Synthesis of and molecular dynamics simulations on a tetrasaccharide corresponding to the repeating unit of the capsular polysaccharide from Salmonella enteritidis

Article abstract of DOI:10.1039/b823428k

Syntheses of two oligosaccharides as methyl glycosides related to the repeating unit of S. enteritidis capsular polysaccharide (CPS) are presented. The trisaccharide corresponds to the backbone of the CPS whereas the tetrasaccharide is a model for the rep

Full text of DOI:10.1039/b823428k

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of and molecular dynamics simulations on a tetrasaccharide
corresponding to the repeating unit of the capsular polysaccharide from
Salmonella enteritidis†
Johan D. M. Olsson,a Jens Landstro¨m,a Jerk Ro¨nnols,a Stefan Oscarsona,b and Go¨ran Widmalm*a
Received 5th January 2009, Accepted 30th January 2009
First published as an Advance Article on the web 4th March 2009
DOI: 10.1039/b823428k
Syntheses of two oligosaccharides as methyl glycosides related to the repeating unit of S. enteritidis
capsular polysaccharide (CPS) are presented. The trisaccharide corresponds to the backbone of the
CPS whereas the tetrasaccharide is a model for the repeating unit which has a branched structure.
Molecular dynamics simulations investigating their flexibility and dynamics revealed that the
oligosaccharides populate several conformational states and indicate that conformational averaging
should be used in describing the accessible conformational space.
We therefore judged it timely to report on our syntheses and MD
simulations of the tri- and tetrasaccharides.
Introduction
Salmonella enterica serovar Enteritidis is a food-borne human
pathogen causing gasteroentritis. Gram-negative bacteria, to
which S. enteritidis belong, carry a lipopolysaccharide (LPS) in
their outer membrane and, in addition, associated extracellular
polysaccharides in some cases. The recent progress in the under-
standing of the importance of a bacterial capsule, e.g., in immune
evasion and survival strategy where it protects the bacterium
from desiccation, has spurred novel interest in the structure of
capsular polysaccharides (CPSs).1 Furthermore, recent studies on
the interaction of an O-antigen-derived octasaccharide with phage
P22 tailspike protein has shed light on the binding process.2 In
S. enteritidis the CPS has the same basic repeating unit as that
of its LPS, consisting of a tetrasaccharide repeating unit with
the following structure: →3)-a-D-Galp-(1→2)[a-Tyvp-(1→3)]-a-
D-Manp-(1→4)-a-L-Rhap-(1→, in which Tyvp is a 3,6-dideoxy-
D-arabino-hexopyranosyl group.3 Structural differences between
the LPS and the CPS occur, and comprise the degree and
positions of glucosylation, the extent of which has been described
as being important for virulence.4 Synthetic organic chemistry
provides an excellent tool to produce well-defined (homogeneous)
oligosaccharides5–7 corresponding to the above repeating unit.
We have therefore synthesized a tetrasaccharide as a methyl
glycoside, since such a derivative is suitable for e.g. solution-
state NMR interaction studies.8 We have also carried out a
molecular simulation on this derivative, to gain information on
its conformational preferences and flexibility, e.g. in comparison
to previously reported studies that utilized HSEA calculations on
similar oligosaccharides.9 After we had completed the synthesis
and during the analysis of the molecular dynamics (MD) simula-
tions, a different synthesis of the same tetrasaccharide appeared.10
Results and discussion
Our synthetic strategy, in contrast to the recently published
synthesis by Son et al.,10 includes synthesis of the a-D-Galp-(1→2)-
a-D-Manp-(1→4)-a-L-Rhap ‘backbone’ trisaccharide starting
from three known monosaccharide units, and subsequent addi-
tion of a 3,6-dideoxy-D-arabino-hexopyranoside donor to com-
plete the synthesis of tetrasaccharide 17. To access mannosyl
donor 2, ethyl 4,6-O-benzylidene-3-O-p-methoxybenzyl-1-thio-a-
D-mannopyranoside (1)11 was benzoylated (Scheme 1).
aDepartment of Organic Chemistry, Arrhenius Laboratory, Stockholm
University, S-106 91, Stockholm, Sweden. E-mail: gw@organ.su.se
bCentre for Synthesis and Chemical Biology, University College Dublin,
Belfield, Dublin 4, Ireland
Scheme 1 Reagents and conditions: i) BzCl, pyridine, 89%; ii) NIS, AgOTf,
† Electronic supplementary information (ESI) available: 1H and 13C NMR
spectra and chemical shift assignments of a constituent trisaccharide and
the target tetrasaccharide. See DOI: 10.1039/b823428k
4 A MS, CH2Cl2, -30 ◦C, 92%; iii) NaOMe, MeOH/CH2Cl2 (2:1), 90%;
˚
iv) NIS, AgOTf, 4 A MS, Et2O, -30 ◦C, 85%.
˚
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NIS-promoted glycosylation at -30 ◦C with methyl 2,3-O-
isopropylidene-a-L-rhamnopyranoside (3)12 gave the desired dis-
accharide 4 in 92% yield. Debenzoylation, followed by a second
NIS-promoted a-glycosylation with ethyl 2,3,4,6-tetra-O-benzyl-
1-thio-b-D-galactopyranoside (6)13 as donor in diethyl ether at
-30 ◦C, gave protected trisaccharide 7 exclusively, in 85% yield.
In the synthesis of the novel tyvelose donor 12, our first
approach was to introduce both deoxy functions in a sin-
gle reaction. In contrast to Bundle et al.,14 the Barton-
McCombie deoxygenation15 of various 3,6-di-O-thiocarbonyl-
imidazole derivatives did not give the desired product in satisfying
yield. Neither did hydride addition using NaBH4 to the corre-
sponding 3,6-di-O-tosylate or 3,6-di-O-trifluoromethanesulfonate
compounds. Therefore, this route was abandoned and the deoxy
functions were installed separately to afford the desired tyvelose
donor. Ethyl 2,3-O-isopropylidene-1-thio-a-D-mannopyranoside
(8) was regioselectively tosylated at O-6, followed by benzoylation
(→9) (Scheme 2).
pleting the synthesis of the 3,6-dideoxy-D-arabino-hexopyranoside
(tyveloside) donor 12 (Scheme 3).
The trisaccharide 7 was fully deprotected to yield 13 or alter-
natively converted into a suitable acceptor for glycosylation with
the 3,6-dideoxy donor. To circumvent the potential problems with
acidic deprotection of the acid-labile dideoxyhexose-containing
tetrasaccharide, both acetal groups were hydrolyzed in acetic acid
and the resulting tetraol was acetylated (→14) prior to cleavage
of the p-methoxybenzyl ether using DDQ to yield acceptor 15
in 63% yield. The synthesis of the tetrasaccharide corresponding
to the repeating unit of the CPS found in S. enteritidis was
completed by MeOTf-promoted glycosylation of trisaccharide 15
with tyveloside donor 12 in the presence of DTBMP to give 16 in
62% yield. A subsequent two-step deprotection gave the desired
tetrasaccharide 17, in 63% yield. 1D- and 2D-NMR experiments,
the CASPER program16 and NMR spin simulations (using the
1
PERCH NMR software17) were used to fully assign the H and
13C chemical shifts as well as JH,H coupling constants of the target
oligosaccharides 13 and 17.
The two oligosaccharides were studied by MD simulations
carried out for 30 ns using a recently developed force field for
carbohydrates and explicit water as solvent. The torsion angle
analyses are given in Table 1 and the conformations populated are
presented as scatter plots (Fig. 1).
At each glycosidic linkage two major conformations are present,
which is also indicated by the large RMSDs for, in particular, the
y torsion angles (Table 1). These correspond for the f torsion
angles (H1–C1–On–Cn, where n is the numbering at the glycosidic
linkage position) to that of an exo-anomeric effect and for the
y torsion angles (C1–On–Cn–Hn) to those alternating between
positive and negative values. Population at the non-exo-anomeric
conformation where f > 0◦ is limited for the galactosyl group
whereas it is higher for the tyvelosyl group (axial hydroxyl group
Scheme 2 Reagents and conditions: i) a) TsCl, Et3N, CH2Cl2 b) BzCl,
pyridine, 91%; ii) NaBH4, DMF, 50 ◦C; iii) 80% AcOH (aq.), 60 ◦C, 69%
(2 steps).
Hydride addition using NaBH4 in DMF yielded the 6-
deoxy sugar, and subsequent acidic hydrolysis of the 2,3-O-
isopropylidene group gave diol 10. Insertion of a 2,3-orthoester
continued by acidic hydrolysis yielded the 2-O-benzoyl derivative
11. This was followed by a Barton-McCombie deoxygenation
of the corresponding 3-O-thiocarbonylimidazole derivative, com-
Scheme 3 Reagents and conditions: i) a) trimethyl orthobenzoate, CSA, CH2Cl2 b) 80% AcOH (aq.), 85%; ii) 1,1¢-thiocarbonyldiimidazole, toluene,
reflux; iii) Bu3SnH, AIBN, benzene, reflux, 75% (two steps); iv) a) 70% AcOH (aq.), 80 ◦C b) Pd/C, H2, EtOH/H2O (2:1), 76%; v) a) 60% AcOH (aq.),
◦
˚
70 C b) Pyridine/Ac2O (1:1), 81%; vi) DDQ, CH2Cl2/H2O (14:1), 63%; vii) MeOTf, DTBMP, 4 A MS, Et2O, 62%; viii) a) Pd/C, H2, EtOAc b) NaOMe,
MeOH, 63%.
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Table 1 Average glycosidic torsion angles for oligosaccharides 13 and 17
from 30 ns MD simulations. Root-mean-square deviations (RMSDs) are
given in parentheses
Trisaccharide
Tetrasaccharide
Sugar residue
f/◦
y/◦
f/◦
y/◦
a-D-Galp-(1→2)
-38 (17)
-29 (30)
46 (22)
—
21 (37)
19 (34)
—
-41 (11)
-31 (26)
46 (21)
17 (34)
26 (34)
—
→2,3)-a-D-Manp-(1→
→4)-a-L-Rhap-OMe
a-Tyvp-(1→3)
—
-48 (16)
7 (30)
at C-2) and significant for the mannosyl residue having the same
configuration at C2 as the tyvelosyl group. Analysis of the ring
conformations of the tyvelosyl group showed that not only was
4
1
the C1 chair conformation present as expected but also the C4
chair conformation as well as other transiently observed ring
conformations. The ring flexibility of a-Tyvp observed in the MD
simulations with the PARM/SU01 force field for carbohydrates,18
which is a CHARMM22 type of force field, agrees well with
the accessible conformations deduced from the potential energy
minimizations using the MM3(92) force field.19 The relative
4
1
0
1
energies for the C1, C4, S2 and S3 conformers were 0, 1.4,
2.4, and 5.2 kcal/mol, respectively, showing that both chair forms
should be anticipated to be populated as well as to some extent
skew-boat conformations.
In the tetrasaccharide an anti-y conformational state (y ª 180 ◦)
is populated during the simulation (Fig. 2). This is reached and
returned from via an intermediate state with y ª 140◦. Only the lat-
ter state is transiently populated in the trisaccharide. Whether the
transition from the anti-y conformation can take more that one
path may be addressed by starting a number of MD simulations
from this conformation, similar to what has been reported for a
disaccharide from a local potential energy well that was higher in
energy on the in vacuo potential energy surface.20 The results for
the MD simulations indicate that the conformational flexibility as
judged by the presence of additional conformations may in fact be
higher in what initially may appear to be a sterically more crowded
and conformationally restricted oligosaccharide in which the
mannosyl residue is vicinally disubstituted, i.e., at O2 and O3, by
the galactosyl and tyvelosyl groups, respectively. These results are
indeed consistent with the computational and NMR experimental
results on a-D-Glcp-(1→3)[b-D-Glcp-(1→4)]-a-D-Glcp-OMe in
which it was concluded that an anti-y conformational state at the
Fig. 1 Scatter plots from the MD simulations of tetrasaccharide 17 (left)
and trisaccharide 13 (right).
(1→4)-linkage should be significantly populated.21 Future analysis
based on experimental NMR data may be able to resolve this issue
for the tetrasaccharide.
In conclusion, we have presented a concise and alternative syn-
thesis of a tetrasaccharide corresponding the repeating unit of S.
enteritidis which also should be possible to apply to other serotypes
such as A and B having paratose and abequose, respectively, as the
3,6-dideoxy sugar residues. The MD simulations show a complex
Fig. 2 Trajectory analysis from the MD simulation of tetrasaccharide 17. The torsion angles in the mannosyl residue for: time vs. y (left) and f vs. y
(right).
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and dynamical behavior at the glycosidic torsion angles of the
oligosaccharides, indicating that the O-antigen polysaccharide
may behave in a similar way.
1.15 mmol, 92%) as a white foam. Rf 0.72 (toluene/EtOAc 3:1).
[a]D -8 (c 1.0, CHCl3). 13C-NMR (100 MHz, CDCl3): d = 17.5,
26.5, 28.2, 54.9, 55.3, 64.0, 64.7, 68.9, 70.6, 71.8, 73.6, 76.1, 76.8,
78.8, 81.2, 98.0 (JCH = 171 Hz), 99.4 (JCH = 173 Hz), 101.7,
109.3, 113.8, 126.3 (2C), 128.2 (2C), 128.5 (2C), 129.0 (2C), 129.3
(2C), 129.8, 130.0 (2C), 130.2, 133.4, 137.7, 159.3, 166.0. 1H-NMR
(400 MHz, CDCl3): d = 1.36 (d, 3H, J = 6.4 Hz), 1.37 (s, 3H),
1.54 (s, 3H), 3.40 (s, 3H), 3.42 (m, 1H), 3.72 (dd, 1H, J = 10.0, 6.0
Hz), 3.78 (s, 3H), 3.90 (m, 1H), 4.11–4.15 (m, 3H), 4.22–4.24 (m,
Materials and methods
General methods
Normal work-up means drying the organic phase with MgSO4 (s)
or Na2SO4 (s), filtering and evaporation of the solvent in vacuo at
2H), 4.34 (dd, 1H, J = 3.2, 9.6 Hz), 4.67 (benzylic d, 1H, Jgem
=
◦
~35 C. CH2Cl2 was distilled over calcium hydride and collected
12.0 Hz), 4.71 (benzylic d, 1H, Jgem = 12.0 Hz), 4.88 (s, 1H), 5.03
(d, 1H, J = 1.6 Hz), 5.55 (dd, 1H, J = 1.6, 3.2 Hz), 5.72 (s, 1H),
6.82 (d, 2H, J = 8.4 Hz, Ar-H), 7.28 (d, 2H, J = 8.4 Hz, Ar-H),
7.38–7.44 (m, 3H, Ar-H), 7.48 (dd, 2H, J = 7.6, 7.6 Hz, Ar-H),
7.57–7.62 (m, 3H, Ar-H), 8.14 (d, 2H, J = 7.6 Hz, Ar-H). HRMS
calcd for C38H44O12: [M + Na]+ 715.2730; Found 715.2748.
˚
onto 4 A predried MS. Thin-Layer Chromatography (TLC) was
carried out on 0.25 mm precoated silica-gel plates (Merck silica-
gel 60 F254); detected with UV-abs (254 nm) and/or by charring
with 8% sulfuric acid or AMC (ammonium molybdate (10 g) and
cerium sulfate (2 g) dissolved in 10% H2SO4 (200 mL)) followed
by heating to ~250 ◦C. FC means Flash Column chromatography
1
using silica gel (Amicon, (0.040–0.063 mm)). H NMR and 13C
Methyl 4,6-O-benzylidene-3-O-p-methoxybenzyl-a-D-mannopy-
ranosyl-(1→4)-2,3-O-isopropylidene-a-L-rhamnopyranoside (5).
Compound 4 (1.67 g, 2.41 mmol) was dissolved in MeOH/CH2Cl2
(12 mL, 2:1). The solution was cooled to 0 ◦C and NaOMe (cat.)
was added. After stirring 6 h at room temperature, the mixture
was neutralized with DOWEX-H+ ion exchange resins, filtered
and concentrated. FC (toluene/EtOAc 6:1 → toluene/EtOAc
3:1) afforded 5 (1.27 g, 2.16 mmol, 90%) as a white foam. Rf
0.34 (toluene/EtOAc 3:1). [a]D +43 (c 1.0, CHCl3). 13C-NMR
(100 MHz, CDCl3): d = 17.6, 26.6, 28.3, 55.0, 55.4, 63.4, 64.8,
69.0, 70.2, 72.9, 75.3, 76.1, 76.9, 79.0, 80.8, 98.1, 100.8, 101.7,
109.3, 114.0, 126.3 (2C), 128.3 (2C), 129.0 (2C), 129.2, 129.6 (2C),
NMR spectra were recorded on Varian 400 or Bruker 500 MHz
instruments at 25 ◦C unless otherwise stated. Chemical shifts are
given in ppm relative to solvent peaks (d 77.17 for 13C and d 7.26
for 1H) in CDCl3. Optical rotation was measured using a Perkin-
Elmer 343 polarimeter.
Experimental procedures
Ethyl 2-O-benzoyl-4,6-O-benzylidene-3-O-p-methoxybenzyl-1-
thio-a-D-mannopyranoside (2). Ethyl 4,6-O-benzylidene-3-O-p-
methoxybenzyl-1-thio-a-D-mannopyranoside (1) was dissolved in
pyridine (30 mL), cooled to 0 ◦C and BzCl (1.18 mL, 10.14 mmol)
was added dropwise. After 14 h, the solution was diluted with
CH2Cl2 (60 mL) and washed with 1 M HCl (aq.) (2 ¥ 100 mL).
Normal work-up, followed by FC (toluene → toluene/EtOAc
19:1) yielded 2 (3.02 g, 5.64 mmol, 89%) as a colourless oil. Rf
0.66 (toluene/EtOAc 9:1). [a]D +25 (c 1.0, CHCl3). 13C-NMR
(100 MHz, CDCl3): d = 15.1, 25.8, 55.4, 64.8, 68.8, 71.9, 72.3,
74.0, 79.0, 83.7, 101.8, 113.9, 126.3 (2C), 128.3 (2C), 128.6 (2C),
129.1 (2C), 129.5 (2C), 129.9, 130.0, 130.1 (2C), 133.4, 137.6, 159.4,
165.9. 1H-NMR (400 MHz, CDCl3): d = 1.31 (t, 3H, J = 7.2 Hz),
2.66 (m, 2H), 3.77 (s, 3H), 3.92 (m, 1H), 4.07 (dd, 1H, J = 3.2,
9.6 Hz), 4.21 (dd, 1H, J = 9.6, 9.6 Hz), 4.26–4.32 (m, 2H), 4.61
(benzylic d, 1H, Jgem = 12.0 Hz), 4.67 (benzylic d, 1H, Jgem = 12.0
Hz), 5.41 (d, 1H, J = 1.2 Hz), 5.65 (dd, 1H, J = 1.2, 3.2 Hz), 5.68
(s, 1H), 6.80 (d, 2H, J = 8.4 Hz, Ar-H), 7.24 (d, 2H, J = 8.4 Hz,
Ar-H), 7.37–7.42 (m, 2H, Ar-H), 7.48 (dd, 2H, J = 7.6, 7.6 Hz,
Ar-H), 7.53 (dd, 2H, J = 1.6, 7.6 Hz, Ar-H), 7.60 (dd, 2H, J =
7.6, 7.6 Hz, Ar-H), 8.12 (d, 2H, J = 7.6 Hz, Ar-H). HRMS calcd
for C30H32O7S: [M + Na]+ 559.1761; Found 559.1759.
1
130.3, 137.8, 159.5. H-NMR (400 MHz, CDCl3): d = 1.26 (d,
3H, J = 6.4 Hz), 1.33 (s, 3H), 1.51 (s, 3H), 2.72 (bs, 1H, -OH),
3.36 (s, 3H), 3.37 (dd, 1H, J = 7.2, 10.0 Hz), 3.64 (dd, 1H, J =
6.4, 10.0 Hz), 3.78–3.83 (m, 4H), 3.88 (dd, 1H, J = 3.6, 9.2 Hz),
4.02 (d, 1H, J = 3.2 Hz), 4.06–4.10 (m, 4H), 4.28 (dd, 1H, J =
3.6, 9.2 Hz), 4.65 (benzylic d, 1H, Jgem = 11.6 Hz), 4.79 (benzylic
d, 1H, Jgem = 11.6 Hz), 4.84 (s, 1H), 4.93 (d, 1H, J = 1.2 Hz), 5.61
(s, 1H), 6.86 (d, 2H, J = 8.4 Hz, Ar-H), 7.16–7.18 (m, 1H, Ar-H),
7.25–7.29 (m, 3H, Ar-H), 7.35–7.40 (m, 2H, Ar-H), 7.50–7.53 (m,
2H). HRMS calcd for C31H40O11: [M + Na]+ 611.2468; Found
611.2495.
Methyl
2,3,4,6-tetra-O-benzyl-a-D-galactopyranosyl-(1→2)-
4,6-O-benzylidene-3-O-p-methoxybenzyl-a-D-mannopyranosyl-
(1→4)-2,3-O-isopropylidene-a-L-rhamnopyranoside (7). Ethyl
2,3,4,6-tetra-O-benzyl-1-thio-b-D-galactopyranoside (6) (0.646 g,
1.10 mmol) and 5 (0.488 g, 0.829 mmol) were dissolved in dry
˚
Et2O (15 mL), 4 A MS were added and the mixture was stirred
under an Ar-atmosphere. After 20 min, the mixture was cooled
to -30 ◦C and NIS (0.270 g, 1.20 mmol) and AgOTf (cat.)
were added. After 30 min, the reaction was quenched by adding
Et3N (0.50 mL) and the resulting mixture filtered through a
pad of Celite, diluted with CH2Cl2 (60 mL), washed with 10%
Na2S2O3 (aq., 80 mL) and subjected to normal work-up. FC
(toluene/EtOAc 19:1 → toluene/EtOAc 9:1) produced 7 (0.783 g,
0.705 mmol, 85%) as a white foam. Rf 0.64 (toluene/EtOAc 3:1).
[a]D +44 (c 1.0, CH2Cl2). 13C-NMR (100 MHz, CDCl3): d = 17.3,
26.5, 28.2, 54.9, 55.3, 64.5, 64.8, 68.8, 69.2, 70.0, 71.4, 73.3, 73.6,
73.7, 73.9, 74.9, 75.2, 76.1, 76.4, 76.7, 76.9, 78.3, 79.3, 80.4, 97.7
(JCH = 172 Hz), 98.0 (JCH = 168 Hz), 101.0 (JCH = 173 Hz), 101.3,
109.2, 113.8, 126.2, 127.3, 127.4, 127.5 (2C), 127.7, 127.8, 127.9
Methyl 2-O-benzoyl-4,6-O-benzylidene-3-O-p-methoxybenzyl-
a-D-mannopyranosyl-(1→4)-2,3-O-isopropylidene-a-L-rhamnopy-
ranoside (4). Methyl 2,3-O-isopropylidene-a-L-rhamnopyrano-
side (3, 0.384 g, 1.76 mmol) and 2 (0.672 g, 1.25 mmol) were
˚
dissolved in dry CH2Cl2 (20 mL), 4 A MS were added and the
mixture was stirred under an Ar-atmosphere. After 30 min, the
mixture was cooled to -30 ◦C and NIS (0.420 g, 1.87 mmol) and
AgOTf (cat.) were added. After 20 min, the reaction was quenched
by adding Et3N (0.600 mL). The mixture was filtered through a pad
of Celite, washed with 10% Na2S2O3 (aq., 60 mL) and subjected
to normal work-up. FC (toluene/EtOAc 9:1) gave 4 (0.796 g,
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(2C), 127.9 (2C), 128.1 (2C), 128.3 (2C), 128.3 (2C), 128.4 (2C),
128.5 (2C), 128.5 (2C), 128.9, 129.5 (2C), 130.8, 138.0 (2C), 138.7,
Ethyl
2,4-di-O-benzoyl-6-deoxy-1-thio-a-D-mannopyranoside
(11). Compound 10 (0.130 g, 0.416 mmol) was dissolved in
dry CH2Cl2 (5 mL), and trimethyl orthobenzoate (0.143 mL,
0.832 mmol) and CSA (0.010 g, 0.04 mmol) were added. After
60 min, the solvent was evaporated and the residue dissolved in
acetic acid (80% aq., 5 mL). After stirring for 45 min, toluene
(10 mL) was added and the solvents were evaporated followed by
co-evaporation with toluene (2 ¥ 10 mL). FC (toluene/EtOAc
19:1 → toluene/EtOAc 9:1) afforded 11 (0.148 g, 0.355 mmol,
85%). Rf 0.53 (toluene/EtOAc 6:1). [a]D +29 (c 1.0, CHCl3).
13C-NMR (100 MHz, CDCl3): d = 15.1, 17.7, 25.9, 67.0, 69.8,
75.3, 75.9, 82.3, 128.6 (2C), 128,7 (2C), 129.5, 129.6, 130.0
1
139.1, 139.2, 159.2. H-NMR (400 MHz, CDCl3): d = 1.23 (d,
3H, J = 6.4 Hz), 1.37 (s, 3H), 1.55 (s, 3H), 3.39 (s, 3H), 3.40 (m,
1H), 3.49–3.56 (m, 2H), 3.62 (dd, 1H, J = 6.4, 10.0 Hz), 3.80 (s,
3H), 3.83 (t, 1H, J = 10.0 Hz), 3.97–4.18 (m, 8H), 4.23–4.27 (m,
2H), 4.32 (dd, 1H, J = 10.0, 10.0 Hz), 4.40 (benzylic d, 1H, Jgem
=
12.0 Hz), 4.45 (benzylic d, 1H, Jgem = 11.2 Hz), 4.50 (benzylic d,
1H, Jgem = 12.0 Hz), 4.60 (benzylic d, 1H, Jgem = 11.6 Hz), 4.68
(benzylic d, 1H, Jgem = 11.2 Hz), 4.77 (benzylic d, 1H, Jgem = 12.0
Hz), 4.81 (benzylic d, 1H, Jgem = 11.6 Hz), 4.87 (benzylic d, 1H,
Jgem = 11.2 Hz), 4.88 (s, 1H), 4.93 (benzylic d, 1H, Jgem = 11.6
Hz), 4.94 (d, 1H, J = 1.2 Hz), 4.98 (benzylic d, 1H, Jgem = 11.6
Hz), 5.49 (s, 1H), 5.70 (d, 1H, J = 3.6 Hz), 6.84 (dd, 2H, J =
8.4, 2.0 Hz, Ar-H), 7.18–7.20 (m, 1H, Ar-H), 7.18–7.47 (m, 25H,
Ar-H), 7.56 (dd, 2H, J = 8.4, 2.0 Hz, Ar-H). HRMS calcd for
C65H74O16: [M + Na]+ 1133.4875; Found 1133.4858.
1
(2C), 130.0 (2C), 133.6 (2C), 166.1, 167.2. H-NMR (400 MHz,
CDCl3): d = 1.33 (d, 3H, J = 6.8 Hz), 1.34 (t, 3H, J = 7.2 Hz),
2.58 (bs, 1H), 2.69 (m, 2H), 4.27 (dd, 1H, J = 3.2, 10.0 Hz), 4.42
(m, 1H), 5.30 (dd, 1H, J = 10.0, 10.0 Hz), 5.45 (s, 1H), 5.49 (dd,
1H, J = 1.2, 3.2 Hz), 7.44–7.61 (m, 6H), 8.08 (d, 2H, J = 8.0 Hz),
8.12 (d, 2H, J = 8.0 Hz). HRMS calcd For C22H24O6S: [M + H]+
417.1372; Found 417.1354.
Ethyl 4-O-benzoyl-2,3-O-isopropylidene-6-O-tosyl-1-thio-a-D-
mannopyranoside (9). To a cooled (0 ◦C) solution of ethyl
2,3-O-isopropylidene-1-thio-a-D-mannopyranoside22 (8, 0.473 g,
1.79 mmol) in dry CH2Cl2 (10 mL) were added Et3N (0.420 mL,
3.04 mmol) and TsCl (0.443 g, 2.32 mmol). After stirring for
24 h, the mixture was washed with 1 M HCl (aq., 20 mL), sat.
NaHCO3 (aq., 20 mL), H2O (20 mL) and subjected to normal
work-up. The crude product was dissolved in pyridine (4 mL) and
BzCl (0.416 mL, 3.58 mmol) was added. After 3 h, the solvents
were evaporated and co-evaporated with toluene (3 ¥ 10 mL). FC
(toluene/EtOAc 19:1 → toluene/EtOAc 9:1) yielded 9 (0.848 g,
1.62 mmol, 91%) as a colourless oil. Rf 0.55 (toluene/EtOAc 9:1).
[a]D +193 (c 2.0, CHCl3). 13C-NMR (100 MHz, CDCl3): d = 14.4,
21.7, 24.2, 26.5, 27.8, 67.0, 68.6, 70.6, 75.6, 76.5, 79.3, 110.3, 128.0
(2C), 128.5 (2C), 129.9 (2C), 130.0 (2C), 130.7, 132.6, 133.6, 144.9,
165.6. 1H-NMR (400 MHz, CDCl3): d = 1.30 (t, 3H, J = 7.2 Hz),
2.35 (s, 3H), 2.54 (dq, J = 7.2, 1.6 Hz), 2.69 (dq, J = 7.2, 1.6 Hz),
4.11–4.08 (m, 2H), 4.21 (dd, 1H, J = 0.8, 5.2 Hz), 4.32 (dd, 1H,
J = 5.2, 7.6 Hz), 4.44 (m, 1H), 5.15 (dd, 1H, J = 7.6, 10.4 Hz),
5.58 (s, 1H), 7.19 (dd, 2H, J = 0.8, 8.4 Hz), 7.42–7.69 (m, 6H), 7.99
(dd, 2H, J = 0.8, 8.4 Hz), 8.16 (dd, 2H, J = 1.2, 8.4 Hz). HRMS
calcd for C25H31O8S2: [M + H]+ 523.1455; Found 523.1442.
Ethyl 2,4-di-O-benzoyl-3,6-dideoxy-1-thio-a-D-arabino-hexopy-
ranoside
(12). 1,1¢-Thiocarbonyldiimidazole
(0.042
g,
0.240 mmol) and 11 (0.050 g, 0.120 mmol) were dissolved
in dry toluene (5 mL) and the mixture was refluxed for 2 h.
After cooling to room temperature, the solvent was evaporated
and FC (toluene/EtOAc 6:1) gave the imidazoylthiocarbonyl
intermediate. The intermediate and Bu3SnH (0.069 mL,
0.255 mmol) were dissolved in dry benzene (6 mL), AIBN
(0.003 g, 0.020 mmol) was added and the mixture was heated to
reflux. After 20 min, the solution was cooled to room temperature
and concentrated. FC (toluene → toluene/EtOAc 19:1) yielded
12 (0.036 g, 0.090 mmol, 75%). Rf 0.69 (toluene/EtOAc 9:1). [a]D
+58 (c 1.0, CHCl3). 13C-NMR (100 MHz, CDCl3): d = 15.3, 18.0,
25.5, 30.9, 67.3, 70.9, 72.3, 82.4, 128.6 (4C), 129.8 (2C), 129.9,
130.0 (2C), 130.1, 133.4, 133.5, 165.8, 165.8. 1H-NMR (400 MHz,
CDCl3): d = 1.32 (d, 3H, J = 6.0 Hz), 1.35 (t, 3H, J = 7.2 Hz),
2.17 (ddd, 1H, J = 2.8, 11.2, 14.0 Hz), 2.47 (ddd, 1H, J = 4.8,
4.8, 14.0 Hz), 2.71 (m, 2H), 4.42 (m, 1H), 5.24 (ddd, 1H, J = 4.8,
9.6, 11.2 Hz), 5.34–5.36 (m, 2H), 7.44–7.61 (m, 6H), 8.04–8.13
(m, 4H). HRMS calcd for C22H25O5S: [M + H]+ 401.1417; Found
401.1416.
Ethyl 4-O-benzoyl-6-deoxy-1-thio-a-D-mannopyranoside (10).
Derivative 9 (0.755 g, 1.45 mmol) and NaBH4 (2.97 g, 11.94 mmol)
were dissolved in DMF (10 mL), and the mixture was heated to
50 ◦C. After 30 h, the mixture was diluted with toluene (30 mL),
washed with H2O (40 mL) and subjected to normal work-up. FC
(toluene → toluene/EtOAc 19:1 → toluene/EtOAc 9:1) afforded
the 6-deoxy derivative, which was dissolved in acetic acid (80% aq.,
25 mL) and the solution stirred at 60 ◦C. After 3 h, the solution was
cooled to r.t, and the solvent evaporated and co-evaporated with
toluene (3 ¥ 20 mL). FC (toluene/EtOAc 3:1 → toluene/EtOAc
1:1) gave 10 (0.312 g, 0.998 mmol, 69%). Rf 0.71 (toluene/EtOAc
1:3). [a]D +225 (c 1.0, CHCl3). 13C-NMR (100 MHz, CDCl3): d =
15.0, 17.6, 25.3, 66.4, 71.0, 72.6, 76.6, 83.9, 128.6 (2C), 129.5, 130.0
(2C), 133.6, 167.6. 1H-NMR (400 MHz, CDCl3): d = 1.28 (d, 3H,
J = 6.4 Hz), 1.31 (t, 3H, J = 7.6 Hz), 2.64 (m, 2H), 3.99 (dd, 1H,
J = 3.2, 9.6 Hz), 4.10 (dd, 1H, J = 1.2, 3.2 Hz), 3.34 (m, 1H), 5.10
(dd, 1H, J = 9.6, 9.6 Hz), 5. 35 (s, 1H), 7.42–7.60 (m, 3H), 8.03
(dd, 1H, J = 1.6, 8.4 Hz). HRMS calcd for C15H20O5S: [M + H]+
313.1110; Found 313.1098.
Methyl
a-D-galactopyranosyl-(1→2)-a-D-mannopyranosyl-
(1→4)-a-L-rhamnopyranoside (13). A solution of 7 (0.038 g,
0.034 mmol) in acetic acid (70% aq., 5 mL) was stirred at
80 ◦C for 6 h. The solvent was evaporated followed by co-
evaporation with toluene (2 ¥ 10 mL). The residue was dissolved
in EtOH/H2O (3 mL, 2:1) and a catalytic amount of Pd/C (10%)
was added. The mixture was stirred under an H2-atmosphere
(100 psi) for 36 h, then filtered through Celite and evaporated.
FC (EtOAc/MeOH/H2O 7:2:1) gave 13 (0.013 g, 0.026 mmol,
76%). Rf 0.22 (EtOAc/MeOH/H2O 7:2:1). [a]D +54 (c 0.7,
H2O). 13C-NMR and 1H-NMR : See Table S1. HRMS calcd For
C19H34O15: [M + Na]+ 525.1790; Found 525.1783.
Methyl
2,3,4,6-tetra-O-benzyl-a-D-galactopyranosyl-(1→2)-
4,6-di-O-acetyl-3-O-p-methoxybenzyl-a-D -mannopyranosyl-
(1→4)-2,3-di-O-acetyl-a-L-rhamnopyranoside (14). A solution
of 7 (0.341 g, 0.307 mmol) in acetic acid (60% aq.) (6 mL) was
stirred at 70 ◦C. After 8 h, the solution was cooled to room
1616 | Org. Biomol. Chem., 2009, 7, 1612–1618
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temperature, and the solvent evaporated and co-evaporated with
˚
0.085 mmol) in dry Et2O (1.5 mL), 4 A MS were added, and the
mixture was stirred under an Ar-atmosphere. After 30 min, MeOTf
(0.023 mL, 0.204 mmol) was added. After 30 h, the reaction was
quenched by adding Et3N (75 mL), an d the mixture diluted with
CH2Cl2 (5 mL), filtered through a pad of Celite and concentrated.
FC (toluene/EtOAc 6:1 → 3:1) afforded 16 (0.029 g, 0.021 mmol,
62%) as an oil. Rf 0.47 (toluene/EtOAc 3:1). [a]D +18 (c 1.0,
CHCl3). 13C-NMR (125 MHz, 30 ◦C, CDCl3): d = 17.9, 18.1, 20.6,
20.7, 20.9, 20.9, 29.3, 55.0, 62.7, 66.9 (2C), 67.4, 67.8, 69.0, 70.2,
70.3 (3C), 72.2, 73.1, 73.4, 74.8 (2C), 75.2, 75.6, 76.0, 77.0, 78.5,
79.1, 98.3 (JCH = 173 Hz), 98.6 (JCH = 170 Hz), 99.1 (JCH = 171 Hz),
100.4 (JCH = 173 Hz), 127.4, 127.5 (2C), 127.6 (2C), 127.6, 127.7
(2C), 127.9, 128.2–128.5, 129.6 (2C), 129.8 (2C), 133.2, 133.3,
137.7, 138.6 (2C), 138.6, 165.4, 165.6, 169.9, 169.9, 170.0, 170.8.
1H-NMR (500 MHz, 30 ◦C, CDCl3): d = 1.25 (d, 3H, J = 6.5 Hz),
1.32 (d, 3H, J = 6.5 Hz), 1.89 (m, 1H), 1.98 (s, 3H), 1.99 (s, 3H),
2.02 (s, 3H), 2.12 (ddd, 1H, J = 4.0, 4.0, 13.5 Hz), 2.23 (s, 3H),
3.35 (s, 3H), 3.46 (dd, 1H, J = 6.0, 9.0 Hz), 3.50 (dd, 1H, J = 6.0,
9.5 Hz), 3.66 (dd, 1H, J = 9.0, 9.0 Hz), 3.77 (dd, 1H, J = 6.0, 10.0
Hz), 3.93 (ddd, 1H, J = 3.0, 3.5, 9.5 Hz), 3.96–3.98 (m, 2H), 4.03
(dd, 1H, J = 2.5, 9.5 Hz), 4.06 (dd, 1H, J = 9.5, 9.5 Hz), 4.13–4.20
(m, 5H), 4.42 (benzylic d, 1H, Jgem = 12.0 Hz), 4.50 (benzylic d,
1H, Jgem = 12.5 Hz), 4.54 (benzylic d, 1H, Jgem = 11.5 Hz), 4.56
(d, 1H, J = 1.5 Hz), 4.74 (benzylic d, 1H, Jgem = 12.0 Hz), 4.79
(benzylic d, 1H, Jgem = 12.0 Hz), 4.82 (benzylic d, 1H, Jgem = 12.0
Hz), 4.91 (benzylic d, 1H, Jgem = 12.5 Hz), 4.94–5.00 (m, 3H), 5.13
(dd, 1H, J = 4.0, 10.5 Hz), 5.16 (d, 1H, J = 1.5 Hz), 5.20 (dd,
1H, J = 1.5, 4.0 Hz), 5.22 (dd, 1H, J = 4.0, 10.0 Hz), 5.39 (d,
1H, J = 3.5 Hz), 5.61 (dd, 1H, J = 10.0, 10.0 Hz), 7.18–7.59 (m,
26H, Ar-H), 7.92 (dd, 2H, J = 1.0, 8.0 Hz), 8.07 (dd, 2H, J = 1.0,
8.0 Hz). HRMS calcd for C75H84O24: [M + Na]+ 1391.5250; Found
1391.5183.
toluene (3 ¥ 10 mL). The crude tetraol was dissolved in pyridine
◦
(5 mL) and cooled to 0 C. Acetic anhydride (5 mL) was added
dropwise to the solution, which was stirred for 12 h. The solvent
was evaporated and co-evaporated with toluene (3 ¥ 10 mL). FC
(toluene/EtOAc 6:1 → toluene/EtOAc 3:1) produced 14 (0.287 g,
0.249 mmol, 81%) as a colourless oil. Rf 0.32 (toluene/EtOAc
3:1). [a]D +11 (c 1.0, CHCl3). 13C-NMR (100 MHz, CDCl3): d =
18.3, 20.8, 21.0, 21.2 (2C), 55.3, 55.5, 62.9, 67.6, 68.1, 69.5, 70.1,
70.3, 70.4, 70.6, 71.8, 72.1, 72.4, 73.6 (2C), 74.9, 75.4, 76.0, 76.3,
77.4, 78.4, 98.1, 98.6, 100.3, 114.1, 127.4, 127.6, 127.6 (2C), 127.8,
127.9 (2C), 128.0, 128.2 (2C), 128.3 (2C), 128.4 (2C), 128.5 (2C),
128.6 (2C), 128.6, 128.6, (2C), 129.6 (2C), 130.1, 138.1, 138.8,
139.0, 139.2, 159.5, 169.5, 170.2 (2C), 171.1. 1H-NMR (400 MHz,
CDCl3): d = 1.22 (d, 3H, J = 5.6 Hz), 1.98 (s, 3H), 1.99 (s,3H),
2.03 (s, 3H), 2.04 (s, 3H), 3.37 (s, 3H), 3.43 (dd, 1H, J = 6.4, 9.2
Hz), 3.47 (dd, 1H, J = 6.4, 9.2 Hz), 3.66–3.70 (m, 2H), 3.78 (s,
3H), 3.80 (m, 1H), 3.90–4.06 (m, 6H), 4.12 (dd, 1H, J = 2.4, 12.0
Hz), 4.17 (dd, 1H, J = 4.0, 12.0 Hz), 4.40 (benzylic d, 1H, Jgem
=
12.0 Hz), 4.44–4.56 (m, 6H), 4.70 (benzylic d, 1H, Jgem = 12.0 Hz),
4.80 (benzylic d, 1H, Jgem = 12.5 Hz), 4.87 (benzylic d, 1H, Jgem
=
12.0 Hz), 4.92 (benzylic d, 1H, Jgem = 11.6 Hz), 5.04 (d, 1H, J =
1.6 Hz), 5.16–5.23 (m, 2H), 5.39 (d, 1H, J = 3.6 Hz), 5.52 (dd,
1H, J = 9.6, 9.6 Hz), 6.81 (d, 2H, J = 8.8 Hz), 7.16 (d, 2H, J =
8.4 Hz), 7.21–7.36 (m, 21H, Ar-H). HRMS calcd for C63H74O20:
[M + Na]+ 1173.4671; Found 1173.4708.
Methyl 2,3,4,6-tetra-O-benzyl-a-D-galactopyranosyl-(1→2)-4,
6-di-O-acetyl-a-D-mannopyranosyl-(1→4)-2,3-di-O-acetyl-a-L-
rhamnopyranoside (15). Compound 14 (0.230 g, 0.200 mmol)
was dissolved in CH2Cl2/H2O (14:1, 15 mL) and DDQ (0.050 g,
0.225 mmol) was added. After 8 h, the solution was diluted with
CH2Cl2 (10 mL), washed with 10% Na2S2O3 (aq.) (30 mL) and
subjected to normal work-up. FC (toluene/EtOAc 3:1) gave 15
(0.130 g, 0.126 mmol, 63%). Rf 0.24 (toluene/EtOAc 3:1). [a]D +1
(c 2.0, CHCl3). 13C-NMR (100 MHz, CDCl3): d = 18.3, 20.8, 20.9,
21.0, 21.1, 55.3, 63.0, 67.4, 68.8, 68.9, 69.4, 69.6, 70.2, 70.4, 70.5,
73.0, 73.4, 74.4, 74.9, 74.9, 76.2, 77.6, 79.3, 81.3, 98.5, 99.8, 102.2,
127.7 (2C), 127.8, 127.9 (3C), 128.0, 128.1, 128.2 (2C), 128.3 (2C),
128.4 (2C), 128.6 (2C), 128.7 (4C), 137.9 (2C), 138.4, 138.6, 170.0,
Methyl-a-D-galactopyranosyl-(1→2)-[3,6-dideoxy-a-D-arabino-
hexopyranosyl-(1→3)]-a-D-mannopyranosyl-(1→4)-a-L-rhamno-
pyranoside (17). A solution of 16 (0.041 g, 0.030 mmol) in
EtOAc (0.8 mL) was applied to an H-cube. The solution was
diluted with additional EtOAc (total volume 15 mL) after injection
and the solution was cycled in the H-cube (40 ◦C, 60 Bar,
Flow 0.3 mL/min) over a Pd/C (10%) cartridge. After cycling
for 45 min, the system was rinsed an additional 15 min with
EtOAc and the solvent was evaporated from the resulting solution.
FC (EtOAc → EtOAc/MeOH 19:1 → 9:1) afforded the tetraol
intermediate, which was dissolved in MeOH (1.5 mL) and NaOMe
(cat.) was added. After stirring 26 h at 25 ◦C, the mixture was
neutralized with DOWEX-H+ ion exchange resins, filtered and
concentrated. FC (EtOAc/MeOH/H2O 7:2:1) gave 17 (0.012 g,
0.019 mmol, 63%). Rf 0.24 (EtOAc/MeOH/H2O 7:2:1). [a]D +66
(c 1.0, H2O). 13C-NMR and 1H-NMR: See Table S2. HRMS calcd
for C25H44O18: [M + Na]+ 655.2425; Found 655.2449.
1
170.1, 170.3, 170.8. H-NMR (400 MHz, CDCl3): d = 1.27 (d,
3H, J = 6.5 Hz), 1.99 (s, 3H), 2.00 (s, 3H), 2.03 (s, 3H), 2.09 (s,
3H), 3.36 (s, 3H), 3.43 (d, 2H, J = 6.4 Hz), 3.58 (d, 1H, J = 12.0
Hz), 3.65–3.81 (m, 4H), 3.89–3.94 (m, 2H), 3.98 (dd, 1H, J = 2.8,
10.5 Hz), 4.03–4.09 (m, 3H), 4.18 (dd, 1H, J = 4.4, 12.4 Hz), 4.39
(benzylic d, 1H, Jgem = 12.0 Hz), 4.47 (benzylic d, 1H, Jgem = 12.0
Hz), 4.53 (benzylic d, 1H, Jgem = 11.2 Hz), 4.56 (d, 1H, J = 1.6
Hz), 4.68 (benzylic d, 1H, Jgem = 11.6 Hz), 4.76 (benzylic d, 1H,
Jgem = 11.6 Hz), 4.82 (benzylic d, 1H, Jgem = 12.0 Hz), 4.85 (d, 1H,
J = 3.6 Hz), 4.91 (benzylic d, 1H, Jgem = 12.0 Hz), 4.93 (benzylic
d, 1H, Jgem = 11.2 Hz), 5.11 (dd, 1H, J = 10.0, 10.0 Hz), 5.17 (dd,
1H, J = 3.6, 9.5 Hz), 5.21 (dd, 1H, J = 1.6, 3.6 Hz), 7.24–7.41 (m,
20H, Ar-H). HRMS calcd for C55H66O19: [M + Na]+ 1053.4096;
Found 1053.4049.
Molecular simulations
Molecular dynamics (MD) simulations used NAMD23,24 (parallel
version, 2.6b1 for the tetrasaccharide and 2.6 for the trisac-
charide) employing a CHARMM22 type of force field, namely
PARM22/SU01 which is a recently modified force field for
carbohydrates. Pdb and psf files of the oligosaccharides were
created using VEGA ZZ (release 2.0.8) and assigned CHARMM
Methyl 2,3,4,6-tetra-O-benzyl-a-D-galactopyranosyl-(1→2)-[2,
4-di-O-benzoyl-3,6-dideoxy-a-D-arabino-hexopyranosyl-(1→3)]-
4,6-di-O-acetyl-a-D-mannopyranosyl-(1→4)-2,3-di-O-acetyl-a-
L-rhamnopyranoside (16). To
a solution of 12 (0.029 g,
0.072 mmol), 15 (0.035 g, 0.034 mmol) and DTBMP (0.017 g
This journal is The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 1612–1618 | 1617
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partial charges. Initial conditions were prepared by placing the
oligosaccharides in a previously equilibrated cubic water box
following minimization and heating. This procedure resulted in
systems with the oligosaccharide and either 1121 or 899 TIP3P
water molecules for the tetra- and trisaccharide respectively. The
MD simulations were carried out with multiple-time-stepping
and 2 fs, 2 fs, and 6 fs were used as the inner, middle and
outer time steps. Following a 1 ns equilibration of the system
the production run was carried out for a 30 ns duration period
performed in the NPT ensemble (P = 1 atm, T = 300 K) with a
References
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˚
cutoff distance for non-bond interactions set at 12 A and periodic
˚
boundary conditions giving a cubic box of approximately 32 A
˚
or 30 A to the side for the tetra- and trisaccharide, respectively.
The smooth particle-mesh Ewald (SPME) method was used to
calculate the full electrostatic interactions. The temperature and
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NMR spectroscopy
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dissolved in D2O (0.5 mL). The NMR experiments for H and
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equipped with a 5 mm PFG triple-resonance CryoProbe. Standard
1D- and 2D-experiments and the CASPER software were used
to assign chemical shifts using internal TSP (0 ppm) as 1H
reference and external dioxane in D2O (67.4 ppm) as 13C reference.
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This work was supported by grants from the Swedish Research
Council (VR), The Knut and Alice Wallenberg Foundation, and
computing resources at the Center for Parallel Computers (PDC),
Stockholm, Sweden.
1618 | Org. Biomol. Chem., 2009, 7, 1612–1618
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