuric acid solution followed by heating. Column chromato-
graphy was performed with silica gel FL-100D (Fuji Silysia Co.). Optical
rotations were measured on a Horiba SEPA-200 polarimeter with sodium
lamp (l ¼ 589 nm). 1H NMR (270 MHz) and 13C NMR (67.5 MHz) spectra
were recorded with a JEOL EX-270 spectrometer in deuterated solvents
1
using tetramethylsilane (d 0.00 for H NMR), CDCl3 (d 77.00 for 13C NMR),
or CD3OD (d 49.00 for 13C NMR) as an internal standard. MALDI-TOF
mass spectra were recorded on a Perceptive Voyager mass spectrometer
with 2,5-dihydroxybenzoic acid as matrix. High-resolution mass spectra
were recorded on a JEOL JMS-700 spectrometer under FAB conditions. Com-
pounds 19a–d were analyzed by LC-MS (Waters 2525 pump system and
Micromass ZQ). The conditions used were as follows. Column: Imtakt Unizon
UK-C18 (4.6 ꢀ 75 mm); flow rate: 1.0 mL/min; column temp.: 408C: elution:
0.1% AcOH in CH3CN (Merck Hyper Grade)/H2O (LC/MS Grade) ¼ 35/65;
ionization: ESI; detection: m/z ¼ 862 [M-H]2 (negative ion mode).
Materials
Compound 17 was synthesized according to the procedure described in
ref. 14.

T. Kanemitsu et al.
368
General Methods for Monitoring of Solid-phase Compounds
with Inverse-gated Decoupling 13C NMR Measurement
The dried resin (ca. 60 mg) was slurried in CDCl3, and the sample was
prepared to contain a relaxation agent, chromium (III) 2,4-pentanedionate
[Cr(AcAc)3, 0.1 M], in an ordinary 5-mm f NMR tube. 13C NMR was
measured on a JEOL EX-270 spectrometer at 67.5 MHz and operated with a
9-s relaxation delay and gated decoupling without NOE (160 transients, 0.6-s
acquisition time). The spectra were referred to resonance for TMS. 13C NMR
spin-lattice relaxation times (T1) were measured by using the inversion
recovery method at 298 K (16 data points, 16 scans per point). T1 values for
methyl and carbonyl groups attached to the resin were shorter than 1 s in
the presence of Cr(AcAc)3.
Procedures
N-(tert-Butoxycarbonyl)-O-acetyl(1-13C)-L-serine.[2] To a solution of
DCC (780 mg, 3.78 mmol) in dry CH2Cl2 (8.0 mL) was added acetic-1-13C acid
(400 mL, 6.87 mmol) at rt and the mixture was stirred for 30 min and then
filtered to remove urea. To the filtrate, a solution of N-(tert-butoxycarbonyl)-
L-serine (800 mg, 3.90 mmol) and DMAP (48 mg, 0.39 mmol) in CH2Cl2
(15.0 mL) and compound 1 were added at rt and stirred for 12 h. The
mixture was concentrated in vacuo. The residue was purified by silica gel
column chromatography using toluene/acetone/AcOH (10 : 5 : 0.2) as an
eluent to provide 2 (784 mg, 92%): [a]D þ15.68 (c 1.0, CHCl3); Rf 0.19
1
(toluene/acetone/AcOH 10 : 5 : 0.2); H NMR (CDCl3) d 5.53–5.41 (m, 1H, Ser
a), 4.60–4.40 (m, 2H, Ser b), 2.08 (d, 3H J ¼ 6.9 Hz, Ac), 1.46 (s, 9H, tBu-
CH3); 13C NMR (CDCl3) d 172.97, 170.76, 155.58, 80.79, 64.17, 52.80, 28.27,
20.66 (d, J ¼ 59.9 Hz). MALDI-TOF MS: Calcd for C9 13CH17NO6 (M): 248.1.
Found m/z: 271.1 (M þ Na)þ; HR-FAB MS: Calcd for C9 13CH17NO6 (M):
248.1089. Found m/z: 247.1026 (M-H)2.
4-Sulfamoylbenzoic acid benzyl ether.[4] 1-Hydroxybenzotriazole (HOBt,
3.7 g, 27.4 mmol) and N, N0-diisopropylcarbodiimide (DIPC, 3.45 g,
27.3 mmol) were added at rt to a solution of 4-sulfamoylbenzoic acid (3)
(5.0 g, 24.9 mmol) and benzylalcohol (6.0 mL, 58.0 mmol) in DMF (12 mL).
After stirring for 12 h, the reaction mixture was concentrated. The residue
was purified by silica gel column chromatography using toluene/acetone
(5 : 1) as an eluent to provide 4 (6.13 g, 85%) as a white mass: [a]D 230.48 (c
0.5, MeOH); Rf 0.35 (toluene/acetone 4 : 1); 1H NMR (CD3OD) d 8.19–7.96
(m, 4H, Ar), 7.48–7.30 (m, 5H, Ar), 5.38 (s, 2H, Bn-CH2); 13C NMR (CD3OD)
d 167.30, 150.01, 138.08, 135.33, 131.97, 130.46, 130.25, 130.17, 128.18,
69.10. Anal calcd for C14H13NO4S: C, 57.72; H, 4.50; N, 4.81; S, 11.01. Found:
C, 57.70; H, 4.48; N, 4.80; S, 10.89.

Solid-Phase Synthesis of Sialyl Tn Antigen
369
N-(tert-Butoxycarbonyl)-O-acetyl(1-13C)-L-seryl-4-sulfamoylbenzoic acid
benzyl ether.[5] To a solution of compound 2 (655 mg, 2.65 mmol) and 4
(1540 mg, 5.3 mmol) in dry CH2Cl2 (15 mL) was added N, N-diisopropylethyla-
mine (iPr2EtN, 0.9 mL, 5.3 mmol) at rt. After stirring for 20 min, the mixture
was cooled to 2208C. PyBop (2100 mg, 4.0 mmol) was added to the mixture
and stirred for 10 h, then diluted with CH2Cl2, which was washed with brine
(three times), dried with Na2SO4, filtered, and concentrated. The residue was
purified by silica gel column chromatography using toluene/acetone (1 : 1) as
an eluent to provide 5 (996 mg, 72 %): [a]D 230.48 (c 0.5, CHCl3); Rf 0.17
1
(toluene/acetone 1 : 1); H NMR (CDCl3) d 8.24–8.11 (m, 4H, Ar), 7.45–7.28
(m, 5H, Ar), 5.40 (s, 2H Bn-CH2), 4.35–4.21 (m, 3H, Ser a and Ser b), 2.02
(d, 3H J ¼ 6.9 Hz, Ac), 1.44 (s, 9H, tBu-CH3); 13C NMR (CDCl3) d 170.80,
167.44, 164.76, 156.12, 142.10, 135.35, 135.13, 130.21, 128.72, 128.57, 128.54,
128.36, 82.14, 67.49. 62.50, 54.13, 28.16, 20.59 (d, J ¼ 59.8 Hz). Anal calcd
for C23 13CH28N2O9S: C, 55.46; H, 5.41; N, 5.37; S, 6.15. Found: C, 55.12; H,
5.36; N, 5.28; S, 6.73.
N-(tert-Butoxycarbonyl)-O-acetyl(1-13C)-L-seryl-4-sulfamoylbenzoic
acid.[6] A mixture of compound 5 (800 mg, 1.53 mmol) in MeOH (10 mL) was
stirred at rt in the presence of 10% Pd/C (50 mg) under an H2 atmosphere
for 12 h. The reaction mixture was filtered through a pad of celite and the
filtrate was concentrated. The residue was purified by Sephadex LH-20
column chromatography using MeOH as an eluent to provide 6 (580 mg
88%): [a]D 248.78 (c 0.5, CHCl3); Rf 0.47 (CH2Cl2/MeOH/AcOH 10 : 1 : 0.5);
1H NMR (CD3OD) d 8.20–8.07 (m, 4 H, Ar), 4.25–4.14 (m, 3H, Ser a and Ser
b), 1.91 (d, 3H J ¼ 6.9 Hz, Ac), 1.39 (s, 9H, tBu-CH3); 13C NMR (CD3OD) d
172.73, 171.31, 168.74, 158.26, 145.07, 137.62, 131.90, 130.19, 81.97, 64.77,
56.18, 29.35, 21.20 (d, J ¼ 59.8 Hz). HR-FAB MS: Calcd for C16 13CH22N2O9S
(M): 431.1080. Found m/z: 454.0977 (M þ Na)þ.
Phenyl 2-azido-2-deoxy-1-thio-6-O-trityl-a,b,-D-galactopyranoside.[8]
A solution of compound 7 (230 mg, 0.77 mmol) and trityl chloride (440 mg,
1.58 mmol) in pyridine (4.0 mL) was stirred at rt for 24 h. The reaction
mixture was concentrated. The residue was purified by silica gel column
chromatography using toluene/acetone (10 : 1) as an eluent to provide 8 as a
1
mixture of a and b anomers (374 mg, 90%): Rf 0.48 (toluene/acetone 4 : 1); H
NMR (CD3OD) d 7.66–7.22 (m, Ar), 5.68 (d, J1,2 ¼ 5.3 Hz, H-1a), 4.45 (ddd,
J4,5 ¼ 1.5, J5,6 ¼ 4.3 Hz, H-5a), 4.41 (d, J1,2 ¼ 9.9 Hz, H-1b), 4.36 (dd,
J2,3 ¼ 10.6 Hz, H-2a), 4.04 (dd, J3,4 ¼ 3.0 Hz, H-4a), 3.93 (dd, J3,4¼ 2.7,
J4,5 ¼ 1.0 Hz, H-4b), 3.80 (dd, H-3a), 3.55–3.40 (m, H-2b, 3b, 5b, 6a, 6b).
MALDI-TOF MS: Calcd for C31H29N3O4S (M): 539.2. Found m/z: 562.5
(M þ Na)þ; Anal calcd for C31H29N3O4S: C, 69.00; H, 5.42; N, 7.79. Found: C,
68.83; H, 5.56; N, 7.65

T. Kanemitsu et al.
370
Phenyl 2-azido-3,4-di-O-benzyl-2-deoxy-1-thio-6-O-trityl-a,b-D-galacto-
pyranoside.[9] To a solution of compound 8 (414 mg, 0.77 mmol) in DMF
(6.0 mL) was added NaH (60%, in mineral oil, 120 mg, 3.0 mmol) at 08C, and
the mixture was stirred for 30 min. Benzyl bromide (360 mL, 3.0 mmol) was
added to the mixture and the temperature of the mixture was allowed to
increase to rt. After stirring for 2 h the excess NaH was carefully destroyed
by adding MeOH. After evaporation, a solution of the residue in CH2Cl2 was
successively washed with H2O (3 times), dried over Na2SO4, filtered, and con-
centrated. The residue was purified by silica gel column chromatography using
hexane/ethyl acetate (10 : 1) as an eluent to provide a mixture of a and b
anomers (495 mg, 90%): Rf 0.47 (hexane/ethyl acetate 6 : 1); 1H NMR
(CDCl3) d 7.56–7.02 (m, Ar), 5.57 (d, J1,2 ¼ 5.3 Hz, H-1a), 4.79–4.64
(m, Bn-CH2), 4.45–4.33 (m, H-1b, 2a, 5a, Bn-CH2), 3.94 (br d, J3,4 ¼ 2.0 Hz,
H-4a), 3.84 (br d, J3,4 ¼ 2.3 Hz, H-4b), 3.79–3.73 (m, H-2b, 3a), 3.55 (dd,
J5,6 ¼ 5.6, J6a,6b ¼ 9.2 Hz, H-6ab), 3.43 (dd, J5,6 ¼ 5.9, J6a,6b ¼ 9.6 Hz,
H-6aa), 3.37–3.30 (m, H-3b, 5b), 3.24–3.16(m, H-6ba, 6bb). MALDI-TOF
MS: Calcd for C45H41N3O4S (M): 719.3. Found m/z: 742.5 (M þ Na)þ; Anal
calcd for C45H41N3O4S: C, 75.08; H, 5.74; N, 5.84. Found: C, 74.84; H, 5.65;
N, 5.81.
Phenyl 2-azido-3,4-di-O-benzyl-2-deoxy-1-thio-a- and - b-D-galactopyr-
anoside.[10] Compound 9 (490 mg, 0.68 mmol) was treated with 10% TFA in
CH2Cl2 at rt for 1 h and then the reaction mixture was concentrated. The
residue was purified by silica gel column chromatography using hexane/
ethyl acetate (2 : 1) as an eluent to provide a and b anomers, respectively:
Data for a anomer: [a]D þ152.68 (c 1.0, CHCl3); Rf 0.39 (hexane/ethyl
acetate 2 : 1); 1H NMR (CDCl3) d 7.50–7.24 (m, 15H, Ar), 5.65 (d, 1H,
J1,2 ¼ 5.3 Hz, H-1), 4.91 and 4.57 (2d, 2H, Jgem ¼ 11.6 Hz, Bn-CH2), 4.78 (dd,
2H, Jgem ¼ 13.8, 11.5 Hz, Bn-CH2), 4.44 (dd, 1H, J2,3 ¼ 10.6 Hz, H-2), 4.25
(ddd, 1H, J5,6 ¼ 6.6 Hz, H-5), 3.94 (br s, 1H H-4), 3.80 (dd, 1H, J3,4 ¼ 2.6 Hz,
H-3), 3.71 (dd, 1H, J6a,6b ¼ 11.5 Hz, H-6a), 3.53 (br m, 1H, H-6b); 13C NMR
(CDCl3) d 137.77, 137.23, 132.99, 132.45, 129.06, 128.59, 128,50, 128.28,
128.09, 128.05, 127.87, 127.75, 87.35, 79.23, 74.61, 73.28, 72.74, 71.88, 62.03,
60.38. MALDI-TOF MS: Calcd for C26H27N3O4S (M): 477.2. Found m/z:
500.5 (M þ Na)þ. Data for b anomer: [a]D 235.68 (c 1.0, CHCl3); Rf 0.22
1
(hexane/ethyl acetate 2:1); H NMR (CDCl3) d 7.59–7.21 (m, 15H, Ar), 4.88
and 4.54 (2d, 2H, Jgem ¼ 11.6 Hz, Bn-CH2), 4.72 (s, 2H, Bn-CH2), 4.40 (d, 1H,
J1,2 ¼ 10.2 Hz, H-1), 3.88–3.79 (m, 3H, H-2, 4, 6a), 3.54 (br m, 1H, H-6b),
3.44–3.39 (m, 2H, H-3, 5); 13C NMR (CDCl3) d 138.04, 137.29, 132.81,
131.77, 128.91, 128.57, 128.39, 128.10, 127.98, 127.91, 127.85, 86.45, 82.64,
78.96, 74.20, 72.72, 72.01, 62.14, 61.69. HR-FAB MS: Calcd for C26H27N3O4S
(M): 477.1722. Found m/z: 478.1797 (M þ H)þ.

Solid-Phase Synthesis of Sialyl Tn Antigen
371
Phenyl 6-O-acetyl(1-13C)-2-azido-3,4-di-O-benzyl-2-deoxy-1-thio-a- and
-b-D-galactopyranoside.[11] Acetic-1-13C acid (15 mL, 0.26 mmol) was added
at rt to a solution of compound 10 (as a mixture of a and b anomers)
(120 mg, 0.25 mmol), DCC (78 mg, 0.38 mmol), and DMAP (6 mg, 0.05 mmol)
in dry CH2Cl2 (5 mL), and the mixture was stirred for 4 h. The reaction
mixture was filtered and the filtrate was diluted with CH2Cl2, washed with
brine (three times), dried over Na2SO4, filtered, and concentrated. The
residue was purified by silica gel column chromatography using hexane/
ethyl acetate (4 : 1) to provide 11 (123 mg, 94%): Data for a anomer: [a]D
1
þ138.68 (c 1.0, CHCl3); Rf 0.61 (hexane/ethyl acetate 2 : 1); H NMR (CDCl3)
d 7.51–7.24 (m, 15H, Ar), 5.65 (d, 1H, J1,2 ¼ 5.3 Hz, H-1), 4.92 and 4.56 (2d,
2 H, Jgem ¼ 11.2 Hz, Bn-CH2), 4.79 (dd, 2H, Jgem ¼ 11.5, 12.9 Hz, Bn-CH2),
4.47–4.41 (m, 2H, H-2, 5), 4.21–4.04 (m, 2H, H-6), 3.92 (br s, 1H H-4), 3.79
(dd, 1H, J2,3 ¼ 10.6, J3,4 ¼ 2.6 Hz, H-3), 1.92 (d, 3H, J ¼ 6.9 Hz, Ac); 13C
NMR (CDCl3) d 170.42, 137.72, 137.22, 133,17, 132.09, 128.93, 128.59,
128.39, 128.14, 128.09, 127.91, 127.85, 127.57, 87.19, 79.16, 74.68, 73.17,
72.80, 69.63, 63.24, 60.25, 20.64 (d, J ¼ 59.8 Hz). MALDI-TOF MS: Calcd for
C27 13CH29N3O5S (M): 520.2. Found m/z: 543.4 (M þ Na)þ. Data for b
1
anomer: [a]D 218.58 (c 1.0, CHCl3); Rf 0.49 (hexane/ethyl acetate 2 : 1); H
NMR (CDCl3) d 7.59–7.18 (m, 15H, Ar), 4.89 and 4.53 (2d, 2 H,
Jgem ¼ 11.6 Hz, Bn-CH2), 4.72 (s, 2H, Bn-CH2), 4.37 (d, 1H, J1,2 ¼ 9.9 Hz,
H-1), 4.25 (dd, 1H, J5,6a ¼ 3.3, J6a,6b¼ 11.2 Hz, H-6a), 4.10 (dd, 1H,
J5,6b ¼ 3.0 Hz, H-6b), 3.84 (dd, 1H, J2,3 ¼ 9.6 Hz, H-2), 3.79 (br d, 1H,
J3,4 ¼ 2.6 Hz, H-4), 3.55 (br dd, 1H, H-5), 3.41 (dd, 1H, H-3), 1.98 (d, 3H,
J ¼ 6.9 Hz, Ac); 13C NMR (CDCl3) d 170.42, 137.86, 137.20, 132.87, 131.73,
128.90, 128.73, 128.48, 128.21, 128.01, 127.89, 127.80, 127.64, 86.31, 82.50,
76.12, 74.20, 72.72, 71.93, 63.22, 61.44, 20.67 (d, J ¼ 58.6 Hz). MALDI-TOF
MS: Calcd for C2713CH29N3O5S (M): 520.2. Found m/z: 543.5 (M þ Na)þ Anal
calcd for C2713CH29N3O5S: C, 64.79; H, 5.61; N, 8.07. Found: C, 64.72; H,
5.60; N, 7.89.
Solid-Phase Synthesis
Resin 13. To a suspension of resin 12 (500 g, 0.13 mmol) and compound 6
(112 mg, 0.26 mmol) in dry DMF (10 mL) were added iPr2EtN (67 mL,
0.39 mmol), HOBt (52 mg, 0.39 mmol) and DIPC (60 mL, 0.39 mmol), at rt.
After shaking for 4 h, the resins were washed with DMF, H2O, MeOH, and
CH2Cl2, then dried in vacuo to give sulfonamide linker-attached resin 12
(97%). The yield was estimated by integral obtained by inverse gated
decoupling as relative to internal 13C marker at 168.9 ppm: 13C NMR
(CDCl3) d 170.0, 168.9.
Deacetylation of Solid-phase Compounds. 13C-enriched acetyl protected
resin-bound compound was treated with 5 mM NaOMe in MeOH-DMF mixed

T. Kanemitsu et al.
372
solvent (1 : 1, v/v) at rt for 12 h, washed with DMF, H2O, DMF-AcOH (10 : 1, v/
v), MeOH, and CH2Cl2, and then dried in vacuo to give the resin-bound com-
pounds with a free hydroxyl group. The deprotection was confirmed by the dis-
appearance of carbonyl signals that originated from the acetyl group in 13C
NMR spectra.
Resin 14. 13C NMR (CDCl3) d 168.9.
Resin 16. 13C NMR (CDCl3) d 169.5.
Glycosylation Reaction on Solid-phase. DMTST (8 equiv.) was added to a
mixture of resin-bound acceptor and glycosyl donor (2 equiv.) in dry CH2Cl2-
Et2O mixed solvent or CH3CN (20 mL/g-resin). The mixture was shaken
under a nitrogen atmosphere at the temperature designated in Scheme 3 for
24 h. The resin was washed with CH2Cl2, MeOH, H2O, DMF, and CH2Cl2,
and this procedure was repeated twice. The resin was then suspended in
CH2Cl2, tBuMe2SiCl, and imidazole added at rt, and the mixture shaken for
24 h. The resins were washed with CH2Cl2, MeOH, water, DMF, and CH2Cl2.
The reaction yields were estimated based on the integrals of signals obtained
by 13C NMR experiments.
Resin 15. 13C NMR (CDCl3) d 171.0, 168.3.
Resin 18. 13C NMR (CDCl3) d 170.0, 52.3.
Cleavage of Compounds from Solid-phase. iPr2EtN (65 mL, 0.37 mmol)
and (trimethylsilyl)diazomethane (2.0 M solution in hexane, 2.0 mL) were
added to a suspension of resin 18 (560 mg) in dry THF (10 mL). The mixture
was shaken in the dark under a nitrogen atmosphere for 24 h. The resins
were washed with THF and DMF. Subsequently, the resin was suspended in
0.05 M NaOH/H2O-THF (1 : 1) and shaken at rt for 12 h. The resins were
washed with DMF and MeOH. The filtrate was neutralized by Amberlite IR-
120 (Hþ), filtered, and concentrated. The residue was passed through a
Sephadex LH-20 gel permeation column with MeOH as a solvent to isolate a
disaccharide-serine fraction. The fraction was purified by repeated silica gel
column chromatography using CH2Cl2/MeOH/H2O/AcOH (20 : 10 : 1 : 1) as
an eluent to provide 19a (17.9 mg), 19b (13.6 mg), 19c (8.6 mg), and 19d
(4.3 mg): Data for 19a: [a]D þ30.28 (c 0.35, MeOH); Rf 0.43 (CH2Cl2/MeOH/
1
H2O/AcOH 20 : 10 : 1 : 1); H NMR (CD3OD) d 7.40–7.27 (m, 10H, Ar), 4.84
(br s, 1H, H-1a), 2.85 (br d, 1H, J ¼ 12.9 Hz, H-3b-eq), 2.01 (s, 3H, Ac), 1.66
(t, 1H, H-3b-ax), 1.45 (s, 9 H, tBu-CH3). MALDI-TOF MS: Calcd for
C39H53N5O17 (M): 863.3. Found m/z: 886.3 (M þ Na)þ; HR-FAB MS: Calcd for
C39H53N5O17 (M): 863.3436. Found m/z: 886.3331 (M þ Na)þ. Data for 19b:

Solid-Phase Synthesis of Sialyl Tn Antigen
373
[a]D þ33.48 (c 0.34, MeOH); Rf 0.41 (CH2Cl2/MeOH/H2O/AcOH 20 : 10 : 1 : 1);
1H NMR (CD3OD) d 7.42–7.25 (m, 10H, Ar), 4.27 (d, 1H, J ¼ 7.6 Hz, H-1a), 2.89
(dd, 1H, J ¼ 11.2, 3.0 Hz, H-3b-eq), 2.01 (s, 3H, Ac), 1.61 (t, 1H, H-3b-ax), 1.42
(s, 9H, tBu-CH3). MALDI-TOF MS: Calcd for C39H53N5O17 (M): 863.3. Found
m/z: 886.3 (M þ Na)þ. Data for 19c: [a]D þ63.28 (c 0.32, MeOH); Rf 0.36
(CH2Cl2/MeOH/H2O/AcOH 20 : 10 : 1 : 1); 1H NMR (CD3OD) d 7.39–7.25
(m, 10H, Ar), 4.89 (br s, 1H, H-1a), 2.41 (br d, 1H, J ¼ 12.1 Hz, H-3b-eq),
2.03 (s, 3H, Ac), 1.63 (t, 1H, H-3b-ax), 1.44 (s, 9H, tBu-CH3). MALDI-TOF
MS: Calcd for C39H53N5O17 (M): 863.3. Found m/z: 886.3 (M þ Na)þ. Data for
19d: [a]D þ3.48 (c 0.44, MeOH); Rf 0.35 (CH2Cl2/MeOH/H2O/AcOH
20 : 10 : 1 : 1); 1H NMR (CD3OD) d 7.38–7.22 (m, 10H, Ar), 4.27 (d, 1H,
J ¼ 8.9 Hz, H-1a), 2.47 (br d, 1H, J ¼ 12.0 Hz, H-3b-eq), 1.98 (s, 3H, Ac), 1.66
(t, 1H, H-3b-ax), 1.43 (s, 9H, tBu-CH3). MALDI-TOF MS: Calcd for
C39H53N5O17 (M): 863.3. Found m/z: 886.5 (M þ Na)þ.
Typical Procedure for Hydrogenolysis and N-Acetylation Reaction.
AcOH (1 drop) and Pd(OH)2 (15 mg) were added at rt to a solution of
compound 19a (17.9 mg, 0.021 mmol) in 4 : 1 MeOH-H2O (5 mL). The suspen-
sion was stirred under an H2 atmosphere for 12 h. The reaction mixture was
filtered through a pad of celite and the filtrate was concentrated in vacuo.
The residue was treated with pyridine (3 mL) and Ac2O (1.5 mL) at rt for
12 h. The reaction mixture was concentrated in vacuo. The residue was then
treated with 0.05 M NaOMe in MeOH (3 mL) at rt for 3 h, neutralized with
Amberlite IR-120 (Hþ), filtered, and concentrated. The residue was purified
by silica gel column chromatography using CH2Cl2/MeOH/AcOH (40 : 1 : 0.5)
as an eluent to provide 20a (8.0 mg 55%): Data for 20a: [a]D þ44.88 (c 0.30,
H2O); Rf 0.45 (CH2Cl2/MeOH/H2O/AcOH 20 : 10 : 1 : 1); 1H NMR (D2O)
d 4.88 (br s, 1H, H-1a), 2.77 (br d, 1H, J ¼ 12.6 Hz, H-3b-eq), 2.04 (s, 3H, Ac),
2.01 (s, 3H, Ac), 1.66 (t, 1H, H-3b-ax), 1.44 (s, 9H, tBu-CH3). MALDI-TOF
MS: Calcd for C27H45N3O18 (M): 699.3. Found m/z: 622.8 (M þ Na)þ; HR-
FAB MS: Calcd for C27H45N3O18 (M): 699.2698. Found m/z: 722.2593
(M þ Na)þ. Data for 20b: [a]D þ42.18 (c 0.21, H2O); Rf 0.44 (CH2Cl2/MeOH/
H2O/AcOH 20 : 10 : 1 : 1); 1H NMR (D2O) d 4.25 (d, 1H, J ¼ 8.0 Hz, H-1a),
2.75 (dd, 1H, J ¼ 12.2, 3.0 Hz, H-3b-eq), 2.04 (s, 3H, Ac), 2.01 (s, 3H, Ac),
1.65 (t, 1H, H-3b-ax), 1.41 (s, 9H, tBu-CH3). MALDI-TOF MS: Calcd for
C27H45N3O18 (M): 699.3. Found m/z: 722.7 (M þ Na)þ. Data for 20c: [a]D
þ50.38 (c 0.28, H2O); Rf 0.44 (CH2Cl2/MeOH/H2O/AcOH 20 : 10 : 1 : 1); 1H
NMR (D2O) d 4.89 (br s, 1H, H-1a), 2.49 (br d, 1H, J ¼ 12.1 Hz, H-3b-eq),
2.05 (s, 3H, Ac), 2.03 (s, 3H, Ac), 1.70 (t, 1H, H-3b-ax), 1.44 (s, 9 H, tBuCH3).
MALDI-TOF MS: Calcd for C27H45N3O18 (M): 699.3. Found m/z: 722.7
(M þ Na)þ. Data for 20d: [a]D þ10.48 (c 0.10, H2O); Rf 0.40 (CH2Cl2/MeOH/
H2O/AcOH 20 : 10 : 1 : 1); 1H NMR (D2O) d 4.27 (d, 1H, J ¼ 8.5 Hz, H-1a),
2.51 (br d, 1H, J ¼ 12.1 Hz, H-3b-eq), 2.04 (s, 3H, Ac), 2.00 (s, 3H, Ac), 1.66

T. Kanemitsu et al.
374
(t, 1H, H-3b-ax), 1.42 (s, 9H, tBu-CH3). MALDI-TOF MS: Calcd for
C27H45N3O18 (M): 699.3. Found m/z: 722.9 (M þ Na)þ.
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