Measurement of interglycosidic 3J(CH) coupling constants of selectively 13C labeled oligosaccharides by 2D J-resolved 1H NMR spectroscopy

Article abstract of DOI:10.1016/S0008-6215(98)00047-0

Tri-, tetra-, and penta-saccharide fragments of the O-specific polysaccharide of Shigella dysenteriae type 1 have been prepared in which a D-galactose residue of each oligosaccharide methyl glycoside derivative contains a ¹³C label at C-1. The interglycosidic coupling constants (³J(CH)) of these ¹³C nuclei with the H-3 nuclei of the adjacent 2- acetamido-2-deoxy-D-glucose residues have been measured by two-dimensional, J-resolved ¹H NMR spectroscopy. The magnitudes of these coupling constants indicate that the trisaccharide is conformationally different to the higher oligosaccharide homologs, in agreement with previous studies of ¹³C chemical shifts and ¹J(CH) values.

Full text of DOI:10.1016/S0008-6215(98)00047-0

Carbohydrate Research 308 (1998) 229±238
Note
3
Measurement of interglycosidic JCH coupling
13
constants of selectively C labeled
1
oligosaccharides by 2D J-resolved H NMR
spectroscopy
Vince Pozsgaya, Nese Sarib, Bruce Coxonb,*
a Laboratory of Developmental and Molecular Immunity, National Institute of Child Health and Human
Development, National Institutes of Health, Bethesda, MD 20892-2720, USA
b Biotechnology Division, National Institute of Standards and Technology, Gaithersburg,
MD 20899, USA
Received 4 September 1997; accepted 16 January 1998
Abstract
Tri-, tetra-, and penta-saccharide fragments of the O-speci®c polysaccharide of Shigella dysen-
teriae type 1 have been prepared in which a d-galactose residue of each oligosaccharide methyl
glycoside derivative contains a 13C label at C-1. The interglycosidic coupling constants (3JCH) of
these 13C nuclei with the H-3 nuclei of the adjacent 2-acetamido-2-deoxy-d-glucose residues have
1
been measured by two-dimensional, J-resolved H NMR spectroscopy. The magnitudes of these
coupling constants indicate that the trisaccharide is conformationally di€erent to the higher oli-
gosaccharide homologs, in agreement with previous studies of 13C chemical shifts and 1JCH values.
# 1998 Elsevier Science Ltd. All rights reserved
Keywords: Trisaccharide; Tetrasaccharide; Pentasaccharide; Oligosaccharides; Carbon-13 labeling; Carbon-
13±proton coupling constants; Shigella dysenteriae type 1; 2D J-resolved NMR spectroscopy
As part of a project to develop improved sacchar-
ide±protein conjugate vaccines [1], we are studying
synthetic approaches to fragments of the O-speci®c
polysaccharide (O-SP) of Shigella dysenteriae type
1 [2±7], an organism that causes dysentery, an
acute invasive disease of the lower intestines, in
many parts of the world [8]. High-resolution NMR
spectroscopy is being used to de®ne the structural
and conformational aspects of the problem [9]. We
had previously observed unexpected changes in
1
some 13C chemical shifts and JC-1,H-1 coupling
constants upon chain extension of the trisaccharide
fragment 1 of the O-SP to higher fragments, such
as 2 and 3 [4,6]. In compound 1, the Gal unit that
1
bears no glycose residue at C-2 shows a JC-1,H-1
value of 171 Hz, which is characteristic of the ꢀ
con®guration [10]. On the other hand, in com-
pounds 2 and 3 the Gal units that have a Rha
residue attached to O-2 of this unit display a
1JC-1,H-1 coupling constant that is 3±4 Hz larger.
* Corresponding author. Tel: 301-975-3135; Fax: 301-
330-3447; e-mail: bruce.coxon@nist.gov
0008-6215/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII S0008-6215(98)00047-0

230
V. Pozsgay et al./Carbohydrate Research 308 (1998) 229±238
This e€ect is mirrored in the 13C chemical shift of
C-3 of the GlcNAc residue to which the Gal unit is
ꢀ-(1!3) linked. Addition of a Rha residue at O-2
of the Gal unit causes the chemical shift of C-3 of
the GlcNAc moiety to decrease from ꢀ78 ppm to
ꢀ75 ppm. A shift in the H-3 signal of 0.07±0.08
ppm was also observed for the GlcNAc residue in
1, relative to 2 and 3. These di€erences have been
interpreted as being due to conformational e€ects,
possibly caused by an increase in steric crowding
on attachment of a Rha residue to O-2 of the Gal
unit [4,6]. This phenomenon has now been investi-
gated further by measurement of the inter-
3
glycosidic JCH coupling constants in the vicinity
of the nuclei that show the unexpected NMR
parameters. Many di€erent methods have been
developed for the measurement of long-range
13C±1H coupling constants, and a number of these
methods have been applied to carbohydrates
[11±27]. Some of the methods are applicable to 13C
at natural abundance, but may have the dis-
advantage of undue complexity, or a requirement
for sophisticated instrumentation. We have chosen
to measure these coupling constants by two-dimen-
1. Results and discussion
Oligosaccharides 1±3 were prepared in a stepwise
fashion using compound 4 [6] as the key starting
material (Scheme 1). The triol 5 obtained from 4
was converted into the diacetate 7 through a
sequence that is shown in Scheme 1, and described
in detail in the Experimental part. The 13C-labeled
galactose donor 11 (Scheme 2) was prepared from
d-(1-13C)galactose, which was ®rst converted into
the diacetate 8 in 51%, in analogy with a procedure
described for the unlabeled analog [4]. Next, the
methylthio group was introduced using Me3SiSMe
as the methanethiol equivalent [29] (!9), and then
1
sional (2D) J-resolved H NMR spectroscopy [28]
of selectively 13C labeled oligosaccharides 1±3,
®rstly, because of the simplicity of the method, and
secondly, because it can be used for older
spectrometers without good inverse detection
capability.
ꢁ
Scheme 1. Reagents and conditions: (a) NaOMe (cat), MeOH, 23 C, 4 h, quant; (b) 7 equiv 4-methoxybenzaldehyde dimethyl-
acetal, 10-camphorsulfonic acid (cat), DMF, 23 ꢁC, 1 h; (c) 1.8 equiv (ClCH2CO)2O, C5H5N, 0 ꢁC, 30 min; (d) HBF4.Et2O, MeOH,
ꢁ
ꢁ
ꢁ
0 C, 1 h; (e) Ac2O, C5H5N, 0 C, 1 h, 61% for four steps; (f) 3.3 equiv thiourea, MeOH±C5H5N, 23 C, 72 h, 64%. Bn=benzyl;
CA=monochloroacetyl.
ꢁ
ꢁ
Scheme 2. Reagents and conditions: (a) Ac2O, HClO4 (cat), 30±40 C, 60 min; (b) HBr, AcOH, 23 C, 90 min; (c) 4 equiv EtOH,
Bu4NBr, 2 equiv 2,4,6-trimethylpyridine, CH2Cl2, 23 ꢁC, 4 d; (d) 4 equiv NaH, DMF, then 4 equiv BnꢁBr; (e) Me3SiOAc, re¯ux, 8 h,
51% for ®ve steps; (f) Me3SiSMe, Me3SiOSO2Me (cat), CH2Cl2 85%; (g) NaOMe (cat), MeOH, 23 C, 48 h, quant; (h) 1.2 equiv
ꢁ
NaH, DMF, then 1.2 equiv MBnCl, 0 C, 3 h, 85%. Bn=benzyl; MBn=4-methoxybenzyl; * Denotes 13C label.

V. Pozsgay et al./Carbohydrate Research 308 (1998) 229±238
231
the 4-methoxybenzyl group was attached through
the intermediacy of the alcohol 10, to provide 11 as
the source of the ꢀ-galactosyl moiety. Thioga-
lactoside 11 was treated with chlorine, and then the
intermediate so obtained was treated, without iso-
lation, with the acceptor 7 under AgOTf activa-
tion, to provide the trisaccharide 12 in 66% yield
(Scheme 3). Oxidative removal of the 4-methoxy-
benzyl group [30] a€orded the acceptor 13, which
was rhamnosylated by using the selectively protected
trichloroacetimidate 14 to give the tetrasaccharide 15
in 89% yield. Next, the mono-chloroacetyl group
was removed (!16) and then the alcohol so obtained
was rhamnosylated with 14. In this reaction, the tar-
get pentasaccharide 17 was obtained in 88% yield.
Conversion of the N3 groups in compounds 13, 16,
and 17 to acetamido groups ((i) PPh3, (ii) Ac2O), and
conventional removal of the O-protecting groups ((i)
NaOMe, (ii) H2/Pd-C) a€orded the unprotected oli-
gosaccharides 1±3, the properties of which matched
those reported for the unlabeled equivalents [6].
The presence of the 13C label in the Gal residue
allowed the measurement of a number of coupling
constants that are not readily detectable in the
unlabeled counterparts. No attempt was made to
determine the signs of these constants. A large
amount of such data is now available for unpro-
tected saccharides and glycosides [31]. However,
comparable data for protected saccharides appears
to be limited. For the acetate 8, both two and
three-bond C±H couplings were seen (Table 1).
The measurement of the corresponding data for
the thiogalactosides 9±11 was hampered by the
1
higher order nature of the H NMR spectra of
these compounds. Nevertheless, the two-bond,
2JC1,H2 coupling constant in 9 could be measured
and is signi®cantly smaller than the value reported
for the corresponding O-galactoside (4.8 versus
6.1 Hz) [32]. A similar pattern was found for the
three-bond 3JC1,SCH coupling constants, which are
3
in the 4.0-4.2 Hz range for 9±11, whereas the cor-
responding value in methyl ꢁ-d-galactopyranoside
is 4.5 Hz. The intra-residual 13C±13C coupling con-
stants (Table 2) for the glycosides 1±3 and 12±17
generally agree with those published for unpro-
tected ꢀ- and ꢁ-d-(1-13C)galactopyranoses [33]
2
except that we did not detect the two bond JC1,C5
coupling in these compounds that was reported for
ꢀ-d-(1-13C)galactopyranose. Sensitivity to the
terminal substituent of the coupling pathway has
been demonstrated previously [31]. Additional
examples of this phenomenon are reported here. In
1
the thiogalactosides 9±11, JC1,C2 is 41±42 Hz and is
Table 1
13C±1H intra-residue coupling constants (Hz) a in galactopyr-
anoses
3
4
3
3
Compound
2JC1,H2 JC1,H3 JC1,H4 JC1,H5 JC1,SCH
3
b
8
9
10
11
6.9
4.8
1.5
0.9
2.4
4.1
4.0
4.2
a
The digital resolution was 0.9 Hz or less.
Spaces indicate that the coupling constant was not deter-
mined.
b
Scheme 3. Reagents and conditions: (a) 0.78 equiv 11, 6.2 equiv 2,6-di-tert-butyl-4-methylpyridine, Cl2 (excess), 23 ꢁC, 2 min, then
hex-1-ene (excess); (b) CF3SO2OAg, CH2Cl2, 78 ꢁC, 3 h, 66% for two steps; (c) 3 equiv (NH4)2Ce(NO3)6, MeCN±H2O, 23 ꢁC, 10
min, then NaHSO3, 80%; (d) 2.5 equiv 14, 0.28 equiv BF3.Et2O, CH2Cl2, 0 ꢁC, 2 h, 89%; (e) 22 equiv thiourea, DMF±C5H5N, 23 ꢁC,
12 h, 91%; (f) 4.8 equiv 14, 0.28 equiv BF3.Et2O, CH2Cl2, 0 ꢁC, 2 h, 88%. Bn=benzyl; CA=monochloroacetyl; *, Denotes 13C label.

232
V. Pozsgay et al./Carbohydrate Research 308 (1998) 229±238
Table 2
13C±13C intra-residue coupling constants (Hz) a in galactopyr-
anoses
Compound
1JC1,C2
2JC1,C3
2JC1,C5
3JC1,C6
b
1
2
3
8
46
47
47
48
42
41
42
47
46
48
47
47.5
3.6
3.2
3.4
5.1
5.1
5.2
5.3
3.9
4.0
3.7
4.0
3.5
4.7
3.4
9
2.3
10
11
12
13
15
16
17
3.5
ꢀ1
a
The digital resolution was 0.9 Hz or less.
b Spaces indicate that the coupling constant was not observed.
signi®cantly smaller by 5±6 Hz than that reported for
2
ꢁ-d-(1-13C)galactopyranose [33]. The JC1,C3 values
for 9±11 are also smaller (3.4, 3.5 versus 4.6 Hz)
3
whereas the opposite was observed for the JC1,C6
couplings (5.1±5.3 versus 4.4 Hz). A global compari-
son of the observed coupling constants indicates their
insensitivity to the environment as long as no direct
substitutions occur at the sugar carbon atoms.
We have now found that the di€erences in the
chemical shifts and the one-bond heteronuclear
coupling constants observed upon chain elongation
of the trisaccharide 1 to 2 and 3 (vide supra) are
also re¯ected in the magnitude of the inter-residue
Fig. 1. 2D J-resolved 1H NMR spectra of the pentasaccharide
3 in deuterium oxide at 500 MHz. (a) full spectrum; the inten-
sities in two regions of the spectrum have been scaled as indi-
cated, to permit an advantageous display on one chart; *
denotes the 13C label; (b) expansion showing the splitting of
3JC-1(C),H-3(B) coupling constant. Values of this
1
coupling constant have been measured for 1±3 by
two-dimensional, J-resolved 1H NMR spectro-
3
the H-3B multiplet (ꢂ 4.066) by the JC-1(C),H-3(B) coupling
constant, in the F2 dimension.
1
scopy, a technique that rotates H±1H splittings
into the second dimension, thus separating them
that bear a rhamnose residue at O-2 of the Gal unit
(Table 3). If it is assumed that these values of the
coupling constants do not result from ensemble
averaging of conformations, but originate from a
single conformation, then application of the Kar-
plus equation
1
from H chemical shifts in the ®rst dimension. In
this work, the heteronuclear 13C±1H splittings
remained in the ®rst dimension, thus distinguishing
them from the 1H±1H splittings in the second
dimension. All of the oligosaccharides studied gave
spectra that were suciently well resolved in the
®rst dimension for the 3JC-1(C),H-3(B) splittings to be
detected. For example, the 2D J-resolved 1H NMR
spectrum of the pentasaccharide derivative 3
shown in Fig. 1, in which the full spectrum appears
3JC;H ˆ 5:7 cos2 0:6 cos ‡ 0:5
ꢀ1†
3
to the JC-1(C),H-3(B) values yields two sets of
interglycosidic dihedral angles
[16]
C
1ꢂC†;H 3ꢂB†
3
in (a) and the JC-1(C),H-3(B) splitting is identi®ed in
(Table 3). Early experience with other oligosacchar-
ides suggests that the smaller set of dihedral angles
may be correct [34], although the larger values could
also be considered [35]. If the smaller set of values is
the spectrum expansion (b). The value of this cou-
pling is 4.9 Hz in trisaccharide 1, but only 4.1±
4.2 Hz in the tetra- and penta-saccharides 2 and 3
taken, then the
values for the higher
C
1ꢂC†;H 3ꢂB†
1 Subscript characters in parentheses denote individual
monosaccharide residues, with A standing for the potential
reducing-end residue.
oligosaccharide homologues 2 and 3 are ꢀ10±11ꢁ
larger than the value for the trisaccharide 1.

V. Pozsgay et al./Carbohydrate Research 308 (1998) 229±238
233
Table 3
Interglycosidic coupling constants (Hz) and dihedral angles of
selectively 13C labeled oligosaccharides
analyses were performed by Atlantic Microlab,
Inc., Norcross, GA.
1
NMR spectroscopy.ÐThe H and the 13C NMR
a
Oligosaccharide
3JC-1(C)-H-3(B)
(‹0.1 Hz b)
C
1ꢂC†;H-3ꢂB†
spectra of the intermediates were recorded at 300
and 75.5 MHz, respectively, by using a Varian
Associates XL-300 spectrometer. Internal refer-
Trisaccharide 1
Tetrasaccharide 2
Pentasaccharide 3
4.9
4.1
4.2
21ꢁ or 146ꢁ
32ꢁ or 138ꢁ
31ꢁ or 139ꢁ
1
ences: Me4Si (0.000 ppm for H NMR of solutions
1
in CDCl3), acetone (2.225 ppm for H NMR and
a
31.00 ppm for 13C NMR of solutions in D2O), and
CDCl3 (77.00 ppm for 13C NMR of solutions in
CDCl3).
Calculated from eq (1). Absolute values are shown.
b Estimated standard uncertainty. According to eq (1), a
0.1 Hz error in the coupling constant results in a 0.9ꢁ±1.6ꢁ
error in the dihedral angle, for the range of angles of interest.
1
The H NMR spectra of solutions of 1±3 (15±
An alternative explanation could be based on a
change in conformational equilibrium. In that case,
the estimated changes of the average dihedral angle
< > would represent the conformational change
of a ``virtual'' conformation [36].
21 mg) in deuterium oxide (0.5 mL) were recorded
at 500 MHz and 300 K by use of a Bruker Instru-
ments AMX-500 NMR spectrometer. The data
were acquired and processed by means of the Bru-
ker XWINNMR program versions 1.2 (Silicon
Graphics Indy R4600, 133 MHz) and 1.3 (Silicon
Graphics Indigo2 R4400, 250 MHz), respectively.
2D J-resolved 1H NMR spectroscopy [28] was
performed by using spectral widths of 2.5 kHz (F2)
and 50 Hz (F1), 90ꢁ and 180ꢁ pulse widths of 9.85
and 19.7 ꢄs, respectively, 80±128 scans per free
induction decay (FID), two dummy scans, a pulse
sequence recycle time of 4.2 s, and data sizes of 16,384
points (F2) and 128 points (F1), zero-®lled or linearly
predicted to 32,768 and 256 points, respectively.
Spectral resolution was enhanced in both dimensions
by Gaussian multiplication of the FID, using a line
broadening of 0.5 Hz and a Gaussian truncation
fraction of 0.3. The digital resolution was 0.08 Hz and
0.2 Hz in the F2 and F1 dimensions, respectively. A
magnitude mode display was used for the 2D J-
1
A correlation of the JC-1,H-1 values of 1±3 with
the dihedral angle ꢃ might also be attempted [35].
However, it has been suggested that this type of
angular dependence is determined mainly by the
orientation of lone pairs of electrons on the glyco-
sidic oxygen atom (O-1) with respect to the C-1±H-
1 bond [35], which may not be entirely appropriate
for a comparison of 1, 2, and 3 that involves
changes in other steric factors and the electro-
negativity of substituents at C-2C.
We conclude that extension of the trisaccharide
1 by attachment of a rhamnose residue at the 2-
position of the Gal unit (! 2, 3) causes a change in
the conformation of the Gal±GlcNAc linkage,
which is re¯ected by enlargement of the
dihedral angle from ꢀ21ꢁ to ꢀ31±
C
1ꢂC†;H 3ꢂB†
32ꢁ, and also changes in the 13C-3B chemical shift
resolved H NMR spectra. For each of the oligo-
1
and 1JC-1(C),H-1(C) coupling constant.
saccharide derivatives 1±3, the 3JC-1(C),H-3(B) coupling
constant was measured as the splitting of the H-3
multiplet of the GlcNAc residue (Scheme 1) in the F2
dimension of the 2D J-resolved 1H NMR spectrum.
Methyl O-(2-azido-2-deoxy-a-d-glucopyrano-
syl)-(1!3)-2,4-di-O-benzyl-a-l-rhamnopyranoside
(5).ÐA catalytic amount of NaOMe was added to
a solution of 4 in MeOH. After 4 h, the solution
was treated with Dowex 50Â2 (H+), ®ltered, and
the ®ltrate concentrated to a€ord 5 (quant) as an
amorphous substance: [ꢀ]d +47ꢁ (c 0.3, CHCl3);
NMR (CDCl3): 1H, ꢂ 4.98 (d, 1 H, J1,2 3.6 Hz, H-1
of GlcN), 4.79, 4.76, 4.68, 4.61 (4 d, 4 H, Jꢀ 12 Hz,
H2C of 2 Bn), 4.67 (d, 1 H, J1,2 2.0 Hz, H-1 of
Rha), 3.29 (s, 3 H, CH3O), 3.20 (dd, 1 H, J2,3
10.2 Hz, H-2 of GlcN), 1.29 (d, 1 H, J5,6 5.6 Hz, H-6
of Rha); 13C, ꢂ 98.1 (C-1 of Rha), 93.7 (C-1 of
GlcN), 79.3, 74.6, 73.3, 71.8, 70.9, 70.7, 67.9, 62.6
2. Experimental
General methods.ÐFor general methods, see ref.
[6]. d-(1-13C)Galactose (13C >99 atom%) was
purchased from Isotec Inc. (Miamisburg, OH).2
Subscript characters
refer to the individual
A±E
monosaccharide residues starting at the reducing
end. Ammonia was used as the ionizing gas for the
chemical ionization (CI) mass spectra. Elemental
2 Certain commercial equipment, instruments, or material
are identi®ed in this paper to specify adequately the experi-
mental procedure. Such identi®cation does not imply recom-
mendation by the National Institute of Standards and
Technology, nor does it imply that the materials are necessa-
rily the best available for the purpose.

234
V. Pozsgay et al./Carbohydrate Research 308 (1998) 229±238
(C-2,3,4,5 of Rha, C-2,3,4,5 of GlcN), 75.4, 72.7
(CH2 of Bn), 61.4 (C-6 of GlcN), 54.8 (CH3O),
17.9 (C-6 of Rha); CI-MS: m/z 563 [(M+NH4)+].
Anal. Calcd for C27H35N3O9: C, 59.44; H, 6.47.
Found: C, 59.34; H, 6.50.
by column chromatographic puri®cation of the
residue (2:1 hexane±EtOAc) gave 7 (8.0 g, 64%) as
an amorphous solid: [ꢀ]d +67ꢁ (c 0.4, CHCl3);
1
NMR (CDCl3): H ꢂ 4.99 (d, 1 H, J1,2 3.5 Hz, H-1
of GlcN), 4.83 (dd, 1 H, J 9.1 Hz, J 10.2 Hz, H-4 of
GlcN), 4.80, 4.78, 4.71, 4.68 (4 d, Jꢀ 11 Hz, H2C of
2 Bn), 4.70 (d, 1 H, J1,2 1.9 Hz, H-1 of Rha), 3.81
(dd, 1 H, J2,3 3.2 Hz, H-2 of Rha), 3.32 (dd, 1 H,
J2,3 10.3 Hz, H-2 of GlcN), 3.30 (s, 3 H, CH3O),
2.03, 1.99 (2 s, 6 H, 2 CH3CO), 1.33 (d, 1 H, J5,6
6.2 Hz, H-6 of Rha); 13C, ꢂ 98.3 (C-1 of Rha), 93.0
(C-1 of GlcN), 79.2, 74.4, 72.9, 71.0, 70.6, 68.0,
67.2, 63.1, (C-2,3,4,5 of Rha, C-2,3,4,5 of GlcN),
75.1, 72.6 (CH2 of 2 Bn), 61.6 (C-6 of GlcN), 54.7
(CH3O), 20.7, 20.6 (2 CH3CO), 17.9 (C-6 of Rha);
CI-MS: m/z 647 [(M+NH4)+]. Anal. Calcd for
C31H39N3O11: C, 59.13; H, 6.24. Found: C, 59.04;
H, 6.27.
1,2-Di-O-acetyl-3,4,6-tri-O-benzyl-b-d-(1-13C)gal-
actopyranose (8).ÐTo a stirred solution of HClO4
(40 ꢄL) in Ac2O (15 mL) was added d-(1-13C)
galactose (3 g, 16.6 mmol) in portions at 30±40 ꢁC
over a period of 60 min. The solution was stirred
for another 10 min then was concentrated at
<30 ꢁC. To a solution of the residual syrup in
AcOH (5 mL) was added a solution of HBr in
AcOH (15 mL of a 33% solution) at 23 ꢁC. After
90 min, the solution was diluted with CHCl3
(100 mL) and was then extracted with H2O
(3Â200 mL). The organic phase was dried
(Na2SO4) and then concentrated. To a solution of
the residue in anhydrous CH2Cl2 (30 mL) were
added 2,4,6-trimethylpyridine (4 mL, 30 mmol),
EtOH (4 mL, 65 mmol), and Bu4NBr (0.4 g) at
23 ꢁC. After 4 days, the mixture was concentrated,
and the residue treated with ether. The solids were
removed by ®ltration, and the residue obtained
after concentration of the ®ltrate was puri®ed by
column chromatography (2:1 hexane±EtOAc). The
fractions containing the product were pooled and
concentrated. Residual trimethylpyridine was
removed at ꢀ0.7 Pa (5 mtorr) to give 4.4 g of a
syrup that was dissolved in dry DMF (40 mL). To
this solution was added under stirring NaH (2.5 g
of a 60% suspension in oil, ꢀ 63 mmol) in portions
at 0 ꢁC, followed by benzyl bromide (7.2 mL,
61 mmol). The mixture was stirred for 4 h then
processed as usual, followed by column chromato-
graphic puri®cation (9:1 hexane±EtOAc) to give a
syrupy residue. A solution of the syrup so obtained
in Me3SiOAc (90 mL) was stirred under re¯ux for
8 h. Concentration of the solution followed by
Methyl O-(4,6-di-O-acetyl-2-azido-3-O-chloro-
acetyl-2-deoxy-a-d-glucopyranosyl)-(1!3)-2,4-di-
O-benzyl-a-l-rhamnopyranoside (6).ÐA solution of
5 (19 g, 35 mmol) in DMF (40 mL) and 4-methox-
ybenzaldehyde dimethyl acetal (30 mL, 205 mmol)
was treated with 10-camphorsulfonic acid (ꢀ 0.5 g)
at 23 ꢁC. After 1 h, the solution was treated with
Et3N (2 mL). The solution was vigorously stirred
with hexane (3Â200 mL) and the supernatant dec-
anted. The residue was equilibrated with CHCl3
and H2O, the organic phase was dried (Na2SO4),
and then concentrated. A solution of the residue
in pyridine (50 mL) was treated with mono-
chloroacetic anhydride (11 g, 64 mmol) at 0 ꢁC.
After 30 min, the solution was treated with MeOH
(20 mL) and then concentrated. A solution of the
residue in MeOH (150 mL) was treated with HBF4
(54% in ether, 1 mL) at 0 ꢁC. After 1 h, the solution
was treated with aq NaHCO3 until pH ꢀ5 was
reached (indicator paper). The solution was con-
centrated to ꢀ100 mL, and then the residue was
equilibrated between CHCl3 and H2O. The organic
phase was dried (Na2SO4) and concentrated. A
solution of the residue in pyridine (30 mL) was
treated with Ac2O (20 mL) at 0 ꢁC. The usual
work-up followed by column chromatography (2:1
hexanes±EtOAc) of the residue a€orded 6 (15.0 g,
61% for four steps) as a colorless syrup: [ꢀ]d
ꢁ
1
+105 (c 0.2, CHCl3); NMR (CDCl3): H, ꢂ 5.57
(t, 1 H, J 9.8 Hz, H-3 of GlcN), 3.36 (dd, 1 H, J2,3
10.2 Hz, H-2 of GlcN), 3.31 (s, 3 H, CH3O), 2.03,
1.90 (2 s, 6 H, 2 CH3CO), 1.37 (d, 1 H, J5,6 6.2 Hz,
H-6 of Rha); 13C, ꢂ 98.1 (C-1 of Rha), 93.0 (C-1 of
GlcN), 79.1, 75.0, 72.7, 72.5, 68.0, 67.8, 67.2, 60.6
(C-2,3,4,5 of Rha, C-2,3,4,5 of GlcN), 75.4, 72.5
(CH2 of 2 Bn), 61.3 (C-6 of GlcN), 54.8 (CH3O),
40.4 (CH2Cl), 20.6, 20.5 (2 CH3CO), 17.9 (C-6 of
Rha). Anal. Calcd for C33H40ClN3O12: C, 56.13;
H, 5.71. Found: C, 56.23; H, 5.79.
Methyl O-(4,6-di-O-acetyl-2-azido-2-deoxy-a-d-
glucopyranosyl)-(1!3)-2,4-di-O-benzyl-a-l-rhamno-
pyranoside (7).ÐA solution of 6 (19 g, 20 mmol) in
MeOH (100 mL) and pyridine (2 mL) was treated
with thiourea (5 g, 66 mmol) at 23 ꢁC. After 72 h,
the mixture was concentrated at <30 ꢁC. The resi-
due was stirred with CHCl3 (100 mL) and was ®l-
tered. Concentration of the mother liquor followed

V. Pozsgay et al./Carbohydrate Research 308 (1998) 229±238
235
equilibration of the residue between CHCl3 and
H2O and crystallization (iPr2O) a€orded 8 (4.5 g,
51% for 5 steps) which had physical properties
identical to those of the unlabeled preparation [37].
NMR (CDCl3): 1H ꢂ 5.57 (dd, 1 H, JH-1,H-2 8.1 Hz,
JH-1,C-1 166 Hz, H-1), 5.48 (ddd, 1 H, JH-2,H-3
10 Hz, JH-2,H-1 6.9 Hz, H-2), 4.95, 4.69, 4.61, 4.52,
4.46, 4.35 (6 d, 6 H, CH2 of 3 Bn), 4.00 (dd, 1 H,
JH-3,H-4 2.8 Hz, JH-4,C-1 0.9 Hz, H-4), 3.71 (dddd, 1
ꢀ1 Hz, C-5), 75.3, 74.4, 73.5, 72.6 (CH2 of 3 Bn,
MBn), 73.6 (C-4), 68.6 (d, JC-1,C-6 5.3 Hz, C-6),
55.3 (CH3O), 12.8 (CH3S); CI-MS: m/z 619
[(M+NH4)+].
Methyl O-[3,4,6-tri-O-benzyl-2-O-(4-methoxy-
benzyl)-a-d-(1-13C)galactopyranosyl]-(1!3)-O-
(4,6-di-O-acetyl-2-azido-2-deoxy-a-d-glucopyrano-
syl)-(1!3)-2,4-di-O-benzyl-a-l-rhamnopyranoside
(12).ÐTo a solution of 11 (150 mg, 0.25 mmol),
and 2,6-di-tert-butyl-4-methylpyridine (300 mg,
1.5 mmol) in CH2Cl2 (5 mL) was added at 23 ꢁC a
solution of Cl2 in CCl4 (excess). After 2 min, 1-
hexene was added until the yellow color dis-
appeared. This solution was transferred by syringe
to a stirred mixture of 7 (200 mg, 0.32 mmol), 2,6-
di-tert-butyl-4-methylpyridine (100 mg, 0.5 mmol),
4A molecular sieves (100 mg), and CH2Cl2 (2 mL).
The mixture was stirred at 23 ꢁC for 15 min and
then cooled to 78 ꢁC and treated with CF3-
SO2OAg (126 mg, 0.49 mmol). After 3 h, the mix-
ture was processed as usual followed by column-
chromatographic puri®cation (2:1 hexane±EtOAc)
to a€ord 12 (194 mg, 66%) as a syrup: [ꢀ]d +65ꢁ
0
H, JH-5,H-6 5.8 Hz, JH-5,H-6 7.1 Hz, JH-5,C-1 2.4 Hz,
H-5), 3.66±3.57 (m, 2 H, H-6,60), 3.58 (ddd, 1 H,
JH-3,C-1 1.5 Hz, H-3), 2.1, 2.0 (2 s, 6 H, 2 CH3CO);
13C, ꢂ 92.6 (C-1), 80.0 (d, JC-1,C-3 4.7 Hz, C-3), 74.6,
73.5, 72.0 (CH2 of 3 Bn), 74.3 (C-5), 72.1 (C-4),
70.3 (d, JC-1,C-2 48 Hz, C-2), 67.8 (d, JC-1,C-6 5.1 Hz,
C-6), 20.9, 20.8 (2 CH3CO); CI-MS: m/z 553
[(M+NH4)+].
Methyl 2-O-acetyl-3,4,6-tri-O-benzyl-1-thio-b-d-
(1-13C)galactopyranoside (9).ÐTreatment of 8 with
MeSSiMe3 as described for the unlabeled com-
pound a€orded 9 which had physical properties
identical to those of the unlabeled preparation [4].
NMR (CDCl3): 1H ꢂ 4.23 (dd, 1 H, JH-1,H-2 9.8 Hz,
JH-1,C-1 152 Hz, H-1), 5.44 (dt, 1 H, JH-2,H-3 9.8 Hz,
JH-2,C-1 4.8 Hz, H-2), 4.95, 4.68, 4.58, 4.54, 4.46,
4.41 (6 d, 6 H, CH2 of 3 Bn), 4.00 (br d, 1 H, JH-
1
(c 0.6, CHCl3); NMR (CDCl3): H ꢂ 5.15 (dd, 1
H, J 10.3 Hz, J 8.8 Hz, H-4 of GlcN), 5.07 (dd, 1
H, JH-1,H-2 3.4 Hz, JH-1,C-1 171 Hz, H-1 of Gal),
5.05 (d, 1 H, J 3.6 Hz, H-1 of GlcN), 3.74 (CH3O
of MBn), 3.37 (dd, 1 H, J2,3 9.9 Hz, H-2 of GlcN),
5.15 (dd, 1H, J 10.3 Hz, J 8.8 Hz, H-4 of GlcN),
3.27 [CH3O (aglycon)], 2.03, 1.75 (2 CH3CO), 1.33
(d, 1 H, J5,6 6.2 Hz, H-6 of Rha); 13C, ꢂ 98.6 (C-1
of Gal), 92.8 (C-1 of GlcN), 79.1, 79.0, 74.8, 74.2,
74.0, 72.7, 70.2, 70.0, 67.9, 67.2, 62.6 (C-2,3,4,5 of
Rha, C-2,3,4,5 of GlcN, C-3,4,5 of Gal), 75.2,
74.9, 73.3, 73.2, 72.8, 72.5 (CH2 of 5 Bn, MBn),
74.8 (d, JC-1,C-2 47 Hz, C-2 of Gal), 68.5 (d, JC-1,C-6
3.9 Hz, C-6 of Gal), 61.6 (C-6 of GlcN), 55.1,
54.8 (2 CH3O), 20.8, 20.6 (2 CH3CO), 17.8 (C-6 of
Rha). FAB-MS: m/z 1156 [(M-N2+H)+]. Anal.
2.8 Hz, H-4), 2.14 (d, 3 H, J
4.1 Hz,
C-1,SCH3
3,H-4
CH3S); 13C, ꢂ 83.0 (C-1), 81.3 (d, JC-1,C-3 3.4 Hz, C-
3), 77.4 (d, JC-1,C-5 2.3 Hz, C-5), 74.4, 73.5, 71.9
(CH2 of 3 Bn), 73.0 (C-4), 68.8 (d, JC-1,C-2 42 Hz,
C-2), 68.3 (d, JC-1,C-6 5.1 Hz, C-6), 21.0 (CH3CO),
10.9 (CH3S); CI-MS: m/z 541 [(M+NH4)+].
Methyl 3,4,6-tri-O-benzyl-1-thio-b-d-(1-13C)-
galactopyranoside (10).ÐTreatment of 9 with
NaOMe±MeOH as described for the preparation
of 5 a€orded 10 (95%) that had physical properties
identical with those of the unlabeled equivalent [4].
NMR (CDCl3): 1H ꢂ 2.19 (d, 3 H, JC-1,SCH 4.0 Hz,
3
CH3S), 13C ꢂ 86.0 (C-1), 83.1 (C-3), 77.5 (C-5),
74.5, 73.5, 72.3 (CH2 of 3 Bn), 73.2 (C-4), 69.0 (d,
JC-1,C-2 41 Hz, C-2), 68.5 (d, JC-1,C-6 5.2 Hz, C-6),
11.6 (CH3S); CI-MS: m/z 499 [(M+NH4)+].
.
Calcd for C66H75 N3O17 EtOAc: C, 65.96; H, 6.50.
Found: C, 65.45; H, 6.21.
Methyl O-[3,4,6-tri-O-benzyl-a-d-(1-13C)galacto-
pyranosyl]-(1!3)-O-(4,6-di-O-acetyl-2-azido-2-de-
oxy-a-d-glucopyranosyl)-(1!3)-2,4-di-O-benzyl-a-
l-rhamnopyranoside (13).ÐTo a solution of 12 (5 g,
4.2 mmol) in MeCN (10 mL) and H2O (10 mL) was
added (NH4)2Ce(NO3)6 (7 g, 12.8 mmol) at 23 ꢁC.
After 10 min, the mixture was treated with aq
NaHSO3 then was concentrated. Equilibration of
the residue between CHCl3 and H2O, followed by
concentration of the organic layer and column
chromatographic puri®cation of the residue
Methyl 3,4,6-tri-O-benzyl-2-O-(4-methoxybenzyl)-
1-thio-b-d-(1-13C)galactopyranoside (11).ÐTreat-
ment of 10 with NaH then with 4-methoxybenzyl
chloride, as described for the unlabeled counter-
part a€orded 11 which had physical properties
identical to those of the unlabeled analog [4].
NMR (CDCl3): 1H ꢂ 4.32 (dd, 1 H, JH-1,H-2 9.6 Hz,
JH-1,C-1 152 Hz, H-1), 2.20 (d, 3 H, JC-1,SCH 4.2 Hz,
3
CH3S); 13C, ꢂ 85.7 (C-1), 84.0 (d, JC-1,C-3 3.5 Hz, C-
3), 77.6 (d, JC-1,C-2 42 Hz, C-2), 77.2 (d, JC-1,C-5

236
V. Pozsgay et al./Carbohydrate Research 308 (1998) 229±238
a€orded 13 as a syrup (3.6 g, 80%): [ꢀ]d +124ꢁ (c
0.1, CHCl3); NMR (CDCl3): H ꢂ 4.97 (dd, 1 H,
ido-2-deoxy-a-d-glucopyranosyl)-(1!3)-2,4-di-O-
benzyl-a-l-rhamnopyranoside (16).ÐTo a solution
of 15 (4.15 g, 2.8 mmol) in pyridine (2 mL) and
DMF (20 mL) was added thiourea (5 g, 63 mmol)
at 23 ꢁC. After 12 h, the solution was concentrated
at <30 ꢁC. To the residue was added CHCl3
(50 mL). The insoluble material was removed by
®ltration and the ®ltrate was extracted with H2O
(3Â50 mL). Concentration of the solution, fol-
lowed by column chromatographic puri®cation of
the residue (3:2 hexane±EtOAc) a€orded 16 as an
amorphous solid (3.58 g, 91%): [ꢀ]d +57ꢁ (c 0.2,
1
J1,2 3.9 Hz, JH-1,C-1 172 Hz, H-1 of Gal), 5.06 (d, 1
H, J 3.6 Hz, H-1 of GlcN), 4.97 (dd, 1 H, J
10.0 Hz, J 9.2 Hz, H-4 of GlcN), 3.30 (CH3O),
2.04, 1.92 (2 CH3CO), 1.35 (d, 1 H, J5,6 6.2 Hz, H-6
of Rha); 13C, ꢂ 101.5 (C-1 of Gal), 98.2 (C-1 of
Rha), 93.3 (C-1 of GlcN), 79.2 (2 C), 76.1, 73.8,
73.7, 72.36, 70.3, 70.2, 67.9, 67.2, 61.3 (C-2,3,4,5 of
Rha, C-2,3,4,5 of GlcN, C-3,4,5 of Gal), 75.2, 74.9,
73.5, 72.40, 72.2 (CH2 of 5 Bn), 68.6 (d, JC-1,C-2
46 Hz, C-2 of Gal), 68.3 (d, JC-1,C-6 4.0 Hz, C-6 of
Gal), 61.5 (C-6 of GlcN), 54.9 (CH3O), 20.9, 20.6
(2 CH3CO), 17.9 (C-6 of Rha). FAB-MS: m/z
1035.47 [(M-N2+H)+], 1061.39 [(M-H2+H)+].
Anal. Calcd for C58H67N3O16: C, 65.59; H, 6.36.
Found: C, 65.17; H, 6.36.
1
CHCl3); NMR (CDCl3): H ꢂ 5.46 (dd, 1 H, J1,2
1.7 Hz, J2,3 3.4 Hz, H-2 of Rhad), 4.71 (dd, 1 H,
J1,2 3.4 Hz, JH-1,C-1 170.5 Hz, H-1 of Gal), 5.36 (d,
1 H, H-1 of Rhad), 5.10 (dd, 1 H, J 10.4 Hz, J
8.9 Hz, H-4 of GlcN), 4.99 (d, 1 H, J 3.9 Hz, H-1
of GlcN), 3.29 (CH3O), 2.01, 1.95 (2 CH3CO),
1.37, 1.28 (2 d, 6 H, J5,6 ꢀ 6 Hz, H-6 of 2 Rha);
13C, ꢂ 96.8 (C-1 of Gal), 93.0 (C-1 of GlcN), 81.4,
79.0 (2 C), 74.7, 74.2 (2 C), 72.7 (2 C), 70.4, 70.1,
69.4, 68.3, 67.9, 66.8, 63.3 (C-2,3,4,5 of 2 Rha, C-
2,3,4,5 of GlcN, C-3,4,5 of Gal), 75.2, 75.0, 74.8,
73.3, 72.9, 72.7 (CH2 of 6 Bn), 71.9 (d, JC-1,C-2
47 Hz, C-2 of Gal), 68.8 (d, JC-1,C-6 4.0 Hz, C-6 of
Gal), 61.5 (C-6 of GlcN), 54.7 (CH3O), 20.9, 20.6
(2 CH3CO), 18.2, 17.8 (C-6 of 2 Rha). FAB-MS:
m/z 1375.60 [(M-N2+H)+]. Anal. Calcd for C78
H87N3O21: C, 66.80; H, 6.25. Found: C, 66.54; H,
6.27.
Methyl O-(2-O-benzoyl-4-O-benzyl-3-O-chloro-
acetyl-a-l-rhamnopyranosyl)-(1!2)-O-[3,4,6-tri-
O-benzyl-a-d-(1-13C)galactopyranosyl]-(1!3)-O-
(4,6-di-O-acetyl-2-azido-2-deoxy-a-d-glucopyrano-
syl)-(1!3)-2,4-di-O-benzyl-a-l-rhamnopyranoside
(15).ÐTo
a
stirred mixture of 13 (3.40 g,
3.2 mmol), 14 (4.60 g, 7.9 mmol), 4A molecular
sieves (2 g), and CH2Cl2 (33 mL) was added at 0 ꢁC
.
BF3 Et2O (270 ꢄL, 2.2 mmol). After 2 h, the mix-
ture was ®ltered and concentrated. Column-chro-
matographic puri®cation of the residue (3:2
hexane±EtOAc) a€orded 15 (4.20 g, 89%) as an
amorphous solid: [ꢀ]d +75ꢁ (c 0.2, CHCl3); NMR
(CDCl3): 1H ꢂ 5.64 (dd, 1 H, J1,2 1.7 Hz, J2,3
3.3 Hz, H-2 of Rhad), 5.40 (dd, 1 H, J3,4 9.7 Hz, H-
3 of Rhad), 5.25 (dd, 1 H, J1,2 3.4 Hz, JH-1,C-1
170.5 Hz, H-1 of Gal), 5.30 (d, 1 H, H-1 of Rhad),
5.15 (dd, 1 H, J 10.0 Hz, J 9.2 Hz, H-4 of GlcN),
4.99 (d, 1 H, J 4.0 Hz, H-1 of GlcN), 4.71 (d, 1
H, H-1 of Rhaa), 3.30 (s, 3 H, CH3O), 1.99, 1.96
(2 CH3CO), 1.33, 1.29 (2 d, 6 H, J5,6 ꢀ 6 Hz, H-6
of 2 Rha); 13C, ꢂ 96.8 (C-1 of Gal), 93.0 (C-1 of
GlcN), 79.0, 78.8, 78.4, 74.6, 74.4, 74.0, 73.6, 70.2
(2 C), 70.0, 69.6, 68.1, 68.0, 66.9, 62.7 (C-2,3,4,5 of
2 Rha, C-2,3,4,5 of GlcN, C-3,4,5 of Gal), 75.1,
74.9, 74.8, 73.3, 72.9, 72.7 (CH2 of 6 Bn), 73.7 (d,
JC-1,C-2 48 Hz, C-2 of Gal), 68.9 (d, JC-1,C-6 3.7 Hz,
C-6 of Gal), 61.5 (C-6 of GlcN), 54.8 (CH3O), 20.9,
20.6 (2 CH3CO), 18.0, 17.9 (C-6 of 2 Rha). FAB-
MS: m/z 1451.53 [(M-N2+H)+]. Anal. Calcd for
C80H88ClN3O22: C, 65.06; H, 6.18. Found: C,
64.80; H, 6.03.
Methyl O-(2-O-benzoyl-4-O-benzyl-3-O-chloro-
acetyl-a-l-rhamnopyranosyl)-(1!3)-O-(2-O-benz-
oyl-4-O-benzyl-a-l-rhamnopyranosyl)-(1!2)-O-
[3,4,6-tri-O-benzyl-a-d-(1-13C)galactopyranosyl]-
(1!3)-O-(4,6-di-O-acetyl-2-azido-2-deoxy-a-d-
glucopyranosyl)-(1!3)-2,4-di-O-benzyl-a-l-rhamno-
pyranoside (17).ÐReaction of 16 (490 mg,
0.35 mmol) and 14 (1 g, 1.7 mmol) as described for
the preparation of 15 a€orded, after column-chro-
matographic puri®cation (3:2 hexane±EtOAc), 17
(560 mg, 88%) as an amorphous solid: [ꢀ]d +59ꢁ (c
1
0.2, CHCl3); NMR (CDCl3): H ꢂ 5.61, 5.55 (2 dd,
2 H, J1,2 1.7 Hz, J2,3 3.3 Hz, H-2 of Rhad, Rhae),
5.46 (d, 1 H, H-1 of Rhae), 5.37 (dd, 1 H, J3,4
9.8 Hz, H-3 of Rhae), 5.21 (d, 1 H, H-1 of Rhad),
5.17 (dd, 1 H, J1,2 3.7 Hz, JH-1,C-1 171 Hz, H-1 of
Gal), 5.10 (dd, 1 H, J 10.9 Hz, J 8.9 Hz, H-4 of
GlcN), 4.98 (d, 1 H, J 3.8 Hz, H-1 of GlcN), 4.70
(d, 1 H, H-1 of Rhaa), 3.29 (s, 3 H, CH3O), 1.82,
1.80 (2 CH3CO), 1.30, 1.29, 1.23 (3 d, 9 H, J5,6
ꢀ6 Hz, H-6 of 3 Rha); 13C, ꢂ 96.8 (C-1 of Gal),
92.7 (C-1 of GlcN), 79.8, 79.0, 78.9, 78.5, 78.1,
Methyl O-(2-O-benzoyl-4-O-benzyl-a-l-rhamno-
pyranosyl)-(1!2)-O-[3,4,6-tri-O-benzyl-a-d-(1-13C)-
galactopyranosyl]-(1!3)-O-(4,6-di-O-acetyl-2-az-

V. Pozsgay et al./Carbohydrate Research 308 (1998) 229±238
237
74.6, 74.4, 73.6, 72.7, 72.5, 72.3, 70.4, 70.2, 70.0,
68.4, 68.3, 68.0, 67.2, 62.6 (C-2,3,4,5 of 3 Rha, C-
2,3,4,5 of GlcN, C-3,4,5 of Gal), 75.4, 75.1, 74.8,
73.9, 73.3, 73.0 (2 C) (CH2 of 7 Bn), 71.9 (d, JC-1,C-
Transformations of compound 17 as described for
the preparation of 1 a€orded 3 as an amorphous
substance having [ꢀ]d identical to that of the unla-
1
beled counterpart [6]; NMR (D2O): H ꢂ 5.59 (dd,
47.5 Hz, C-2 of Gal), 68.8 (d, JC-1,C-6 3.5 Hz, C-6
1 H, J1,2 3.6 Hz, JH-1,C-1 174 Hz; 13C, ꢂ 74.5 (d, JC-
1,C-2 47 Hz, C-2 of Gal), 61.5 (d, JC-1,C-6 3.4 Hz, C-6
of Gal).
2
of Gal), 61.4 (C-6 of GlcN), 54.8 (CH3O), 20.8,
20.4 (2 CH3CO), 18.2, 17.9 (2 C) (C-6 of 3 Rha).
FAB-MS: m/z 1791.82 [(M-N2+H)+]. Anal. Calcd
for C100H108ClN3O27: C, 66.02; H, 5.98. Found: C,
66.13; H, 6.05.
Acknowledgements
Methyl O-a-d-(1-13C)galactopyranosyl-(1!3)-
O-(2-acetamido-2-deoxy-a-d-glucopyranosyl)-(1!3)-
O-a-l-rhamnopyranoside (1).ÐA solution of 13
(650 mg, 0.55 mmol) and Ph3P (600 mg, 2.3 mmol)
in CH2Cl2 (6 mL) was stirred at 23 ꢁC for 24 h, and
was then treated with H2O (0.5 mL). After another
36 h, the solution was cooled to 0 ꢁC and treated
with Ac2O (0.5 mL). The solution was extracted
with H2O, and was then concentrated. Column
chromatographic puri®cation of the residue (2:1
hexane±EtOAc) gave an amorphous solid (463 mg).
A solution of the intermediate in dry MeOH
(5 mL) was treated at 23 ꢁC with NaOMe
(ꢀ10 mg). After 72 h, the solution was treated with
Dowex 50Â8±100 (H+), and then concentrated.
The residue was dissolved in a mixture of EtOH
(10 mL) and AcOH (1 mL) and was hydro-
genolyzed over Pd-C (10%, ꢀ0.2 g) at 1.4 MPa
(200 psi) for 24 h. The mixture was ®ltered and the
®ltrate concentrated. The residue was puri®ed
through Biogel P-2 using 0.05 N pyridine±AcOH
as the eluant. The fractions containing the product
were pooled and the combined solution was freeze-
dried to a€ord 1 as an amorphous solid (220 mg)
having [ꢀ]d identical with that of the unlabeled
equivalent [6]; NMR (D2O): 13C, ꢂ 77.9 (br, C-3 of
GlcN), 71.5 (br, C-5 of Gal), 69.2 (d, JC-1,C-2 46 Hz,
C-2 of Gal), (d, JC-1,C-6 3.6 Hz, C-6 of Gal).
We thank Dr. Lewis Pannell and Mr. Noel
Whittaker for the mass spectra.
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Methyl O-a-l-rhamnopyranosyl-(1!2)-O-a-d-
(1-13C)galactopyranosyl-(1!3)-O-(2-acetamido-
2-deoxy-a-d-glucopyranosyl)-(1!3)-O-a-l-rhamno-
pyranoside (2).ÐTransformations of compound 16
as described for the preparation of 1 a€orded 2 as
an amorphous substance having [ꢀ]d identical to
that of the unlabeled equivalent [6]; NMR (D2O):
1H ꢂ 5.59 (dd, 1 H, J1,2 3.6 Hz, JH-1,C-1 174 Hz; 13C,
ꢂ 74.4 (d, JC-1,C-2 47 Hz, C-2 of Gal), 61.5 (d, JC-1,C-
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3.2 Hz, C-6 of Gal).
6
Methyl O-a-l-rhamnopyranosyl-(1!3)-O-a-l-
rhamnopyranosyl-(1!2)-O-a-d-(1-13C)galactopyr-
anosyl-(1!3)-O-(2-acetamido-2-deoxy-a-d-gluco-
pyranosyl)-(1!3)-O-a-l rhamnopyranoside (3).Ð

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