Synthesis of a disialylated hexasaccharide of Type VIII Group B Streptococcus capsular polysaccharide

Article abstract of DOI:10.1016/S0008-6215(99)00103-2

As part of our program to design, develop and prepare protective vaccines against the bacterial pathogens Group B Streptococcus, we report the synthesis of a disialylated hexasaccharide. This hexasaccharide represents a portion of the serotype-specific ca

Full text of DOI:10.1016/S0008-6215(99)00103-2

www.elsevier.nl/locate/carres
Carbohydrate Research 319 (1999) 1–16
Synthesis of a disialylated hexasaccharide of Type VIII
Group B Streptococcus capsular polysaccharide
Eva Eichler, Harold J. Jennings, Michel Gilbert, Dennis M. Whitfield *
National Research Council, 100 Sussex Dri6e, Ottawa, Ontario, K1A 0R6 Canada
Received 28 January 1999; accepted 18 April 1999
Abstract
As part of our program to design, develop and prepare protective vaccines against the bacterial pathogens Group
B Streptococcus, we report the synthesis of a disialylated hexasaccharide. This hexasaccharide represents a portion of
the serotype-specific capsular polysaccharide of Type VIII that has the tetrasaccharide repeat unit {b-
L
-Rhap-(1“4)-
b-D
-Glcp-(1“4)-[a-Neu5Ac-(2“3)]-b-
D
-Galp-(1“4)}n. A tetrasaccharide corresponding to this repeat unit has
been synthesized by us [E. Eichler, H.J. Jennings, D.M. Whitfield, J. Carbohydr. Chem., 16 (1997) 385–411]. Since
the protective epitopes are believed to involve several repeat units, methods to extend this tetrasaccharide were
examined. This objective requires a glycosylation of the unreactive OH-4 of the b-
by coupling a -Galp glycosyl trichloroacetimidate donor with a b- -Rhap-(1“4)-
coupling of this trisaccharide as a donor to an a-Neu5Ac-(2“3)- -Galp disaccharide acceptor gave a pentasaccha-
ride. The pentasaccharide was deprotected and enzymatically sialylated using an a-(2“3)-sialyltransferase from
Campylobacter jejuni to give the title hexasaccahride a-Neu5Ac-(2“3)-b- -Galp-(1“4)-b- -Rhap-(1“4)-b- -Glcp-
(1“4)-[a-Neu5Ac-(2“3)]-b- -Galp-(1“O)-(CH2)3N3. © 1999 Elsevier Science Ltd. All rights reserved.
L
-Rhap, which was accomplished
D
L
D
-Glcp acceptor. Subsequent
D
D
L
D
D
Keywords: Oligosaccharide synthesis; Sialyltransferase; Chemoenzymatic; Group B Streptococcus
1. Introduction
Japan where it is now a prevalent strain [3].
From our experience with other serotypes, it is
anticipated that the protective antigen will be
a conformational epitope that consists of at
least two repeating units [4]. Previously we
have synthesized tetrasaccharides that corre-
spond to the repeating unit [5]. In order to
extend such tetrasaccharides, it was necessary
As part of our institute’s program to design,
develop and prepare protective vaccines
against bacterial pathogens, we report the syn-
thesis of a disialylated hexasaccharide. This
hexasaccharide represents a portion of the
serotype-specific capsular polysaccharide of
Group B Streptococcus (GBS) Type VIII that
to form the b-
This objective requires the glycosylation of the
unreactive OH-4 of the -Rhap. The b-
Galp-(1“4)-a- -Rhap bond has been success-
D
-Galp-(1“4)-b- -Rhap bond.
L
has the tetrasaccharide repeat unit {b-
(1“4)-b- -Glcp-(1“4)-[a-Neu5Ac-(2“3)]-
b- -Galp-(1“4)}n (Fig. 1) [1]. GBS has long
L
-Rhap-
D
L
D-
D
L
been recognized as a major cause of neonatal
sepsis and meningitis [2]. Type VIII has been
identified among disease-causing isolates in
fully synthesized to form a disaccharide [6,7].
This work describes the successful achieve-
ment of this goal and the subsequent elabora-
tion of the molecule into disialylated hexa-
saccharides 1 and 2.
* Corresponding author. Tel.: + 1-613-993-5265; fax: +1-
613-952-9092.
0008-6215/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(99)00103-2

2
E. Eichler et al. / Carbohydrate Research 319 (1999) 1–16
Rhap OH-4, had been developed in the design
stage of the synthesis (see Scheme 1). The
good yield of this glycosylation contrasted
greatly with the previously unsuccessful results
with tetrasaccharide donors.
Since trisaccharide 6 has an alkylthio sub-
stituent at its reducing termini, it was easily
activated under NIS/TfOH conditions [13,14].
Thus, 6 was glycosylated with the linkers 2-
trimethylsilylethanol [15,16] or 3-azidopropan-
1-ol [17] to give the trisaccharides 7 and 9 (see
Scheme 2). The acetates were cleaved by mild
treatment with sodium methoxide in methanol
to yield the triols 8 and 10. Subsequent at-
tempts to sialylate these triols with the sialyl
donor 11 [18,19] did lead to the expected O-3
regioselectivity, but the resulting tetrasacchar-
ides 12 and 13 were isolated as inseparable
mixtures of a,b anomers (see Scheme 3). This
contrasts with the high stereoselectivity ob-
served in the formation of disaccharide 14 (see
below) under very similar conditions. The fac-
tors that control these differences are not
known.
Fig. 1. Tetrasaccharide repeat unit of Group B Streptococcus
(GBS) Type VIII.
2. Results and discussion
Our initial strategy was to use the tetrasac-
charide donor we had developed in the previ-
ous synthesis to glycosylate the OH-4 of the
L
-Rhap. However, this could not be accom-
plished, so several alternate routes were exam-
ined. Thus, known trichloroacetimidate
D
-Galp donor 3 [8] was reacted with b-
L
-
Rhap-(1“4)- -Glcp disaccharide 5 under
D
promotion with triethylsilyl trifluoromethane-
sulfonate [9,10] to yield trisaccharide 6 in 67%
isolated yield. The OH-4 of 5 was liberated
from known disaccharide 4 by carefully con-
trolled methanolysis with 2% HCl in methanol
[11,12]. The protecting group pattern of 4,
The direction of chain extension was there-
fore reversed, and trisaccharide 6 was reacted
with known disaccharide diol 14 [5,20] to yield
pentasaccharide 15 with high regioselectivity
and stereoselectivity (see Scheme 4). An im-
which allows for chain extension at the
L
-
Scheme 1.

E. Eichler et al. / Carbohydrate Research 319 (1999) 1–16
3
1
added after conversion to the hemiacetal 20
and activation as the trichloroacetimidate 21
to give 22 in an overall yield of 37% from 15.
Then, 22 was deprotected to yield 23 (see
Scheme 6). Both 18 and 23 were completely
characterized by 1H and 13C NMR spec-
troscopy in D2O. The assignments of the 1D
pure fraction with H NMR resonances simi-
lar to those of 15 was isolated, which by
analogy to previous results is likely to be the
1,2-isomer 16. The 1,4 regiochemistry of 15
follows from observation of
a
COSY
crosspeak between the OH-2II and H-2II at lH
2.25 and 3.55 ppm, respectively. After hydro-
genation to cleave the O-benzyl protecting
groups, cf. 17, the O-acyl groups were cleaved
under Zemple´n conditions, followed by basic
hydrolysis of the methyl ester, to give the
deprotected pentasaccharide 18 (see Scheme
5). Alternatively 17 was reacetylated to give
19. Then, the linker, 3-azido-propan-1-ol, was
1
1H and 13C spectra are based on 2D H-1H
13
1
COSY, 2D C-1H COSY, 2D H-1H ROESY,
2D H-1H TOCSY and 1D-selTOCSY experi-
1
ments at 600.1 and 150.9 MHz, respectively
(see Tables 1 and 2). The resonances of 18 and
23 resembled those of the tetrasaccharide
without
D
-GalpV except for the resonances of
Scheme 2.
Scheme 3.

4
E. Eichler et al. / Carbohydrate Research 319 (1999) 1–16
Scheme 4.
L
-Rhap H-4IV, H-5IV, C-4IV and C-3IV. These
with water on a Bio-Rad P-2 gel permeation
column. Hexasaccharide 1 was repurified on a
changes reflect the successful glycosylation of
L
-Rhap O-4IV. (For the I, II to VI nomencla-
C18 reversed-phase Sep-Pak, and only enough
1
pure compound to characterize by H NMR
ture see Section 3 and Scheme 7.)
spectroscopy was isolated. For hexasaccharide
2 the fractions from the P-2 column were
nearly pure (see Fig. 2), and the yield from 23
was nearly quantitative. The 1H and 13C NMR
spectra in D2O of 2 could be completely as-
signed (see Table 1). A comparison of the
resonances of the Neu5AcI attached to the
In order to install the terminal sialic acid of
1 and 2, pentasaccharides 18 and 23 were
enzymatically glycosylated using b-Neu5Ac-
(2“CMP) as donor as catalyzed by a recently
cloned and expressed a-Neu5Ac-(2“3)-trans-
ferase from C. jejuni (M. Gilbert, A.-M
Cunningham,
M.F.
Karwaski,
W.W.
reducing
D
-GalpII in 2 versus 23 (Neu5AcI)
Wakarchuk, unpublished observations). As
judged by TLC, the reactions went to comple-
tion to yield 1 and 2 (see Scheme 7). It should
be noted that the a-Neu5Ac-(2“3)-trans-
ferase from Neisseria meningitidis was unable
to catalyze this transformation. This trans-
ferase sialylates a number of Galp derivatives
[21]. In fact, to the best of our knowledge, this
is the first observation of the transfer of
shows only small changes. The biggest changes
are for H-4I and C-4I, possibly suggesting a
small reorientation of this part of the
molecule.
Thus, a hexasaccharide representing one-
and-a-half repeat units of the capsular polysac-
charide of Group B Streptococcus (GBS) Type
VIII has been synthesized and characterized.
The combined chemoenzymatic synthesis
opens the way to efficiently prepare multiple
repeat units of this polysaccharide.
Neu5Ac to a terminal
D
-Galp linked to an
L
-Rhap. The products were purified by eluting

E. Eichler et al. / Carbohydrate Research 319 (1999) 1–16
5
Scheme 5.
3. Experimental
NMR spectra to the central peak of CDCl3
77.0 ppm. In D2O spectra were referenced to
internal acetone at 2.225 and 31.55 ppm, for
1H and 13C NMR spectra, respectively. As-
signments were made by standard 1H-1H-
General methods.—Optical rotations were
measured (u=589 nm) at room temperature
(rt) using a Perkin–Elmer 243 polarimeter in a
1
13
10-cm 1-mL cell. The H and C NMR spec-
tra were recorded at 500.1 and 125 MHz or
600.1 and 150.9 MHz on Bruker or Varian
spectrometers, respectively, in either CDCl3 or
COSY
and
1H-coupled
13C-1H-COSY
experiments. For 1, 2, 18, and 23, additional
1
2D gradient-enhanced H, decoupled 13C-1H-
COSY, 1H-1H-TOCSY,1H-1H-ROESY and
1
1
D2O. H NMR spectra in CDCl3 were refer-
1D gradient-enhanced H sel TOCSY mea-
13
enced to residual CHCl3 at 7.24 ppm, and C
surements were made at 298 K using standard

6
E. Eichler et al. / Carbohydrate Research 319 (1999) 1–16
Scheme 6.
Varian pulse sequences. The results are pre-
sented in Table 1 (1H) and 2 (13C). For NMR
assignments, all carbohydrate residues in
products larger than monosaccharides have
been designated as follows: rhamnose IV, glu-
cose III, galactose II, nonreducing terminal
galactose V, neuraminic acid I and non-reduc-
ing terminal neuraminic acid VI. The mass
spectral analysis was done on a Jeol JMS-
AX505-H forward mass spectrometer. Fast-
atom bombardment mass spectrometry
(FABMS) was performed using xenon atoms
at 6 kV as the source. Thioglycerol or a
mixture of glycerol and thioglycerol was used
as the FAB matrix. Typically 10–15 full-
range, low-resolution MS scans were averaged
to yield a low-resolution mass spectrum. For
high-resolution MS, the electric sector was
scanned over the range of interest. Typically
polyethylene glycol or polypropylene glycol
was used as an internal mass standard, and
between 75 and 150 scans were averaged. The
electrospray-ionization mass spectra (ESIMS)
were recorded on a Fisons VG-Quattro spec-
trometer in the loop injection mode using 1:1
acetonitrile–water with 0.4% acetic acid as
matrix in the positive-ion mode. MALDIMS
spectra were taken on a Voyager-De STR
Biochemistry Workstation, from PerSeptive
Biosystems, Framingham, MA, USA. TLC
was performed on E. Merck Silica Gel 60 F254
plates. Preparative silica gel chromatography
used E. Merck Silica Gel 60 (70–230 mesh),
and MPLC used E. Merck Silica Gel 60 (230–

E. Eichler et al. / Carbohydrate Research 319 (1999) 1–16
7
Table 1
1H NMR spectral data of pentasaccharides 17 and 23 and hexasaccharides 1 and 2 a
1
IV
III
4.80
3.30 (8.3)
3.67
II
V
I
VI
H-1
H-2
H-3
H-3%
H-4
H-5
H-6
H-6%
H-7
H-8
H-9
H-9%
4.87
4.09
3.83
4.52 (7.8)
3.63
4.17
4.73 (7.8)
3.58
4.12
2.72 (3.4)
1.84 (12.2)
3.69
3.81
3.58
2.76 (3.4)
1.80 (12.2)
3.69
3.85
3.65
3.62
3.50
1.38 (5.8)
3.63
3.52
3.83
3.92
4.13
3.95
b
3.72
b
b
b
b
b
b
b
b
b
b
b
b
Other resonances:
OCH2CH2CH2Si(CH3)3 4.03, 3.76; 0.95; 1.05, −0.02 (9)
CH3CON 2.02 (3)
2
IV
III
4.82
3.29 (8.3)
3.66
II
V
I
VI
H-1
H-2
H-3
H-3%
H-4
H-5
H-6
H-6%
H-7
H-8
H-9
H-9%
4.88
4.10
3.82
4.50 (7.8)
3.65
4.18
4.73 (7.8)
3.52
4.12
2.72 (3.4)
1.82 (12.2)
3.72
3.82
3.58
2.76 (3.4)
1.80 (12.2)
3.70
3.84
3.64
3.63
3.48
1.38 (5.8)
3.61
3.51
3.83
3.91
4.15
3.73
3.71
3.76
3.94
3.67
3.73
3.77
3.59
3.79
3.63
3.84
3.59
3.87
3.63
3.86
Other resonances:
OCH2CH2CH2N3 3.98; 3.76; 3.46 (2); 1.92 (2)
CH3CON 2.02 (3)
17
IV
4.86
4.07 (3.2)
3.82
III
II
V
I
H-1
H-2
H-3
H-3%
H-4
H-5
H-6
H-6%
H-7
H-8
H-9
H-9%
4.80 (8.0)
3.29 (8.4)
3.65
4.49 (8.0)
3.62 (2.6)
4.16 (9.8)
4.64 (7.9)
3.52
3.64
2.71 (4.5)
1.84 (12.2)
3.68
3.81
3.57
3.62
3.45
1.40 (6.1)
3.61
3.51
3.81
3.90
4.12
3.90
3.72
3.66
3.69 c
3.75 c
3.69 c
3.75 c
3.61
3.80
3.64
3.87
Other resonances:
OCH2CH2Si(CH3)3 4.03; 3.76; 1.08; 0.97; −0.02 (9)
CH3CON 2.02; (3)
23
IV
III
II
V
I
H-1
H-2
H-3
H-3%
H-4
H-5
H-6
H-6%
4.87
4.09
3.83
4.82 (8.4)
3.30 (8.5)
3.67
4.50 (8.0)
3.66
4.19 (9.8)
4.66 (7.7)
3.52
3.68
2.72 (4.4)
1.84 (12.8)
3.70
3.83
3.57
3.63
3.49
1.40 (5.8)
3.62
3.52
3.83
3.93
4.14
3.71
3.70
3.74
3.91
3.67
3.72
3.76

8
E. Eichler et al. / Carbohydrate Research 319 (1999) 1–16
Table 1 (Continued)
H-7
H-8
H-9
H-9%
3.59
3.79
3.62
3.84
Other resonances:
OCH2CH2CH2N3 3.86; 3.76; 3.45 (2); 1.93 (2)
CH3CON 2.02 (3)
a Chemical shifts in ppm and (coupling constants) in Hz in D2O.
b Assignments are uncertain.
c Assignments are tentative.
Table 2
13C NMR spectral data of pentasaccharides 17 and 23 and hexasaccharide 2 a
2
IV
III
II
IV
I
VI
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
101.33
71.39
73.55
81.73
71.61
17.69
103.45
74.18
76.35
77.49
75.03
61.79
103.31
70.23
76.11
75.65
75.03
61.53
104.41
70.90
76.59
68.27
75.80
61.69
174.32
101.40
39.38
70.44
52.52
73.73
68.86
72.87
63.45
175.86
100.66
40.52
69.32
52.52
73.73
68.97
72.67
63.45
Other resonances:
OCH2CH2CH2N3 69.06; 48.76; 22.87
CH3CON 29.08, 173.8
17
IV
III
II
V
I
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
101.31
71.51
73.48
81.54
71.64
17.36
103.50
74.15
76.35
77.47
74.96
61.77
102.57
70.19
76.24
75.70
74.97
61.72
104.56
72.48
73.58
69.35
75.99
61.42
178.0
101.43
39.29
69.40
52.48
73.74
68.89
72.92
63.49
Other resonances:
OCH2CH2Si(CH3)3 69.08; 18.40; −1.70
CH3CON 22.87; 176.2
23
IV
III
II
V
I
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
101.31
71.51
73.48
81.50
71.64
17.36
103.45
74.05
76.30
77.40
74.97
61.72
103.30
70.10
76.05
75.60
74.97
61.71
104.57
72.48
73.58
69.40
75.99
61.51
175.40
101.42
39.40
69.39
52.40
73.65
68.90
72.90
63.35
Other resonances:
OCH2CH2CH2N3 68.08; 48.75; 29.09
CH3CON 22.81, 173.90
a Chemical shifts in ppm in D2O.
400 mesh). Detection was effected by exami-
nation under UV light and by charring with
5% sulfuric acid in water. Solutions were
evaporated at or below 40 °C at aspirator
pressure. Microanalyses were carried out by
the analytical services of this department, and
all samples submitted for elemental analyses
were dried overnight under vacuum with

E. Eichler et al. / Carbohydrate Research 319 (1999) 1–16
9
phosphorus pentoxide at 56 °C (refluxing
acetone).
methylsilyl)ethyl 6-O-benzyl-b-
D
-galactopyran-
oside [5] (1.3 g, 3.5 mmol) was dissolved in dry
pyridine (20 mL) and chilled to 0 °C in an ice
bath under an argon atmosphere. Acetic
2,3,4-Tri-O-acetyl-6-O-benzyl-h,i-
D-galac-
topyranosyl trichloroacetimidate (3). 2-(Tri-
Scheme 7.
1
Fig. 2. H NMR spectrum of hexasaccharide 2 in D2O at 23 °C.

10
E. Eichler et al. / Carbohydrate Research 319 (1999) 1–16
1 H, J 12.0 Hz, CH2Ph), 4.05 (t, 1 H, H-5),
3.56 (dd, 1 H, J6,6% 9.5 Hz and J5,6 5.7 Hz, H-6),
3.50 (dd, 1 H, J5,6% 7.4 Hz, H-6%), 2.07 (s, 3 H,
OAc), 2.01 (s, 3 H, OAc), 2.00 (s, 3 H, OAc);
13C NMR (CDCl3): l 96.45 (C-1), 161.36
(CꢀN), 91.0 CCl3. Anal. Calcd for
C21H24Cl3NO9 (540.78): C, 46.64; H, 4.47; N,
2.59. Found: C, 46.63; H, 4.48; N, 2.62.
anhydride (8 mL) was added by syringe, and
the mixture was stirred overnight. The temper-
ature was allowed to rise to rt. The solution
was concentrated in the morning, and co-con-
centrated three times with toluene. Crude 2-
(trimethylsilyl)ethyl 2,3,4 - tri - O - acetyl - 6 - O-
benzyl-b- -galactopyranose (1.75 g, ꢀ100%)
D
was carried on as such to the next step. The
syrup was dissolved in CH2Cl2 (72 mL), and
trifluoroacetic acid (8 mL) was added. The
mixture was stirred at rt for 5 h. Ethyl acetate
(5 mL) was added to terminate the reaction.
After stirring for a few min, the solvents were
concentrated, then co-concentrated three times
with ethyl acetate. The residue was purified on
column chromatography using 3:7 ethyl ac-
etate–hexanes to obtain a syrupy material
(1.32 g, 97%). 2,3,4-Tri-O-acetyl-6-O-benzyl-
Ethyl
osyl - (1“4) - 2,3 - di - O - benzoyl - 6 - O - benzyl-
1-thio-i- -glucopyranoside (5). Ethyl 4-O-
acetyl-2,3-di-O-benzyl-b- -rhamnopyranosyl-
(1“4)-2,3-di-O-benzoyl-6-O-benzyl-1-thio-
b- -glucopyranoside (4) [5] (850 mg, 0.95
2,3-di-O-benzyl-i-
L
-rhamnopyran-
D
L
D
mmol) was dissolved in dry MeOH (85 mL)
and chilled to 0 °C in an ice bath. To this was
added a 3% solution of HCl in MeOH (30
mL). The mixture was left stirring overnight
and allowed to warm to rt. The mixture was
chilled before neutralizing it with solid sodium
hydrogencarbonate. Inorganic material was
filtered off, and the filtrates were concentrated.
The residue was purified by chromatography
using 47:3 toluene–ethyl acetate to obtained
an amorphous solid 5 (720 mg, 85%): [h]D +
D-galactopyranose was dissolved in dry
CH2Cl2 (15 mL), and the solution was chilled
under an argon atmosphere to 0 °C. While
stirring, trichloroacetonitrile (4 mL) was
added, followed by 50 mL of 1,8-diazabi-
cylco[5.4.0]undec-7-ene (DBU). The mixture
was stirred for 4 h, then co-concentrated.
Purification of the residue by column chro-
matography using 4:1 hexanes–ethyl acetate
as eluant afforded the a isomer (360 mg, 20%),
a mixture of a and b isomers (535 mg, 29%),
and the b isomer (662 mg, 37%) for a total
yield of 86% of 3 as amorphous solids. Data
for the a isomer: [h]D +68.7° (c 0.38, chloro-
form); 1H NMR (CDCl3): l 8.62 (s, 1 H, NH),
7.34 (m, 5 H, Ph), 6.58 (d, 1 H, J1,2 3.6 Hz,
H-1), 5.63 (d, 1 H, J3,4 3.1 Hz, H-4), 5.42 (dd,
1 H, J2,3 10.8 Hz, H-3), 5.34 (dd, 1 H, H-2),
4.53 (d, 1 H, J 11.9 Hz, CH2Ph), 4.39 (m, 2 H,
CH2Ph and H-5), 3.54 (dd, 1 H, J6,6% 9.6 Hz
and J5,6 5.8 Hz, H-6), 3.46 (dd, 1 H, J5,6% 7.2
Hz, H-6%), 2.06 (s, 3 H, OAc), 2.01 (s, 3 H,
1
76.2° (c 0.45, chloroform); H NMR (CDCl3):
l 7.95–6.88 (m, 25 H, 5Ph), 5.75 (t, 1 H, J3,4
9.5 Hz, H-3III), 5.40 (t, 1 H, J2,3 9.8 Hz, H-2III),
4.82 (d, 1 H, J 12.3 Hz, CH2Ph), 4.72 (d, 1 H,
J1,2 9.9 Hz, H-1III), 4.63 (d, 1 H, J 12.1 Hz,
CH2Ph), 4.57 (d, 1 H, J 12.1 Hz, CH2Ph), 4.53
(d, 1 H, J 12.3 Hz, CH2Ph), 4.42 (s, 1 H,
H-1IV), 4.05 (d, 1 H, J 10.5 Hz, CH2Ph), 3.99
(t, 1 H, J3,4 and J4,5 9.3 Hz, H-4III), 3.83 (d, 1
H, J 11.6 Hz, CH2Ph), 3.79–3.75 (m, 2 H,
H-5III and H-6III), 3.52–3.48 (m, 3 H, H-2IV,
H-4IV and H-6%III), 3.09 (dt, 1 H, J4,5 6.0 and
J5,6 6.1 Hz, H-5IV), 2.78–2.73 (m, 2 H,
CH3CH2S), 2.65 (d, 1 H, J3,4 9.3 Hz and J2,3
2.9 Hz, H-3IV), 2.06 (d, 1 H, J4,OH 1.9 Hz,
OH-4IV), 1.28–1.24 (m, 6 H, CH3CH2S, and
H-6IV). Anal. Calcd for C49H52O11S (849.02):
C, 69.32; H, 6.17; S, 3.77. Found: C, 69.67; H,
5.92.
13
OAc), 2.00 (s, 3 H, OAc); C NMR (CDCl3):
l 93.94 (C-1), 161.27 (CꢀN), 91.07 (CCl3).
Anal. Calcd for C21H24Cl3NO9 (540.78): C,
46.64; H, 4.47; N, 2.59. Found: C, 46.27; H,
4.29; N, 3.09. Data for the b isomer: [h]D
−0.6° (c 0.32, chloroform); 1H NMR
(CDCl3): l 8.70 (s, 1 H, NH), 7.26–7.35 (m, 5
H, Ph), 5.82 (d, 1 H, J1,2 8.3 Hz, H-1), 5.54
(dd, 1 H, J3,4 3.3 Hz and J4,5 0.8 Hz, H-4), 5.46
(dd, 1 H, J2,3 10.4 Hz, H-2), 5.12 (dd, 1 H,
H-3), 4.56 (d, 1 H, J 12.0 Hz, CH2Ph), 4.43 (d,
Ethyl
galactopyranosyl-(1“4)-2,3-di-O-benzyl-i-
-rhamnopyranosyl-(1“4)-2,3-di-O-benzoyl-
6-O-benzyl-1-thio-i- -glucopyranoside (6). Al-
2,3,4-tri-O-acetyl-6-O-benzyl-i-
D-
L
D
cohol 5 (485 mg, 0.57 mmol) was dissolved in
CH2Cl2 (50 mL), and 2,3,4-tri-O-acetyl-6-O-
benzyl- -galactopyranosyl trichloroacetimid-
D

E. Eichler et al. / Carbohydrate Research 319 (1999) 1–16
11
were filtered off through Celite. The residue
was purified by chromatography using 1:3
ethyl acetate–hexanes as eluant to obtain a
white amorphous solid 7 (170 mg, 90%):
ate 3 (462 mg, 0.85 mmol) was added, fol-
,
lowed by activated 4 A molecular sieves
(1.5 g). The suspension was stirred at rt for 1.5
h under an argon atmosphere, then chilled to
−5 °C in a CaCl2–ice bath, and triethylsilyl
trifluoromethanesulfonate (13 mL in 1.3 mL
CH2Cl2) was added. Triethylamine was added
to terminate the reaction, and after further
dilution with CH2Cl2, some solids were filtered
off through Celite. The residue was purified by
chromatography using 3:7 ethyl acetate–hex-
anes as eluant to give 6 as an amorphous solid
(469 mg, 67%): [h]D +11.8° (c 0.44, chloro-
1
[h]D +7.17° (c 0.46, chloroform); H NMR
(CDCl3): l 7.95–6.88 (m, 30 H, 6Ph) 5.66 (t, 1
H, J3,4 9.6 Hz, H-3III), 5.39 (d, 1 H, J3,4 3.2
Hz, H-4V), 5.34 (dd, 1 H, J2,3 10.0 Hz, H-2III),
5.02 (dd, 1 H, J2,3 10.3 Hz, H-2V), 4.90 (dd, 1
H, H-3V), 4.82 (d, 1 H, J1,2 8.0 Hz, H-1V), 4.73
(d, 1 H, J 12.3 Hz, CH2Ph), 4.70 (d, 1 H, J1,2
7.8 Hz, H-1III), 4.62 (d, 1 H, J 12.1 Hz,
CH2Ph), 4.57–4.48 (m, 3 H, 3CH2Ph), 4.38–
4.36 (m, 2 H, H-1IV and CH2 Ph), 4.04–3.98
(m, 2 H, CH2Ph and CH2CH2Si), 3.93 (t, 1 H,
J4,5 9.2 Hz, H-4III), 3.75–3.70 (m, 5 H, H-5III,
H-6III, H-6III%, H-5V and CH2Ph), 3.65–3.58
(m, 2 H, CH2CH2Si and H-4IV), 3.44–3.39 (m,
3 H, H-6V, H-6%II and H-2IV), 3.14–3.10 (m, 1
H, H-5IV), 2.75 (dd, 1 H, J3,4 9.2 Hz and J2,3
2.8 Hz, H-3IV), 2.02 (s, 3 H, OAc), 1.93 (s, 3
H, OAc), 1.77 (s, 3 H, OAc), 1.25 (d, 3 H, J5,6
1
form); H NMR (CDCl3): l 7.95–7.06 (m, 30
H, 6 Ph), 5.69 (t, 1 H, J2,3 and J3,4 9.5 Hz,
H-3III), 5.40–5.37 (m, 2 H, H-2III and H-4V),
5.02 (dd, 1 H, J2,3 10.4 Hz, H-2V), 4.9 (dd, 1
H, J3,4 3.4 Hz, H-3V), 4.8 (d, 1 H, J1,2 7.9 Hz,
H-1V), 4.7 (m, 2 H, H-1III, and CH2Ph), 4.6 (d,
1 H, J 12.0, CH2Ph), 4.56–4.48 (m, 3 H,
3CH2Ph), 4.37 (m, 2 H, H-1IV and CH2Ph),
4.02 (d, 1 H, J 9.8 Hz, CH2Ph), 3.96 (t, 1 H,
J4,5 9.4 Hz, H-4III), 3.76–3.72 (m, 5 H, H-5V,
H-5III, CH2Ph, H-6III and H-6%III), 3.63 (t, 1 H,
J3,4 and J4,5 9.3 Hz, H-4IV), 3.44 (m, 3 H,
H-2IV, H-6II and H-6V), 3.10 (dd, 1 H, J4,5 11.9
Hz and J5,6 5.8 Hz, H-5IV), 2.77–2.71 (m, 3 H,
SCH2CH3 and H-3IV), 2.01 (s, 3 H, OAc), 1.92
(s, 3 H, OAc), 1.77 (s, 3 H, OAc), 1.29–1.22
(m, 6 H, SCH2CH3; and H-6IV). Anal. Calcd
for C68H74O19S (1227.39): C, 66.54; H, 6.07; S,
2.61. Found: C, 66.21; H, 5.83.
6.0 Hz, H-6IV), 0.91–0.84 (m,
2
H,
CH2CH2Si), −0.08 (s, 9 H, Si(CH3)3);
13C NMR (CDCl3): l 102.39 (J 156.3 Hz,
C–H-1IV), 100.49 (J 170.8, C–H-1V), 100.14
(J 160.6, C–H-1III). Anal. Calcd for
C17H82O20Si (1283.52): C, 66.44; H, 6.44; Si,
2.19. Found: C, 66.25; H, 6.47.
2-(Trimethylsilyl)ethyl
6-O-benzyl-i-
D
-
galactopyranosyl-(1“4)-2,3-di-O-benzyl-i-
L
-
rhamnopyranosyl-(1“4)-2,3-di-O-benzoyl-6-
O-benzyl-i- -glucopyranoside (8). To a solu-
2-(Trimethylsilyl)ethyl 2,3,4-tri-O-acetyl-6-
D
O-benzyl-i-
di-O-benzyl-i-
di-O-benzoyl-6-O-benzyl-i-
D
-galactopyranosyl-(1“4)-2,3-
-rhamnopyranosyl-(1“4)-2,3-
-glucopyranoside
tion of 7 (190 mg, 0.148 mmol) in dry MeOH
(25 mL), under an argon atmosphere, 1 M
sodium methoxide was added dropwise until
pH 9. The reaction was monitored by TLC.
After 4 h, additional MeOH was added, and
the mixture was neutralized with Rexyn 101
(H+) ion-exchange resin, filtered, and the
filtrate was concentrated. The residue was
purified on a short column of silica gel using
3:1 ethyl acetate–hexanes as eluant to obtain
syrupy 8 (165 mg, 96%): [h]D +30.4° (c 0.33,
L
D
(7). To a solution of 6 (180 mg, 0.147 mmol)
and 2-(trimethylsilyl)ethanol (50 mL, 0.37
,
mmol) in dry CH2Cl2 (30 mL) was added 4 A
molecular sieves (700 mg), and the mixture
was stirred at rt for 2 h under an argon
atmosphere, at the end of which time NIS (83
mg, 0.37 mmol) was added. After the reaction
mixture was stirred for 15 min, a solution of
TfOH (0.21 mL in 5 mL of dichloromethane)
was added (0.15 mL). When a TLC was taken
5 min after the addition of acid, it showed
total consumption of the thioglycoside 6. Tri-
ethylamine (0.15 mL) was then added to neu-
tralize the reaction, and after further dilution
with dichloromethane (100 mL), some solids
1
chloroform); H NMR (CDCl3): l 7.94–6.95
(m, 30 H, 6 Ph), 5.69 (t, 1 H, J3,4 9.7 Hz,
H-3III), 5.34 (dd, 1 H, J2,3 9.8 Hz, H-2III), 4.8
(d, 1 H, J 12.2 Hz, CH2Ph), 4.71 (d, 1 H, J1,2
7.9 Hz, H-1III), 4.6 (d, 1 H, J 12 Hz, CH2Ph),
4.57–4.52 (m, 2 H, CH2PH), 4.38 (brs, 1 H,
H-1IV), 4.28 (brd, 1 H, J1,2 7.8 Hz, H-1V),

12
E. Eichler et al. / Carbohydrate Research 319 (1999) 1–16
3-Azidopropyl 6-O-benzyl-i-
nosyl-(1“4)-2,3-di-O-benzyl-i-
ranosyl-(1“4)-2,3-di-O-benzoyl-6-O-benzyl-
i- -glucopyranoside (10). Trisaccharide 9 (330
D
-galactopyra-
4.06–4.02 (m, 2 H, CH2Ph and CH2CH2Si),
3.98–3.92 (m, 2 H, H-4III and H-4V), 3.85 (d,
1 H, J 11.4 Hz, CH2Ph), 3.76–3.67 (m, 4 H,
CH2Ph, H-5III, H-6III and H-6V), 3.64–3.53
(m, 6 H, CH2Ph, H-5V, H-6%V, H-4IV, H-2IV
and CH2CH2Si), 3.44 (m, 3 H, H-2V, H-3V and
H-6%III), 3.14 (m, 1 H, H-5IV), 2.91 (dd, 1 H,
J3,4 9.7 Hz and J2,3 2.7 Hz, H-3IV), 1.33 (d, 3
H, J5,6 6.1 Hz, H-6IV), 0.90–0.84 (m, 2 H,
L
-rhamnopy
D
mg, 0.26 mmol) was treated as for the conver-
sion of 7 to 8. The product was purified by
silica gel chromatography using 3:2 ethyl
acetate–hexanes as an eluant to obtain an
amorphous solid 10 (220 mg, 62%): [h]D +
1
13
32.6° (c 0.17, chloroform); H NMR (CDCl3):
CH2CH2Si), 0.08 (s, 9 H, Si(CH3)3); C NMR
(CDCl3): l 100.02 (J 159.6 Hz, C-H-1III),
102.24 (J 156.0 Hz, C–H-1IV), 104.85 (J 160.6
Hz, C–H-1V). Anal. Calcd for C65H76O17Si
(1157.39): C, 67.45; H, 5.52; Si, 2.42. Found:
C, 66.97; H, 6.13.
l 7.94–6.95 (m, 30 H, 6 Ph), 5.69 (t, 1 H, J3,4
9.6 Hz, H-3III), 5.34 (dd, 1 H, J2,3 10.1 Hz,
H-2III), 4.80 (d, 1 H, J 12.2 Hz, CH2Ph), 4.68
(d, 1 H, J1,2 7.9 Hz, H-1III), 4.62 (d, 1 H, J
12.1 Hz, CH2Ph), 4.56–4.52 (m, 4 H,
4CH2Ph), 4.49 (s, 1 H, H-1IV), 4.37 (m, 1 H,
H-1V), 4.02 (d, 1 H, J 9.7 Hz, CH2Ph), 3.99–
3.95 (m, 2 H, H-4III and OCH2–CH2CH2N3),
3.92 (brs, 1 H, H-4V), 3.86 (d, 1 H, J 11.4 Hz,
CH2Ph), 3.76–3.66 (m, 5 H, H-5V, H-6III,
H-6%III, H-6V and H-6%II), 3.63–3.53 (m, 4 H,
H-5III, H-2IV, H-4IV, and OCH2CH2CH2N3),
3.43–3.42 (m, 2 H, H-2V and H-3V), 3.25–3.19
(m, 2 H, CH2N3), 3.15–3.12 (m, 1 H, H-5IV),
2.90 (dd, 1 H, J3,4 9.6 Hz and J2,3 2.9 Hz,
H-3IV), 2.57 (brs, 1 H, OH), 2.42 (brs, 1 H,
OH), 1.80–1.73 (m, 2 H, CH2CH2CH2N3),
1.33 (d, 3 H, J5,6 6.1 Hz, H-6IV); 13C NMR
(CDCl3): l 104.86 (J 160.1 Hz, C–H-1V),
102.18 (J 155.0, C–H-1IV), 100.65 (J 161.2 Hz,
C–H-1III). Anal. Calcd for C63H69N3O17
(1140.262): C, 66.36; H, 6.09; N, 3.68. Found:
C, 66.50; H, 6.26; N, 3.60.
3-Azidopropyl 2,3,4-tri-O-acetyl-6-O-ben-
zyl-i-
benzyl-i-
O-benzoyl-6-O-benzyl-i-
D
-galactopyranosyl-(1“4)-2,3-di-O-
L
-rhamnopyranosyl-(1“4)-2,3-di-
-glucopyranoside (9).
D
Trisaccharide 6 (450 mg, 0.36 mmol) was re-
acted with 3-azidopropan-1-ol (110 mg, 1.08
mmol) as for the conversion of 6 to 7. The
product was purified by silica gel chromatog-
raphy using 7:3 hexanes–ethyl acetate as elu-
ant to give a white foamy solid 9 (430 mg,
78%): [h]D+11.8° (c 0.6 chloroform); 1H
NMR (CDCl3): l 7.96–7.00 (m, 30 H, 6 Ph),
5.67 (t, 1 H, J3,4 9.7 Hz, H-3III), 5.39 (d, 1 H,
J3,4 3.3 Hz, H-4V), 5.33 (dd, 1 H, J2,3 10.0 Hz,
H-2III), 5.03 (dd, 1 H, J2,3 10.4 Hz, H-2V), 4.88
(dd, 1 H, J3,4 3.4 Hz, H-3V), 4.82 (d, 1 H, J1,2
7.9 Hz, H-1V), 4.72 (d, 1 H, J 12.3 Hz,
CH2Ph), 4.68 (d, 1 H, J1,2 7.8 Hz, H-1III), 4.61
(d, 1 H, J 12.1 Hz, CH2Ph), 4.56–4.48 (m, 3
H, 3CH2Ph), 4.38 (s, 1 H, H-1IV), 4.37 (d, 1 H,
J 11.1 Hz, CH2Ph), 4.03 (d, 1 H, J 9.8 Hz,
CH2Ph), 3.99–3.93 (m, 2 H, H-4III and
OCH2CH2CH2N3), 3.76–3.65 (m, 5 H, H-5III,
H-6III, H-6III%, H-5V and CH2Ph), 3.63 (t, 1 H,
J4,5 9.3 Hz, H-4IV), 3.61–3.57 (m, 1 H,
OCH2CH2CH2N3), 3.48–3.39 (m, 3 H, H-2IV,
H-6V and H-6%V), 3.24–3.20 (m, 2 H, CH2N3),
3.14–3.11 (m, 1 H, H-5IV), 2.77 (dd, 1 H, J3,4
9.4 Hz and J2,3 3.0 Hz, H-3IV), 2.01 (s, 3 H,
OAc), 1.93 (s, 3 H, OAc), 1.84–1.69 (m, 5 H,
OAc and CH2CH2CH2), 1.27 (d, 3 H, J5,6 6.1
Hz, H-6IV); 13C NMR (CDCl3): l 102.6 (J
156.7 Hz, C–H-1V), 101.10, (J 161.0 Hz, C–
H-1III), 100.75 (J 163.7 Hz, C–H-1IV). Anal.
Calcd for C69H75N3O20 (1266.37): C, 65.44; H,
5.97; N, 3.31. Found: C, 65.72; H, 5.62; N,
2.73.
2-(Trimethylsilyl)ethyl [methyl 5-acetamido-
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
D
-glycero-h-
D
-galacto-2-nonulopyranosylonate-(2“3)] -6-
O-benzyl-i-
D
-galactopyranosyl-(1“4)-2,3-
- rhamnopyranosyl - (1“4)-
-glucopyra-
di - O - benzyl - i -
L
2,3-di-O-benzoyl-6-O-benzyl-i-
D
noside (12). A suspension of 8 (120 mg, 0.103
mmol), phenyl (methyl 5-acetamido-4,7,8,9-
tetra-O-acetyl-3,5-dideoxy-2-thio-
a-
D
-glycero-
D
-galacto-2-nonulopyranoside)onate (11)
[18,19] (121 mg, 0.21 mmol), and activated 3
A molecular sieves (600 mg) in CH3CN (3
,
mL) was stirred at rt for 3 h under an argon
atmosphere, at the end of which time NIS
(140 mg, 0.62 mmol) was added. The reaction
mixture was stirred for 15 min and chilled to
−40 °C, then a solution of TfOH (0.4 mL in
10 mL of dichloromethane) (0.45 mL) was
added. The mixture was stirred at −40 °C for

E. Eichler et al. / Carbohydrate Research 319 (1999) 1–16
13
stirred for 2 h, at the end of which time NIS
(206 mg, 0.915 mmol) was added. After the
reaction mixture was stirred for 15 min, a
solution of TfOH (0.38 mL in 10 mL of
CH2Cl2) was added (0.7 mL). TLC analysis
after 5 min showed total consumption of the
thioglycoside 6. Triethylamine (1 mL) was
added to neutralize the reaction, and after
further dilution with CH2Cl2 (100 mL), some
solids were filtered off through Celite. The
filtrate was washed (2×10% Na2S2O4, 3×0.05
M HCl, 2×NaHCO3, water, and brine), dried
(MgSO4), filtered and concentrated. The
residue was purified by silica gel chromatogra-
phy, using a gradient of 65“75 ethyl acetate–
35“25 hexanes as eluent, to give a foamy solid
15 (251 mg, 34%) and impure 16 (178 mg, 24%)
that was not further characterized. Data for 15:
[h]D +4.6° (c 0.24, CHCl3); 1H NMR (CDCl3):
l 7.9–6.9 (m, 35 H, 7 Ph), 5.64 (t, 1 H, J3,4 and
J2,3 9.6 Hz, H-3III), 5.38 (m, 2 H, H-4V, and
H-8I), 5.27–5.20 (m, 3 H, H-2III, H-7I and NH),
5.08 (d, 1 H, J1,2 7.9 Hz, H-1III), 5.02 (m, 1 H,
H-2V), 4.96 (m, 1 H, H-4I), 4.89 (dd, 1 H, J2,3
10.3 Hz and J3,4 3.4 Hz, H-3V), 4.82 (d, 1 H, J1,2
8.0 Hz, H-1V), 4.75 (d, 1 H, J 12.2 Hz, CH2Ph),
4.58–4.41 (m, 8 H, CH2Ph), 4.39–4.34 (m, 2 H,
H-1IV and CH2Ph), 4.28 (d, 1 H, J1,2 7.7 Hz,
H-1II), 4.17 (dd, 1 H, J9,9% 12.6 Hz and J8,9 2.6
Hz, H-9I), 4.02 (dd, 1 H, J8,9% 5.1 Hz, H-9%I),
3.98–3.90 (m, 6 H, H-5V, H-4III, H-5I, N-6I,
H-3V and CH2CH2Si), 3.86–3.83 (m, 3 H,
H-4II, H-6II and H-6V), 3.75–3.66 (m, 5 H,
OMe, H-5III and H-5II), 3.63–3.56 (m, 4 H,
H-4IV, H-6%II, H-6%II% and CH2CH2Si), 3.48–
3.35 (m, 4 H, H-6III, H-6III%, H-2IV, and H-2II),
3.12 (dd, 1 H, J4,5 9.1 Hz and J5,6 6.1 Hz,
H-5IV), 2.77 (dd, 1 H, J3,4 9.5 Hz and J2,3 3.0
Hz, H-3IV), 2.69 (dd, 1 H, J3,3% 13.2 Hz and J3,4
4.6 Hz, H-3I), 2.25 (brs, 1 H, OH-2II), 2.06 (s,
3 H, OAc), 2.05 (s, 3 H, OAc), 2.04 (s, 3 H,
OAc), 2.01 (s, 3 H, OAc), 1.98 (s, 3 H, OAc),
1.92 (s, 3 H, OAc), 1.88 (s, 3 H, OAc), 1.85 (t,
1 H, J3%,4 13.3 Hz, H-3%I), 1.76 (s, 3 H, NAc),
1.27 (d, 3 H, H-6IV), 1.03–0.85 (m, 2 H,
CH2CH2Si), −0.05 (s, 9 H, Si(CH3)3); 13C
NMR (CDCl3): l 100.70 (J 167.5 Hz, C–H-
1III), 100.73 (J 169.0 Hz, C–H-1V), 102.22 (J
159.8 Hz, C–H-1II), 102.76 (J 157.1 Hz, C–H-
1IV); FABMS (positive-ion): Anal. Calcd for
C104H125ONNaSi: 2032.19. Found: m/z
2032.20 [M+Na].
2 h, then overnight at +4 °C. Triethylamine
(0.5 mL) was added in the morning to neutral-
ize the reaction, and after further dilution with
CH2Cl2 (60 mL), some solids were filtered off
through Celite. The residue was purified first
on a silica gel column, then on a Sephadex LH
20 column using 4:3 CHCl3 –MeOH as eluant
to give 12 (26 mg, 19%) as a 5:3 a:b mixture:
1H NMR (CDCl3): l 2.55 (dd, J3,3% 13.7 Hz and
J3,4 4.7 Hz, H-3Ia), 2.15 (m, H-3I%b); FABMS
(positive-ion) Anal. Calcd for C85H102-
NNaO29Si: 1652.81. Found: m/z 1652.62 [M+
Na].
3-Azidopropyl [5-acetamido-3,5-dideoxy-
glycero-h- -galacto-2-nonulopyranosylonate-
(2“3)] - 6 - O - benzyl - i - - galactopyranosyl-
(1“4)-2,3-di-O-benzyl-i- -rhamnopyranosyl-
(1“4)-2,3-di-O-benzoyl-6-O-benzyl-i- -glu-
D
-
D
D
L
D
copyranoside (13). A suspension of 10 (100 mg,
0.088 mmol) was treated as above for 12. After
dry silica gel column chromatography using 4:1
ethyl acetate–toluene as the eluant, the
product was further purified on a Sephadex LH
20 column, using 4:3 CHCl3–MeOH as eluant,
and again on silica gel using 11:11:1 ethyl
acetate–hexanes–EtOH to yield still impure
fractions that were not further characterized.
2-(Trimethylsilyl)ethyl 2,3,4-tri-O-acetyl-6-
O-benzyl-i-
di-O-benzyl-i-
di-O-benzoyl-6-O-benzyl-i-
(1“2)-[methyl-5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy- -glycero-h- -galacto-2-
nonulopyranosylonate-(2“3)]-6-O-benzyl-i-
-galactopyranoside (16) and 2-(Trimethylsi-
D
-galactopyranosyl-(1“4)-2,3-
-rhamnopyranosyl-(1“4)-2,3-
-glucopyranosyl-
L
D
D
D
D
lyl)ethyl 2,3,4-tri-O-acetyl-6-O-benzyl-i-
D
-
galactopyranosyl-(1“4)-2,3-di-O-benzyl-i-
L
-
rhamnopyranosyl-(1“4)-2,3-di-O-benzyl-6-
O-benzyl-i- -glucopyranosyl-(1“4)-[methyl
5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dide-
oxy- -glycero-h- -galacto-2-nonulopyranosy-
lonate-(2“3)]-6-O-benzyl-i- -galactopyrano-
D
D
D
D
side (15). Trisaccharide 6 (450 mg, 0.37 mmol)
and 2-(trimethylsilyl)ethyl (methyl 5-ac-
etamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
glycero-a-
(2“3)-6-O-benzyl-b-
D
-
D
-galacto-2-nonulopyranosylonate)-
-galactopyranoside (14)
[5,20] (325 mg, 0.385 mmol) were dissolved in
D
,
CH2Cl2 (40 mL), and powdered 4 A molecular
sieves (1.1 g) were added. The mixture was
maintained under an argon atmosphere and

14
E. Eichler et al. / Carbohydrate Research 319 (1999) 1–16
2-(Trimethylsilyl)ethyl 2,3,4-tri-O-acetyl-i-
-galactopyranosyl-(1“4)-i- -rhamnopyran-
-glucopyran-
osyl-(1“4)-[methyl 5-acetamido-4,7,8,9-tetra-
O-acetyl-3,5-dideoxy- -glycero-h- -galacto-2-
nonulopyranosylonate-(2“3)]-i- -galactopy-
ranoside (17). Pentasaccharide 15 (165 mg,
0.082 mmol) in glacial acetic acid (15 mL) was
hydrogenated over 10% Pd/C (200 mg). After
filtration through Celite and evaporation of
the filtrates, the residue was co-concentrated
with toluene (2×) to yield a sandy-looking
amorphous solid 17 (130 mg, 100%) that was
used as such for the next step.
was obtained, 2-(trimethylsilyl)ethyl 2,3,4,6-te-
tra - O - acetyl - b - - galactopyranosyl - (1“4)-
2,3-di-O-acetyl-b- -rhamnopyranosyl-(1“4)-
6-O-acetyl-2,3-di-O-benzoyl-b- -glucopyran-
osyl-(1“4)-[methyl 5-acetamido-4,7,8,9-tetra-
O-acetyl-3,5-dideoxy- -glycero-a- -galacto-2-
nonulopyranosylonate-(2“3)]-b- -galactopy-
ranoside (19, 145 mg, 96%). A solution of 19
(140 mg, 0.077 mmol) in dry CH2Cl2 (4.5 mL)
was cooled to 0 °C under an argon atmo-
sphere, and trifluoroacetic acid (0.5 mL) was
added. TLC analysis indicated that the reac-
tion was complete after stirring for 3 h at
0 °C. Ethyl acetate (5 mL) was added, and the
reaction mixture was concentrated, then co-
concentrated twice more with ethyl acetate.
Purification by silica gel column chromatogra-
phy of the residue with ethyl acetate as an
D
L
D
osyl-(1“4)-2,3-di-O-benzoyl-i-
D
L
D
D
D
D
D
D
D
2-(Trimethylsilyl)ethyl i-
D
-galactopyrano-
syl - (1“4) - i -
L
- rhamnopyranosyl - (1“4)-i-
D
-glucopyranosyl - (1“4) - [5 - acetamido - 3,5-
dideoxy-
D
-glycero-h-
D
-galacto-2-nonulopyra-
-galactopyranoside
nosylonic acid-(2“3)]-i-
D
eluent gave 2,3,4,6-tetra-O-acetyl-b-
D
-galac-
(18). To a solution of 17 (21 mg, 0.014 mmol)
in MeOH (6 mL) was added 1 M sodium
methoxide (1 mL). This solution was stirred at
rt for 6 h, then chilled to 0 °C, at which point
water (0.5 mL) and 1 M sodium hydroxide
(0.1 mL) were added. The mixture was stirred
and allowed to come to rt overnight. Addi-
tional methanol was added, the mixture was
neutralized with Rexyn 101 (H+) ion-ex-
change resin and filtered, and the filtrate was
concentrated. The residue was placed on a
Bio-Gel P-2 column and eluted with water.
The fractions were monitored by RI detection.
Freeze-drying of the pure fractions gave 18
(11 mg, 78%) as its sodium salt: [h]D +1.9° (c
0.09, water); MALDIMS: Anal. Calcd for
C40H71O28NNaSi: 1065.07. Found: m/z
1065.07 [M+Na].
topyranosyl-(1“4)-2,3-di-O-acetyl-b-
L
-rham-
nopyranosyl-(1“4)-6-O-acetyl-2,3-di-O-ben
zoyl-b- -glucopyranosyl-(1“4)-[methyl 5-
acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
- glycero - a - - galacto - 2 - nonulopyranosyl-
onate-(2“3)]-2,6-di-O-acetyl- -galactopyr-
D
D
D
D
anose (20, 119 mg, 89%) as an amorphous
solid. To a solution of 20 (60 mg, 0.035 mmol)
in CH2Cl2 (15 mL) and trichloroacetonitrile
(0.2 mL) under argon atmosphere was added
DBU (5 mg) at −5 °C, and the mixture was
stirred for 3 h. It was then concentrated, and
the residue was purified by silica gel column
chromatography using 9:1 ethyl acetate–hex-
anes as eluant to obtain an amorphous solid,
2,3,4,6-tetra-O-acetyl-b-
(1“4) - 2,3 - di - O - acetyl - b -
osyl-(1“4)-6-O-acetyl-2,3-di-O-benzoyl-b-
glucopyranosyl-(1“4)-[methyl 5-acetamido-
4,7,8,9-tetra-O-acetyl-3,5-dideoxy- -glycero-
a- -galacto-2-nonulopyranosylonate-(2“3)]-
2,6-di-O-acetyl- -galactopyranosyl trichloro-
D
-galactopyranosyl-
L
- rhamnopyran-
D-
3-Azidopropyl 2,3,4,6-tetra-O-acetyl-i-
D
-
-
galactopyranosyl-(1“4)-2,3-di-O-acetyl-i-
L
D
rhamnopyranosyl-(1“4)-6-O-acetyl-2,3-di-O-
benzoyl-i- -glucopyranosyl-(1“4)-[methyl 5-
acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
-glycero-h- -galacto-nonulopyranosylonate-
(2“3)]-2,6-di-O-acetyl-i- -galactopyranoside
D
D
D
acetimidate (21) (57 mg, 87%). To a solution
of 21 (57 mg, 0.03 mmol) and 3-azidopropan-
1-ol [17] (10 mg, 0.1 mmol), in dry CH2Cl2 (4
D
D
D
,
(22). To a solution of 17 (130 mg, 0.08 mmol)
in pyridine (10 mL) at 0 °C under argon was
added dropwise acetic anhydride (5 mL). This
mixture was stirred at 0 °C for 3 h, then
allowed to come to rt overnight. The mixture
was stirred for 40 h, then concentrated and
co-concentrated with toluene (2×). After fur-
ther drying in vacuo a sandy-looking solid
mL) were added 4 A molecular sieves (170
mg), and the mixture was stirred at rt for 1.5
h, then cooled to 0 °C under an argon atmo-
sphere. Boron trifluoride etherate (50 mL) was
added, and after the mixture had been stirred
for 1 h at 0 °C, the reaction was complete.
Triethylamine (0.1 mL) was then added,
and the suspension was further diluted with

E. Eichler et al. / Carbohydrate Research 319 (1999) 1–16
15
CH2Cl2, then filtered through Celite. The
filtrate was concentrated, and the residue was
purified by silica gel chromatography using 9:1
ethyl acetate–hexanes as an eluant to obtain 22
sodium salt: [h]D +17.1° (c 0.33, water);
MALDIMS Anal. Calcd for C38H64N4NaO28:
1047.94. Found: m/z 1047.17 [M+Na].
3-Azidopropyl (5-acetamido-3,5-dideoxy-
glycero - h - - galacto - 2 - nonulopyranosylonic
acid)-(2“3)-i- -galactopyranosyl-(1“4)-i-
-rhamnopyranosyl-(1“4)-i- -glucopyrano-
syl - (1“4) - [5 - acetamido - 3,5 - dideoxy -
glycero - h - - galacto - 2 - nonulopyranosylonic
acid-(2“3)]-i- -galactopyranoside (2). Pen-
D-
1
(42 mg, 77%): [h]D +18.9° (c 1.1, CHCl3); H
D
NMR (CDCl3): l 7.93–7.29 (m, 10 H, 2 Ph),
5.50 (t, 1 H, J3,4 9.6 Hz, H-3III), 5.45 (m, 1 H,
H-8I), 5.30 (m, 2 H, H–Nu 7 and H-4V), 5.21 (t,
1 H, J2,3 9.8 Hz, H-2III), 5.04–5.01 (m, 3 H, H-
2V, H-2IV and NH), 4.89–4.83 (m, 4 H, H-1III,
H-4I, H-2II and H-3V), 4.56–4.53 (m, 2 H, H-
3IV and H-1IV), 4.50–4.47 (m, 2 H, H-1V and H-
4II), 4.38 (d, 1 H, J1,2 8.0 Hz, H-1II), 4.34 (bdd,
1 H, H-9I), 4.23–4.02 (m, 8 H, H-3II, H-6III, H-
4III, H-6II, H-6%III, H-6%II, H-6V and H-6%II),
3.98–3.92 (m, 2 H, H-5I and H-9%I), 3.85–3.82
(m, 4 H, OMe, and H-5V), 3.73–3.70 (m, 2 H,
H-6I and OCH2CH2CH2N), 3.59 (m, 2 H, H-
5III and H-5V), 3.46 (m, 1 H, CH2CH2CH2N),
3.42 (t, 1 H, J3,4 and J4,5 9.3 Hz, H-4IV), 3.28–
3.21 (m, 3 H, H-5IV and CH2CH2CH2N), 2.57
(dd, 1 H, J3,3% 12.6 Hz and J3,4 4.8 Hz, H-3I),
2.12 (brs, 9 H, 3OAc), 2.09 (s, 3 H, OAc), 2.06
(s, 3 H, OAc), 2.04 (s, 3 H, OAc), 2.03 (s, 3 H,
OAc), 2.02 (s, 3 H, OAc), 2.00 (brs, 6 H,
2OAc), 1.93 (s, 3 H, OAc), 1.92 (s, 3 H, OAc),
1.87 (s, 3 H, OAc), 1.85 (s, 3 H, NAc), 1.79–
1.63 (m, 2 H, CH2CH2CH2), 1.59 (t, 1 H, J3%,4
12.4, H-3%I), 1.31 (d, 3 H, J5,6 6.1 Hz, H-6IV); 13C
NMR (CDCl3): l 101.09 (J 162.0 Hz, C–H-
1V), 100.63, (J 165.1 Hz, C–H-1II) 100.42, (J
165.2 Hz, C–H-1III), 97.92 (J 157.8 Hz, C–H-
1IV); ESIMS (positive-ion) Anal. Calcd for
C79H100N4NaO43: 1815.6. Found: m/z 1815.5
[M+Na].
D
L
D
D
-
D
D
tasaccharide 23 (2.0 mg; mmol) was sialylated
using 0.45 U of an a-2,3-sialyltransferase
(CST-06) from C. jejuni. The reaction mixture
included 100 mM of Tris pH 7, 10 mM of
MgCl2, 8 mM of b-Neu5AcCMP, and 10 units
of alkaline phosphatase. The reaction was per-
formed at 37 °C for 3 h in a final volume of 0.3
mL. Product formation was followed by TLC
on silica using 2-propanol–butanol–0.1 M
HCl (2:1:1) as the developing solvent (Rf 0.2).
The residue was placed on a Bio-Gel P2
column and eluted with water (RI detection).
Freeze-drying of the pure fractions gave 2 (2.3
mg, 85%) as its disodium salt: [h]D +22.1° (c
0.1, water); ESIMS (positive-ion) Anal. Calcd
for C49H81N5Na3O36: 1382.20. Found: m/z
1382.29 [M+3Na].
2 - (Trimethylsilyl)ethyl 5 - acetamido - 3,5-
dideoxy-
D
-glycero-h-
acid-(2“3)-i-
- rhamnopyranosyl - (1“4) - i - -
D
-galacto-2-nonulopyrano-
sylonic
D
-galactopyranosyl-
(1“4) - i -
L
D
glucopyranosyl - (1“4) - [5 - acetamido - 3,5-
dideoxy- -glycero-h- -galacto-2-nonulopyrano-
sylonic acid-(2“3)]-i- -galactopyranoside (1).
D
D
D
Pentasaccharide 18 (2.0 mg; 1.9 mmol) was
treated as above for 23 to 2. 1H NMR analysis
of the fractions from Bio-Gel P2 indicated
some CMP-derived impurities that were re-
moved by passing through a C18 Sep-Pak car-
tridge loading in water and eluting successively
with 10% CH3OHaq, 50% CH3OHaq and 100%
CH3OH to yield 1 (0.7 mg, 26%) as its dis-
odium salt: [h]D +17.1° (c 0.06, water); ESIMS
(positive-ion) Anal. Calcd for C51H87N2-
NaO36Si: 1355.34. Found: m/z 1355.36 [M+
Na], 1377.13 [M+2Na], 1398.92 [M+3Na].
3-Azidopropyl i-
i- -rhamnopyranosyl-(1“4)-i -
ranosyl-(1“4)-[5-acetamido-3,5-dideoxy-
glycero - h - - galacto - 2 - nonulopyranosylonic
acid-(2“3)]-i- -galactopyranoside (23). To a
D
-galactopyranosyl-(1“4)-
L
D
- glucopy-
D-
D
D
solution of 22 (26 mg, 0.0145 mmol) in dry
MeOH (5 mL) was added 1 M sodium methox-
ide in MeOH (1 mL). This solution was stirred
at rt for 6 h, then chilled to 0 °C, and water (1
mL) was added, followed by 1 M NaOH (0.2
mL). This solution was stirred overnight, then
further diluted with MeOH, and the reaction
was neutralized with Rexyn 101 (H+) ion-ex-
change resin and concentrated. The residue was
placed on a Bio-Gel P2 column and eluted with
water (RI monitoring). Freeze-drying of the
pure fractions gave 23 (12 mg, 81%) as its
Acknowledgements
The authors would like to thank Dr W.
Wakarchuk for helpful discussions. The au-

16
E. Eichler et al. / Carbohydrate Research 319 (1999) 1–16
[8] W. Stahl, U. Prengard, G. Kretzschmar, D.W. Schmidt,
H. Kunz, J. Prakt. Chem., 337 (1995) 441–445.
[9] R.R. Schmidt, W. Kinzy, Ad6. Carbohydr. Chem.
Biochem., 50 (1994) 21–123.
[10] R.R. Schmidt, Angew. Chem., Int. Ed. Engl., 25 (1986)
212–235.
[11] B.M. Pinto, D.G. Morissette, D.R. Bundle, J. Chem.
Soc., Perkin Trans 1, (1987) 9–14.
[12] T.L. Lowary, E. Eichler, D.R. Bundle, J. Org. Chem., 60
(1995) 7216–7327.
[13] G.H. Veeneman, S.H. van Leeuwen, J.H. van Boom,
Tetrahedron Lett., 31 (1990) 1331–1334.
[14] P. Konradsson, D.R. Mootoo, R.E. McDevitt, B.
Fraser-Reid, J. Chem. Soc., Chem. Commun., (1990)
270–272.
thors are also grateful to Dr J.R. Brisson for
help with the NMR spectroscopy, Mr K.
Chan and Mr D. Krajcarski for the mass
spectral analyses, and Ms A. Webb for ele-
mental analyses. The authors also thank Ms
E. Roux, an NRC summer student, who pre-
pared some of the intermediates. This is NRC
paper 42379.
References
[15] K. Jansson, S. Ahlfors, T. Frejd, J. Kihlberg, G. Mag-
nusson, J. Org. Chem., 53 (1988) 5629–5647.
[16] G. Magnusson, Trends Glycosci. Glycotech., 4 (1992)
357–367.
[17] J. Szmuszkovicz, M.P. Kane, L.G. Laurian, C.G.
Chidester, T.A. Scahill, J. Org. Chem., 46 (1981) 3562–
3564.
[18] J. Rothermel, H. Faillard, Biol. Chem. Hoppe-Seyler, 370
(1989) 1077–1084.
[19] S. Cao, S.J. Meunier, F.O. Andersson, M. Letellier, R.
Roy, Tetrahedron: Asymmetry, 5 (1994) 2303–2312.
[20] M. Kiso, A. Hasegawa, Methods Enzymol., 242 (1994)
173–183.
[1] G. Kogan, D. Uhr´ın, J.-R. Brisson, L.C. Paoletti, A.E.
Blodgett, D.L. Kasper, H.J. Jennings, J. Biol. Chem., 271
(1996) 8786–8798.
[2] C.J. Baker, M.S. Edwards, Ann. NY Acad. Sci., 459
(1988) 193–202.
[3] T. Murai, Y. Inazumi, M. Sugiyama, Y. Nishiyama,
Zentralbl. Bakteriol. Mikrobiol. Hyg. Abt. 1 Suppl., 22
(1992) 467–469.
[4] W. Zou, J.R. Brisson, Q.L. Yang, M. van der Zwan, H.J.
Jennings, Carbohydr. Res., 295 (1996) 209–228.
[5] E. Eichler, H.J. Jennings, D.M. Whitfield, J. Carbohydr.
Chem., 16 (1997) 385–411.
[6] G.M. Bebault, G.G.S. Dutton, N.A. Funnell, Can. J.
Chem., 52 (1974) 3844–3846.
[7] V.I. Betaneli, M.V. Ovchinnikov, L.V. Backinowsky,
N.K. Kochetkov, Carbohydr. Res., 76 (1979) 252–256.
[21] M. Gilbert, A.-M. Cunningham, D.C. Watson, A. Mar-
tin, J.C. Richards, W.W. Wakarchuk, Eur. J. Biochem.,
249 (1997) 187–194.
.