Synthesis of a hexasaccharide fragment of the capsular polysaccharide of streptococcus pneumoniae type 3

Article abstract of DOI:10.1139/v01-190

In the framework of the development of a new generation of neoglycoconjugate vaccines against Streptococcus pneumoniae, the synthesis is described of a spacer-containing hexasaccharide fragment related to the capsular polysaccharide of S. pneumoniae type

Full text of DOI:10.1139/v01-190

76
Synthesis of a hexasaccharide fragment of the
capsular polysaccharide of Streptococcus
pneumoniae type 3
Dirk J. Lefeber, Eneko Aldaba Arévalo, Johannis P. Kamerling, and
Johannes F.G. Vliegenthart
Abstract: In the framework of the development of a new generation of neoglycoconjugate vaccines against Streptococ-
cus pneumoniae, the synthesis is described of a spacer-containing hexasaccharide fragment related to the capsular
polysaccharide of S. pneumoniae type 3. Hexasaccharide β-D-GlcpA-(1→4)-β-D-Glcp-(1→3)-β-D-GlcpA-(1→4)-β-D-Glcp-
(1→3)-β-D-GlcpA-(1→4)-β-D-Glcp-(1→O-(CH2)3NH2) (1), comprised of three repeating units, was synthesized via a
blockwise strategy employing suitably protected disaccharide building blocks. Carboxylic groups were introduced by
selective oxidation with TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) in the last reaction steps. Deprotection afforded
target hexasaccharide 1.
Key words: oligosaccharide synthesis, Streptococcus pneumoniae type 3, TEMPO oxidation.
Résumé : Dans le cadre du développement d’une nouvelle génération de vaccins néoglycoconjugués actifs contre le
Streptococcus pneumoniae et faisant appel à une stratégie de synthèse par blocs à l’aide de disaccharides protégés de
façon appropriée, on a réalisé la synthèse d’un fragment hexasaccharidique apparenté au polysaccharide capsulaire du
S. pneumoniae de type 3 et contenant un espaceur, soit le β-D-GlcpA-(1→4)-β-D-Glcp-(1→3)-β-D-GlcpA-(1→4)-β-D-
Glcp-(1→3)-β-D-GlcpA-(1→4)-β-D-Glcp-(1→O-(CH2)3NH2) (1) qui trois motifs. Les groupes carboxyliques ont été in-
troduits en procédant à une oxydation sélective à l’aide de TEMPO (2,2,6,6-tétraméthyl-1-pipéridinyloxy) dans les der-
nières étapes réactionnelles. La déprotection fournit l’hexasaccharide recherché, 1.
Mots clés : synthèse d’oligosaccharide, Streptococcus pneumoniae de type 3, oxydation par TEMPO.
[Traduit par la Rédaction] Lefeber et al. 81
Introduction
mutant of diphtheria toxin (CRM197) (4). While immuniza-
tion experiments with these conjugates in mice showed that
increasing IgG antibody titres were found with increasing
oligosaccharide chain length, no difference was found with
respect to the oligosaccharide loading (5).
The use of conjugates that contain larger oligosaccharide
fragments than in the study described above might lead to an
improved immunogenicity. Furthermore, human antibodies
can better recognize larger epitopes on the polysaccharide
than do animal antibodies, as shown for neoglycoproteins
consisting of synthetic oligosaccharides related to the capsular
polysaccharide of S. pneumoniae type 23F (6). For these rea-
sons, a program was started for the synthesis of a
S. pneumoniae type 3 related hexasaccharide fragment (1),
comprised of three cellobiuronic acid repeating units (Fig. 1).
Diseases caused by encapsulated bacteria can be prevented
by vaccination with neoglycoconjugate vaccines as proven for
Haemophilus influenzae type b (1), Neisseria meningitidis
type C (2), and Streptococcus pneumoniae serotypes (3).
These vaccines are prepared by conjugation of an isolated
polysaccharide or a mixture of polysaccharide-derived oligo-
saccharides to a protein carrier. For a detailed investigation
of the immune response to these types of vaccines, neo-
glycoproteins with a well-defined carbohydrate structure
need to be available.
Neoglycoconjugates related to S. pneumoniae type 3 have
been prepared by coupling di-, tri-, or tetrasaccharide frag-
ments in several carbohydrate–protein ratios to a nontoxic
Received 13 July 2001. Published on the NRC Research
Press Web site at http://canjchem.nrc.ca on 21 January 2002.
Results and discussion
Dedicated to the memory of Ray Lemieux, one of the most
inspiring glycoscientists.
The synthesis for tetrasaccharide acceptor 11 was previ-
ously described (4). Two disaccharide donors (6 and 10,
Scheme 1) were tested for the ability to couple with 11 to af-
ford protected hexasaccharide 12 (Scheme 2).
For the synthesis of disaccharide thioethyl donor 6, first
monosaccharide acceptor 4 was prepared by debenzyl-
idenation of 2 (7) using trifluoroacetic acid (→3, 88%) and
subsequent selective benzoylation using benzoyl chloride
D.J. Lefeber, E. Aldaba Arévalo, J.P. Kamerling, and
J.F.G. Vliegenthart.1 Bijvoet Center, Department of Bio-
Organic Chemistry, Utrecht University, P.O. Box 80.075,
NL-3508 TB Utrecht, The Netherlands.
1Corresponding author (fax: (+31) 30 2 540 980; e-mail:
j.f.g.vliegenthart@chem.uu.nl).
Can. J. Chem. 80: 76–81 (2002)
DOI: 10.1139/V01-190
© 2002 NRC Canada

Lefeber et al.
77
Fig. 1. Target hexasaccharide 1 comprising three repeating units of the capsular polysaccharide of S. pneumoniae type 3.
COOH
OH
COOH
OH
COOH
O
OH
HO
HO
O
O
HO
O
O
O
HO
O
O
O
O
HO
O
HO
HO
HO
HO
O
NH2
HO
HO
HO
HO
f
e
d
c
b
a
1
Scheme 1. Synthesis of disaccharide donors 6 and 10. Reagents and conditions: (a) CF3COOH, H2O, CH2Cl2, 88%; (b) PhCOCl, Et3N,
CH2Cl2, 50%; (c) 10% TMSOTf, CH2Cl2, 75%; (d) 11% TMSOTf, CH2Cl2, 83%; (e) (i) (PPh3)3Rh(I)Cl, DABCO, CH3C6H5–EtOH–
CH2Cl2 (10:5:1), (ii) NIS, H2O, THF, 68%; (f) Cl3CCN, DBU, CH2Cl2, 86%.
R2O
R1O
O
c
Ph
OBz
O
O
O
BzO
BzO
SEt
O
BzO
O
BzO
Ph
O
O
BzO
SEt
R2
R1
O
BzO
BzO
O
CCl3
BzO
6
2
3
PhCH
a
HN
5
H
H
H
b
4
Bz
OBz
Ph
OBz
O
O
O
BzO
d
O
O
HO
+
5
O
BzO
BzO
OAll
OAll
BzO
BzO
BzO
8
7
e
Ph
OBz
O
O
O
O
O
BzO
BzO
R1
BzO
BzO
R2
R1
H,OH
H OC(NH)CCl3
R2
9
f
10
Scheme 2. Synthesis of hexasaccharide 1. Reagents and conditions: (a) 10% TMSOTf, CH2Cl2, 63%; (b) CF3COOH, H2O, CH2Cl2,
75%; (c) TEMPO, aqueous NaOCl, KBr, Bu4NBr, aqueous NaCl, aqueous NaHCO3, CH2Cl2, 70%; (d) (i) NaOMe, MeOH (pH 10),
(ii) NaBH4, 10% Pd/C, 0.05 M NaOH, 61%.
Ph
OBz
O
O
Ph
OBz
O
O
O
O
O
10
+
O
O
BzO
HO
O
BzO
O
BzO
O
N3
BzO
BzO
OBz
11
a
Ph
O
BzO
OBz
Ph
O
O
OBz
O
O
O
Ph
OBz
O
O
O
O
O
O
BzO
O
O
BzO
O
O
O
BzO
BzO
BzO
BzO
O
N3
BzO
BzO
OBz
12
b
R
OBz
R
OBz
O
R
OBz
HO
BzO
O
O
HO
O
BzO
O
O
BzO
HO
O
O
O
BzO
O
O
BzO
BzO
BzO
BzO
O
N3
BzO
OBz
R
CH2OH
COOH
13
14
c
d
1
(→4, 50%). Coupling of 4 with imidate donor 5 (4) using
10% TMSOTf as a promoter gave 6 in a yield of 75%. For
the synthesis of disaccharide imidate donor 10, first 8 was
prepared in 83% yield by coupling of acceptor 7 (4) and do-
nor 5 with 11% TMSOTf as a promoter. Then, deallylation
of 8 using tris(triphenylphosphine)rhodium(I) chloride and
N-iodosuccinimide (→9, 68%) and subsequent trichloro-
acetimidation gave 10 (86%).
For the preparation of protected hexasaccharide 12
(Scheme 2), tetrasaccharide acceptor 11 (4) was coupled
© 2002 NRC Canada

78
Can. J. Chem. Vol. 80, 2002
1
Table 1. 500 MHz H NMR data of 1 at 305 K (in ppm).
H-1 H-2 H-3 H-4 H-5 H-6a H-6b
4.50 (7.8) 3.33 3.64 3.61 n.d.a 3.98
were recorded at 27°C with a Bruker AC 300 spectrometer;
indicated ppm values for δ are relative to the signal of
1
CDCl3 (δ = 76.9). The H NMR spectrum of 1 was recorded
Glca
3.80
3.80
3.80
in D2O at 32°C with a Bruker AMX 500 spectrometer, and
the δ values are given in ppm relative to the signal for inter-
nal acetone (δ = 2.225). Two-dimensional TOCSY and
ROESY spectra were recorded using a Bruker AMX 500 ap-
paratus (500 MHz) to assign the spectra of compounds 1, 12,
13, and 14. Residues a–f (Fig. 1) were assigned on the basis
of interglycosidic ROESY cross-peaks. Matrix-assisted laser
desorption ionization time-of-flight (MALDI-TOF) spectra
were obtained on a Voyager-DE™ mass spectrometer using
2,4-dihydroxybenzoic acid (DHB) in H2O as a matrix. Ele-
mental analyses were carried out by H. Kolbe Mikro-
analytisches Laboratorium (Mülheim an der Ruhr, Germany).
GlcAb 4.53 (8.1) 3.56 3.79 n.d.
4.82 (8.0) 3.38 3.65 3.62 n.d.
GlcAd 4.53 (8.0) 3.56 3.79 n.d.
n.d.
Glcc
3.98
3.98
n.d.
Glce
GlcAf
4.82 (8.0) 3.38 3.65 3.62 n.d.
4.50 (8.2) 3.36 3.51 3.51 3.75
Note: The signals for the 3-aminopropyl spacer are: δ = 4.05 and 3.83
(OCH2CH2CH2NH2), 3.16 (OCH2CH2CH2NH2), 2.01 (OCH2CH2CH2NH2).
J1,2 couplings are presented in parentheses.
an.d. = not determined.
with donor 6 using N-iodosuccinimide and triflic acid as a
promoter system. Low yields of product (<8%) were ob-
tained when the reaction was performed at 0°C or lower
temperatures. Hexasaccharide 12 could, however, be ob-
tained in a yield of 63% by coupling 11 with disaccharide
imidate donor 10 using 10% TMSOTf as a promoter at room
temperature. After debenzylidenation of 12 using trifluoro-
acetic acid (→13, 75%), the free primary hydroxyl functions
were oxidized. Treatment of 13 with TEMPO and sodium
hypochlorite (8) was found to be efficient for the oxidation
of the three primary hydroxyl groups in the presence of sec-
Ethyl 2,3-di-O-benzoyl-1-thio-β-D-glucopyranoside (3)
To a solution of ethyl 2,3-di-O-benzoyl-4,6-O-benzylidene-
1-thio-β-D-glucopyranoside (2) (7) (1.37 g, 2.63 mmol) in
CH2Cl2 (36 mL) were added CF3COOH (1.5 mL) and H2O
(0.2 mL). The mixture was stirred for 16 h, diluted with
CH2Cl2, washed with 10% (w/v) aqueous NaHCO3 until
neutral pH and 10% (w/v) aqueous NaCl, and the organic
layer was dried, filtered, and concentrated. The residue was
purified by column chromatography (CH2Cl2–MeOH, 98:2)
to obtain 3 (1.0 g, 88%). [α]2D0 +76 (c = 1). Rf = 0.05
(CH2Cl2–EtOAc, 9:1). 1H NMR (CDCl3) δ: 7.97–7.93 and
7.53–7.34 (2m, 10H, 2PhCO), 4.74 (d, J1,2 = 9.8 Hz, 1H, H-1),
2.76–2.73 (m, 2H, SCH2CH3), 1.26 (t, 3H, SCH2CH3). 13C
NMR (CDCl3) δ: 167.4 and 165.2 (2PhCO); 129.1 and 128.7
(2PhCO, quaternary C); 83.6 (C-1); 79.9, 78.2, 70.1, and
69.9 (C-2–C-5); 62.3 (C-6); 24.3 (SCH2CH3); 14.8
(SCH2CH3). Anal. calcd. for C22H24O7S (432.4): C 61.11,
H 5.59; found C 60.99, H 5.74.
1
ondary functions, and 14 was obtained in 70% yield. H
NMR analysis after methylation with diazomethane showed
three singlets at δ = 3.39, 3.30, and 3.23 (COOCH3).
Debenzoylation of 14 with sodium methoxide and reduction
of the azide using sodium borohydride and 10% Pd/C af-
forded target hexasaccharide 1 in 61% yield. The identity of
the compound was established by 1H NMR spectroscopy
(Table 1) and mass spectrometry.
In conclusion, a versatile synthetic route was developed
using a block wise strategy for the preparation of hexa-
saccharide fragment 1, representing three repeating units of
the capsular polysaccharide of S. pneumoniae type 3. The
carboxylic acids were efficiently introduced by selective oxi-
dation using TEMPO.
Ethyl 2,3,6-tri-O-benzoyl-1-thio-β-D-glucopyranoside (4)
To a solution of 3 (0.95 g, 2.2 mmol) in CH2Cl2 (50 mL)
and Et3N (0.37 mL) at 0°C was added dropwise a solution of
benzoyl chloride (0.27 mL, 2.3 mmol) in CH2Cl2 (10 mL).
After stirring for 24 h, the mixture was diluted with CH2Cl2,
washed with 10% (w/v) aqueous NaCl, and the organic layer
was dried, filtered, and concentrated. The crude residue was
purified by column chromatography (CH2Cl2–EtOAc, 95:5)
to give 4 (0.6 g, 50%). [α]2D0 +60 (c = 1). Rf = 0.51 (CH2Cl2–
Experimental section
General
All chemicals were of reagent grade and were used with-
out any further purification. Reactions were monitored by
TLC on Silica gel 60 F254 (Merck); compounds were visu-
alized, after examination under UV light, by heating with
10% (v/v) ethanolic H2SO4, orcinol (2 mg mL–1) in 20%
(v/v) methanolic H2SO4, or ninhydrin (1.5 mg mL–1) in
BuOH–H2O–HOAc (38:1.75:0.25). In the work-up proce-
dures of the reaction mixtures, the organic solutions were
washed with appropriate amounts of the indicated aqueous
solutions, then dried (MgSO4), and concentrated under re-
duced pressure at 20–40°C on a water bath. Column chro-
matography was performed on Silica gel 60 (Merck, 0.063–
0.200 mm). Optical rotations were measured in CHCl3, un-
less stated otherwise, with a PerkinElmer 241 polarimeter,
1
EtOAc, 9:1). H NMR (CDCl3) δ: 8.05–7.91 and 7.46–7.28
(2m, 15H, 3PhCO), 5.60 and 5.47 (2t, each 1H, H-2,3), 4.82
(d, J1,2 = 9.9 Hz, 1H, H-1), 2.82–2.64 (m, 2H, SCH2CH3),
1.24 (t, 3H, SCH2CH3). 13C NMR (CDCl3) δ: 166.8, 166.7,
and 165.2 (3PhCO); 83.4 (C-1); 78.2, 77.2, 70.2, and 69.3
(C-2–C-5); 63.6 (C-6); 24.1 (SCH2CH3); 14.8 (SCH2CH3).
Anal. calcd. for C29H28O8S (536.5): C 64.92, H 5.26; found
C 65.05, H 5.31.
Ethyl (2,3-di-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl)-(1→4)-2,3,6-tri-O-benzoyl-1-thio-β-D-
glucopyranoside (6)
A solution of 4 (50 mg, 0.09 mmol) and 2,3-di-O-benzoyl-
4,6-O-benzylidene-D-glucopyranosyl trichloroacetimidate 5
(4) (87 mg, 0.14 mmol) in dry CH2Cl2 (2 mL), containing
4 Å molecular sieves, was stirred under Ar for 1 h. Then,
TMSOTf (2.5 µL, 14 µmol) was added. After 30 min, the
1
using a 10 cm, l mL cell. H NMR spectra in CDCl3 were
recorded at 27°C with a Bruker AC 300 spectrometer; the δ
values are given in ppm relative to the signal for internal
Me4Si (δ = 0). 13C (APT, 75 MHz) NMR spectra in CDCl3
© 2002 NRC Canada

Lefeber et al.
79
mixture was neutralized with dry pyridine, filtered over
cotton, diluted with CH2Cl2, and washed with 10% (w/v)
aqueous NaCl. The organic layer was dried, filtered, and
concentrated. The residue was purified by column chroma-
tography (toluene–EtOAc, 95:5) to obtain 6 (67 mg, 75%).
aqueous NaHSO3 (2×) and 10% (w/v) aqueous NaCl, and
the organic layer was dried, filtered, and concentrated. The
crude residue was purified by column chromatography. Im-
purities were eluted with toluene–EtOAc (95:5), and 9 was
obtained as a light brown syrup by elution with toluene–
EtOAc (9:1) (0.99 g, 68%). Rf = 0.17α/0.08β (toluene–
1
[α]2D0 +41 (c = 1). Rf = 0.76 (CH2Cl2–EtOAc, 9:1). H NMR
1
(CDCl3) δ: 8.05–7.85 and 7.49–7.31 (2m, 30H, PhCH,
5PhCO), 5.74 and 5.61 (2t, J1,2 = 9.9 Hz, each 1H, H-2,3),
5.44 (dd, J1′,2′ = 7.7, J2′,3′ = 9.5 Hz, 1H, H-2′), 5.42 (t, 1H,
H-3′), 5.20 (s, 1H, PhCH), 4.83 (d, 1H, H-1′), 4.68 (d, 1H,
H-1), 4.49 (dd, J5′,6a′ = 2.1, J6a′,6b′ = 12.2 Hz, 1H, H-6a), 4.40
(dd, J5′,6b′ = 4.8 Hz, 1H, H-6b), 2.70-2.56 (m, 2H,
SCH2CH3), 1.16 (t, 3H, SCH2CH3). 13C NMR (CDCl3) δ:
136.4 (PhCH, quaternary C); 101.7 and 101.1 (PhCH, C-1′);
83.3 (C-1); 67.5 (C-6′); 62.5 (C-6); 24.2 (SCH2CH3); 14.8
(SCH2CH3). Anal. calcd. for C56H50O15S (995.0): C 67.59,
H 5.06; found C 67.56, H 5.01.
EtOAc, 8:2). H NMR (CDCl3) δ: 8.08–7.85 and 7.49–7.23
(2m, 30H, PhCH, 5PhCO), 5.63 (t, 1H, H-3′), 5.46 (dd,
J1′,2′ = 7.7, J2′,3′ = 9.5 Hz, 1H, H-2′), 5.23 (s, 1H, PhCH),
4.89 (d, 1H, H-1′), 4.49 (dd, J5,6a = 2.0, J6a,6b = 12.3 Hz, 1H,
H-6a), 4.40 (dd, J5,6b = 3.8 Hz, 1H, H-6b), 3.63 (dd, J5′,6a′
=
4.9, J6a′,6b′ = 10.6 Hz, 1H, H-6a′), 3.32 (dt, J5′,6b′ = 10.4 Hz,
1H, H-5′), 2.94 (t, 1H, H-6b′). 13C NMR (CDCl3) δ: 165.8,
165.4, 165.0 (2C), and 164.9 (5PhCO); 136.5 (PhCH, qua-
ternary C); 101.9 and 101.1 (PhCH, C-1′); 95.5 and 90.1
(C-1α,1β); 67.7 (C-6′); 62.0 (C-6). Anal. calcd. for
C54H46O16 (951.0): C 68.21, H 4.87; found C 68.16, H 5.02.
Allyl (2,3-di-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl)-(1→4)-2,3,6-tri-O-benzoyl-β-D-
glucopyranoside (8)
(2,3-Di-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranosyl)-
(1→4)-2,3,6-tri-O-benzoyl-α-D-glucopyranosyl
trichloroacetimidate (10)
A solution of allyl 2,3,6-tri-O-benzoyl-β-D-glucopyranoside
7 (4) (0.48 g, 0.93 mmol) and 2,3-di-O-benzoyl-4,6-O-
To a solution of 9 (1.0 g, 1.1 mmol) in dry CH2Cl2
(14 mL) were added Cl3CCN (1.2 mL) and DBU (40 µL).
After stirring for 16 h, the mixture was concentrated and the
residue was purified by column chromatography (toluene–
EtOAc, 88:12) to yield 10 (0.99 g, 86%). [α]2D0 +83 (c = 1).
Rf = 0.32 (toluene–EtOAc, 9:1). 1H NMR (CDCl3) δ: 8.55 (s,
1H, NH), 8.08–7.85 and 7.47–7.16 (2m, 30H, PhCH,
benzylidene-D-glucopyranosyl trichloroacetimidate
5 (4)
(1.02 g, 1.64 mmol) in dry CH2Cl2 (15 mL), containing 4 Å
molecular sieves, was stirred under Ar for 1 h. Then,
TMSOTf (35 µL, 0.19 mmol) was added. After 45 min, the
mixture was neutralized with dry pyridine, filtered over cot-
ton, diluted with CH2Cl2, and washed with 10% (w/v) aque-
ous NaCl. The organic layer was dried, filtered, and
concentrated. The residue was purified by column chroma-
tography (toluene–EtOAc, 95:5) to obtain 8 (0.76 g, 83%).
5PhCO), 6.68 (d, J1,2 = 3.7 Hz, 1H, H-1), 6.12 (dd, J2,3
=
10.2, J3,4 = 8.6 Hz, 1H, H-3), 5.66 (t, J2′,3′ = J3′,4′ = 9.6 Hz,
1H, H-3′), 5.49 (dd, 1H, H-2), 5.48 (dd, J1′,2′ = 7.7 Hz, 1H,
H-2′), 5.25 (s, 1H, PhCH), 4.95 (d, 1H, H-1′), 4.54 (dd,
1
[α]2D0 +27 (c = 1). Rf = 0.37 (toluene–EtOAc, 9:1). H NMR
J5,6a = 2.0, J6a,6b = 12.2 Hz, 1H, H-6a), 4.44 (dd, J5,6b =
(CDCl3) δ: 8.05–7.85 and 7.50–7.25 (2m, 30H, PhCH,
5PhCO), 5.78–5.65 (m, 1H, OCH2CH=CH2), 5.72 and 5.60
(2t, J2,3 = 9.6, J2′,3′ = 9.6 Hz, each 1H, H-3,3′), 5.43 and
5.40 (2dd, J1,2 = 7.7, J1′,2′ = 7.8 Hz, each 1H, H-2,2′), 5.21
(s, 1H, PhCH), 5.17–5.10 and 5.08–5.03 (2m, each 1H,
OCH2CH=CH2), 4.83 and 4.72 (2d, each 1H, H-1,1′), 4.49
3.8 Hz, 1H, H-6b), 4.30 (ddd, J4,5 = 10.0 Hz, 1H, H-5), 4.22
(dd, 1H, H-4), 3.69 (t, J4′,5′ = 9.5 Hz, 1H, H-4′), 3.64 (dd,
J5′,6a′ = 4.8, J6a′,6b′ = 10.5 Hz, 1H, H-6a′), 3.34 (dt, J5′,6b′
=
10.3 Hz, 1H, H-5′), 2.99 (t, 1H, H-6b′). 13C NMR (CDCl3)
δ: 165.4, 165.3 (2C), and 164.8 (2C) (5PhCO); 160.3
(OC(NH)CCl3); 136.4 (PhCH, quaternary C); 101.8 and
101.1 (PhCH, C-1′); 92.8 (C-1), 67.6 (C-6′); 61.6 (C-6).
(dd, J5,6a = 2.1, J6a,6b = 12.1 Hz, 1H, H-6a), 4.39 (dd, J5,6b
=
4.5 Hz, 1H, H-6b), 4.27–4.20 and 4.07–3.99 (2m, each 1H,
OCH2CH=CH2), 4.11 and 3.63 (2t, each 1H, H-4,4′), 3.76
(ddd, 1H, H-5), 3.61 (dd, J5′,6a′ = 4.6, J6a′,6b′ = 10.6 Hz, 1H,
H-6a′), 3.31 (ddd, 1H, H-5′), 2.83 (t, J5′,6b′ = 10.6 Hz, 1H,
H-6b′). 13C NMR (CDCl3) δ: 165.4, 165.2, 165.0, 164.9, and
164.7 (5PhCO); 136.4 (PhCH, quaternary C); 117.5
(OCH2CH=CH2); 101.7, 101.0, and 99.2 (PhCH, C-1,1′);
69.7 (OCH2CH=CH2); 67.4 (C-6′); 62.2 (C-6). Anal. calcd.
for C57H50O16 (991.0): C 69.08, H 5.08; found C 69.16,
H 5.05.
3-Azidopropyl (2,3-di-O-benzoyl-4,6-O-benzylidene-β-D-
glucopyranosyl)-(1→4)-(2,3,6-tri-O-benzoyl-β-D-
glucopyranosyl)-(1→3)-(2-O-benzoyl-4,6-O-benzylidene-β-
D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-benzoyl-β-D-
glucopyranosyl)-(1→3)-(2-O-benzoyl-4,6-O-benzylidene-β-
D-glucopyranosyl)-(1→4)-2,3,6-tri-O-benzoyl-β-D-
glucopyranoside (12)
A solution of 10 (70 mg, 63 µmol) and 11 (4) (65 mg,
35 µmol) in dry CH2Cl2 (1 mL), containing 4 Å molecular
sieves, was stirred under Ar for 1 h. Then, TMSOTf (1.1 µL,
6 µmol) was added. After 30 min, the mixture was neutral-
ized with dry pyridine, filtered over cotton, diluted with
CH2Cl2, and washed with 10% (w/v) aqueous NaCl. The or-
ganic layer was dried, filtered, and concentrated. The residue
was purified by column chromatography (toluene–EtOAc,
95:5) to obtain 12 (59 mg, 63%). [α]2D0 +59 (c = 1). Rf = 0.46
(toluene–EtOAc, 9:1). 1H NMR (CDCl3) δ: 5.56 (t, 1H, H-3a),
5.45 (t, 1H, H-3f), 5.38 (t, 1H, H-3e), 5.29 (t, 1H, H-3c),
5.26 (2H, H-2a,2f), 5.16 (1H, H-2e), 5.15 (1H, H-2b),
5.14 (1H, H-2c), 5.12 (2H) and 5.09 (2s, 3PhCH), 5.06
(2,3-Di-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranosyl)-
(1→4)-2,3,6-tri-O-benzoyl-D-glucopyranose (9)
To a solution of 8 (1.5 g, 1.5 mmol) in absolute EtOH
(40 mL), toluene (80 mL), and CH2Cl2 (8 mL) were added a
catalytic amount of diazabicyclo[2.2.2]octane (DABCO) and
tris(triphenylphosphine)rhodium(I) chloride (0.4 g). After
stirring at reflux for 4 h, the mixture was concentrated. The
residue was dissolved in THF (75 mL), and water (10 mL)
and NIS (0.6 g) were added. After 20 min, the mixture was
concentrated, diluted with CH2Cl2, washed with 10% (w/v)
© 2002 NRC Canada

80
Can. J. Chem. Vol. 80, 2002
(1H, H-2d), 4.67 (d, J1,2 = 7.8 Hz, 1H, H-1e), 4.64 (d, J1,2
=
tered, and concentrated. The residue was purified by column
chromatography. Impurities were eluted with CH2Cl2–ace-
tone (8:2), and 14 (35 mg, 70%) was eluted with CH2Cl2–
acetone–HOAc, (8:2:0.5). Rf = 0.05 (CH2Cl2–acetone–
HOAc, 8:2:1). MALDI-TOF-MS m/z: 2490 [M + Na]+, 2512
[M – H + 2Na]+, 2534 [M – 2H + 3Na]+, 2556 [M – 3H +
4Na]+.
7.8 Hz, 1H, H-1c), 4.59 (d, J1,2 = 7.8 Hz, 1H, H-1f), 4.54 (d,
J1,2 = 7.8 Hz, 1H, H-1a), 4.50 (d, J1,2 = 7.8 Hz, 1H, H-1b),
4.34 (d, J1,2 = 7.8 Hz, 1H, H-1d), 3.97 (t, 1H, H-4e), 3.90 (t,
2H, H-4a,3b), 3.85 (t, 1H, H-3d), 3.84 (t, 1H, H-4c), 3.77
and 3.45 (OCH2CH2CH2N3), 3.55 (H-5a), 3.25 (H-5e), 3.21
(H-5c), 3.12 (OCH2CH2CH2N3), 3.05 (H-5b), 2.93 (H-5d),
2.66 (H-6f), 2.65 (H-6b), 2.53 (H-6d), 1.72-1.57 (m, 2H,
OCH2CH2CH2N3). 13C NMR (CDCl3) δ: 136.4 (PhCH,
quaternary C); 101.4, 101.3 (2C), 101.1, 100.9 (2C),
100.6, 99.8, and 99.7 (3PhCH, C-1a,1b,1c,1d,1e,1f); 66.2
A small amount was methylated with diazomethane in
methanol for analysis. 1H NMR (CDCl3) δ: 5.55 (t, 1H, H-3e),
5.54 (t, 1H, H-3a), 5.39 (t, 1H, H-3c), 5.36 (2H, H-2f,3f),
5.26 (dd, J1,2 = 7.8, J2,3 = 9.6 Hz, 1H, H-2e), 5.20 (dd, J1,2
=
7.8, J2,3 = 9.5 Hz, 1H, H-2a), 5.15 (dd, J2,3 = 9.7 Hz, 1H, H-2c),
5.08 (2t, each 1H, H-2b,2d), 4.83 (d, J1,2 = 7.6 Hz, 1H, H-1f),
(OCH2CH2CH2N3);
47.6
(OCH2CH2CH2N3);
28.7
(OCH2CH2CH2N3). MALDI-TOF-MS m/z: 2713 [M + Na]+.
4.65 (d, 1H, H-1e), 4.61 (dd, 1H, H-6e), 4.53 (d, J1,2
8.0 Hz, 2H, H-1b,1c), 4.52 (d, 1H, H-1a), 4.48 (d, J1,2
=
=
3-Azidopropyl (2,3-di-O-benzoyl-β-D-glucopyranosyl)-
(1→4)-(2,3,6-tri-O-benzoyl-β-D-glucopyranosyl)-(1→3)-(2-
O-benzoyl-β-D-glucopyranosyl)-(1→4)-(2,3,6-tri-O-benzoyl-
β-D-glucopyranosyl)-(1→3)-(2-O-benzoyl-β-D-
glucopyranosyl)-(1→4)-2,3,6-tri-O-benzoyl-β-D-
glucopyranoside (13)
7.9 Hz, 1H, H-1d), 4.38 (dd, 1H, H-6aa), 4.33 (dd, 1H, H-6ac),
4.28 (dd, 1H, H-6be), 4.17 (dd, 1H, H-6ba), 4.13 (dd, 1H,
H-6bc), 4.10 (t, 1H, H-4e), 4.04 (t, 1H, H-4a), 3.91 (H-4f),
3.90 (t, 1H, H-4c), 3.81 (H-5e), 3.78 and 3.44 (OCH2
CH2CH2N3), 3.76 (H-4d), 3.74 (H-4b), 3.69 (H-5f), 3.63 (t,
1H, H-3d), 3.57 (t, 1H, H-3b), 3.56 (H-5c), 3.55 (H-5a),
3.51 (d, J4,5 = 9.6 Hz, 1H, H-5d), 3.49 (d, J4,5 = 9.6 Hz,
1H, H-5b), 3.39, 3.30, and 3.23 (3s, each 3H, COOCH3), 3.09
(OCH2CH2CH2N3), 1.70–1.59 (m, 2H, OCH2CH2CH2N3).
To a solution of 12 (84 mg, 31 µmol) in CH2Cl2 (0.85 mL)
were added CF3COOH (75 µL) and H2O (75 µL). The mix-
ture was stirred for 4 h, diluted with CH2Cl2, washed with
10% (w/v) aqueous NaHCO3 until neutral pH and 10% (w/v)
aqueous NaCl, and the organic layer was dried, filtered, and
concentrated. The residue was purified by column chroma-
tography (toluene–EtOAc, 6:4→4:6) to obtain 13 (55 mg,
3-Aminopropyl (β-D-glucopyranosyluronic acid)-(1→4)-(β-
D-glucopyranosyl)-(1→3)-(β-D-glucopyranosyluronic acid)-
(1→4)-(β-D-glucopyranosyl)-(1→3)-(β-D-glucopyranosyluronic
acid)-(1→4)-β-D-glucopyranoside (1)
1
75%). [α]2D0 +45 (c = 1). Rf = 0.21 (toluene–EtOAc, 1:1). H
NMR (CDCl3) δ: 5.57 (t, 1H, H-3e), 5.50 (t, 1H, H-3a), 5.39
(t, 1H, H-3c), 5.33 (dd, 1H, H-2f), 5.31 (dd, 1H, H-2a), 5.30
(dd, 1H, H-2e), 5.25 (t, 1H, H-3f), 5.23 (dd, 1H, H-2c), 5.03
(br t, 2H, H-2b,2d), 4.76 (d, J1,2 = 7.7 Hz, 1H, H-1f), 4.63
(2H, H-1e,6e), 4.53 (d, 1H, H-1a), 4.52 (d, 1H, H-1c), 4.43 (d,
J1,2 = 8.0 Hz, 1H, H-1b), 4.42 (d, J1,2 = 8.0 Hz, 1H, H-1d),
4.31 (3H, H-6a,6ac,6ae), 4.18 (dd, 1H, H-6ba), 4.14 (dd, 1H,
H-6bc), 3.99 (t, 1H, H-4e), 3.98 (t, 1H, H-4a), 3.83 (t, 1H,
H-4c), 3.82 (H-5e), 3.78 and 3.45 (OCH2CH2CH2N3), 3.73
(H-4f), 3.58 (t, 1H, H-3d), 3.56 (H-5c), 3.54 (H-5a), 3.51 (t,
1H, H-3b), 3.36 (H-4d), 3.32 (H-4b), 3.23 (H-5f), 3.15–3.09
(m, 2H, OCH2CH2CH2N3), 3.00 (H-5d), 2.96 (H-5b), 1.72–
1.55 (m, 2H, OCH2CH2CH2N3). 13C NMR (CDCl3) δ: 101.1
(2C), 100.6 (3C), and 100.5 (C-1a,1b,1c,1d,1e,1f); 66.2
(OCH2CH2CH2N3); 62.2, 62.1, 62.0 (2C), 61.8, and 61.3
(C-6a,6b,6c,6d,6e,6f); 47.6 (OCH2CH2CH2N3); 28.7
(OCH2CH2 CH2N3). MALDI-TOF-MS m/z: 2440 [M + Na]+.
To a solution of 14 (20 mg, 8 µmol) in MeOH (3 mL) was
added NaOMe until pH 10, and after 5 h, water (1 mL) was
added. After stirring for 16 h, TLC (EtOAc–MeOH–H2O,
12:5:3) showed the formation of a new spot (Rf = 0.1), and
the mixture was neutralized with Dowex H+, filtered, and
concentrated. A solution of the residue in water was washed
with CH2Cl2 (3×), and the aqueous layer was concentrated.
Then, a solution of the residue in 0.1 M NaOH (0.4 mL) was
added dropwise to a suspension of 10% Pd/C (0.8 mg) and
NaBH4 (3.5 mg) in doubly distilled water (0.3 mL). After
2 h, when TLC (EtOAc–MeOH–H2O, 12:5:3) showed the
formation of a ninhydrin-positive spot on the baseline, the
mixture was acidified with Dowex H+ (pH 4), then loaded
on a short column of Dowex 50 W × 2 (H+, 200–400 mesh).
After elution of contaminants with water, elution with 1%
NH4OH afforded 1 after concentration and lyophilization
from water (2×) (5.4 mg, 61%). [α]2D0 = –17 (c = 0.4, H2O).
MALDI-TOF-MS m/z: 1091 [M + H]+, 1112 [M + Na]+,
3-Azidopropyl (2,3-di-O-benzoyl-β-D-glucopyranosyluronic
acid)-(1→4)-(2,3,6-tri-O-benzoyl-β-D-glucopyranosyl)-
(1→3)-(2-O-benzoyl-β-D-glucopyranosyluronic acid)-
(1→4)-(2,3,6-tri-O-benzoyl-β-D-glucopyranosyl)-(1→3)-(2-
O-benzoyl-β-D-glucopyranosyluronic acid)-(1→4)-2,3,6-tri-
O-benzoyl-β-D-glucopyranoside (14)
To a solution of 13 (50 mg, 21 µmol) in CH2Cl2 (0.22 mL)
were added TEMPO (catalytic amount), and 40 µL of a solu-
tion of KBr (2.4 mg) and Bu4NBr (3.2 mg) in saturated
aqueous NaHCO3 (0.4 mL). The mixture was stirred vigor-
ously at 0°C, when 122 µL (total) of a solution of saturated
aqueous NaCl (0.44 mL), saturated aqueous NaHCO3
(0.22 mL), and aqueous NaOCl (13% Cl active, 0.56 mL)
was added dropwise. After 45 min, the mixture was acidified
with 4 M HCl (pH 4), diluted with CH2Cl2, and the organic
layer was washed with 10% (w/v) aqueous NaCl, dried, fil-
1
1134 [M – H + 2Na]+, 1156 [M – 2H + 3Na]+. For H NMR
data, see Table 1.
Acknowledgments
This work was supported by the European Union program
VACNET (grant ERB BIO 4CT960158).
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