Convergent synthesis and conformational analysis of the hexasaccharide repeating unit of the o-antigen of shigella flexneri serotype 1d

Article abstract of DOI:10.1002/ejoc.201402392

A hexasaccharide repeating unit of the O-antigen of the cell wall of Shigella flexneri type 1d has been synthesized using a stereoselective [3+3] block glycosylation approach. Recently developed glycosylation conditions were used in the synthesis. A thioglycoside was used as an orthogonal glycosyl donor during the synthesis. The synthesized hexasaccharide was subjected to detailed NMR spectroscopic and molecular modeling studies to determine its conformational behavior in water. The NOE-based two-dimensional NOSEY experiment and all-atom explicit molecular dynamics (MD) simulation studies suggest that the oligosaccharide is not very flexible, that it remains rigid with respect to the glycosidic linkages between the sugar moieties. Copyright

Full text of DOI:10.1002/ejoc.201402392

FULL PAPER
DOI: 10.1002/ejoc.201402392
Convergent Synthesis and Conformational Analysis of the Hexasaccharide
Repeating Unit of the O-Antigen of Shigella flexneri Serotype 1d
Debashis Dhara,[a] Rajiv Kumar Kar,[b] Anirban Bhunia,*[b] and Anup Kumar Misra*[a]
Keywords: Synthetic methods / Carbohydrates / Oligosaccharides / Glycosylation / Conformation analysis / Molecular
dynamics
A hexasaccharide repeating unit of the O-antigen of the cell
wall of Shigella flexneri type 1d has been synthesized using
molecular modeling studies to determine its conformational
behavior in water. The NOE-based two-dimensional NOSEY
a stereoselective [3+3] block glycosylation approach. Re- experiment and all-atom explicit molecular dynamics (MD)
cently developed glycosylation conditions were used in the
synthesis. A thioglycoside was used as an orthogonal glyc-
osyl donor during the synthesis. The synthesized hexasac-
charide was subjected to detailed NMR spectroscopic and
simulation studies suggest that the oligosaccharide is not
very flexible, that it remains rigid with respect to the glyc-
osidic linkages between the sugar moieties.
A recent thrust in drug discovery has been towards the
development of drugs relying on alternative mechanisms for
controlling bacterial infections, because of the emergence of
multi-drug-resistant bacteria.[8] Similarly to other bacterial
infections, the development of therapeutics against drug-
resistant Shigella strains is also highly desirable. As an alter-
native to small-molecule therapeutics, the development of
glycoconjugate vaccines based on the cell-wall O-antigen of
Shigella species has been attempted in the recent past.[9–12]
However, extraction from the living organism of the oligo-
saccharides required for the preparation of glycoconjugate
derivatives is quite tedious, and does not provide sufficient
quantities of material for comprehensive biological studies.
Besides, the isolation of oligosaccharides without biological
contamination is quite challenging. Therefore, it is often
preferable or mandatory to find solutions using synthetic
organic chemistry.[13,14] In this context, a convergent syn-
thesis of the hexasaccharide repeating unit of the O-antigen
of Shigella flexneri serotype 1d is reported in this paper as
its 2-aminoethyl glycoside. To understand the conforma-
tional behavior of the hexasaccharide repeating unit in
water, a detailed NMR spectroscopic and molecular model-
ing study of the synthesized hexasaccharide was under-
taken, and these results are also presented.
Introduction
Shigellosis is a serious health concern, and is caused by
Shigella infections.[1] Shigella is a major cause of diarrheal
outbreaks worldwide that cause millions of deaths annu-
ally.[2] Shigella is a well-studied human pathogen associated
with dysentery, which is diagnosed by bloody diarrhea, ab-
dominal cramps, fever, etc.[3] Shigella are mainly divided
into four species, which are Shigella dysenteriae (group A),
Shigella flexneri (group B), Shigella boydii (group C), and
Shigella sonnei (group D).[4] These species are further classi-
fied into several serotypes on the basis of their O-antigen
structures. In general, Shigella O-antigens are acidic in na-
ture (with some exceptions), as they contain acidic sugars
or functional groups.[5] Since cell-wall O-antigens are the
main virulence factors for Shigella infections, several O-an-
tigenic structures related to different strains of Shigella have
been isolated and characterized to date.[5] Recently, the
structure of the repeating unit of the cell-wall O-antigen of
Shigella flexneri serotype 1d, isolated from diarrheal
patients in China,[6] was reported by Shashkov et al.[7] The
repeating unit consists of a neutral oligosaccharide with a
linear backbone composed of l-rhamnose moieties, with a
branch at the reducing end.
[a] Bose Institute, Division of Molecular Medicine, 1/12, C.I.T.
Scheme VII-M,
Results and Discussion
Kolkata 700054, India
E-mail: akmisra69@gmail.com
http://www.boseinst.ernet.in/
[b] Bose Institute, Department of Biophysics, 1/12, C.I.T. Scheme
VII-M,
The target 2-aminoethyl-substituted hexasaccharide 1
was synthesized using a convergent [3+3] block glycosyl-
ation strategy. For the construction of the target molecule,
suitably derivatized monosaccharide intermediates 5, 6,[15]
7, 8,[16] 9,[17] and 10[18] were prepared from commercially
available reducing sugars using earlier reported reaction
Kolkata 700054, India
E-mail: anirbanbhunia@gmail.com
http://www.boseinst.ernet.in
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201402392.
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D. Dhara, R. K. Kar, A. Bhunia, A. K. Misra
FULL PAPER
conditions (Figure 1). A regioselective reaction was used for
the preparation of trisaccharide acceptor 12. For this, re-
cently developed glycosylation conditions using catalytic
nitrosyl tetrafluoborate (NOBF4)[19] were used to activate
glycosyl trichloroacetimidate derivative 8, and this was
followed by iodonium-ion-promoted glycosylation using
thioglycoside 7, and removal of the PMB (p-meth-
oxybenzyl) ether in one pot[20] to give 12 in a minimum
number of reaction steps. The preparation of trisaccharide
thioglycoside donor 15 was achieved using a high-yielding
NOBF4-catalyzed orthogonal glycosylation[21] of l-
rhamnosyl thioglycoside acceptor 6 with d-glucopyranosyl
trichloroacetimidate donor 9. The key features of our syn-
thetic strategy are the use of an in-situ-removable protecting
group (PMB ether), a minimum number of steps, and con-
vergent block glycosylations. The presence of the anomeric
2-aminoethyl linker could be useful for the preparation of
glycoconjugate derivatives by providing ready access to an
amino group for linking with a protein.
Scheme 1. Reagents: (a) HO(CH2)2NHCbz (Cbz = benzyloxy-
carbonyl), BF3·OEt2, (CH2Cl)2, 55 °C, 16 h, 76 %; (b) CH3ONa
(0.05 m), CH3OH, room temperature, 1 h; (c) PhCH(OCH3)2,
pTsOH, DMF, room temperature, 12 h, 77 % over two steps;
(d) Et3SiH, I2, CH3CN, 5 °C, 30 min, 72 %; (e) p-methoxybenzyl
chloride, NaOH, DMF, room temperature, 3 h, 88%.
solvent mixture gave disaccharide acceptor 11 in 73% yield,
together with a minor quantity (about 7%) of its re-
gioisomer, which was removed by column chromatography.
The regioselective formation of compound 11 was con-
firmed by analysis of its spectra [signals at δ = 5.10 (d, J =
1
8.0 Hz, 1A-H), 4.62 (br. s, 1B-H) in the H NMR spectrum,
and δ = 101.7 (C-1B), 98.7 (C-1A) in the 13C NMR spec-
trum]. Regioselective 4-OH glycosylation of 2-deoxy-2-N-
phthalimido-d-glucosamine 3,4-diol derivative has also
been observed earlier.[27] Stereoselective glycosylation of
compound 11 with l-rhamnosyl thioglycoside donor 7 in
the presence of a combination of N-iodosuccinimide (NIS)
and triflic acid (TfOH)[28,29] in CH2Cl2/Et2O (2:1) followed
by in situ removal[20] of the PMB group from the glycos-
ylated product in one pot by raising the reaction tempera-
ture gave trisaccharide acceptor 12 in 71% yield. Analysis
of the spectra of compound 12 confirmed its formation [sig-
nals at δ = 5.43 (d, J = 3.0 Hz, 1B-H), 5.25 (br. s, 1C-H),
Figure 1. Structures of the hexasaccharide repeating unit and its
synthetic intermediates.
1
5.20 (d, J = 8.5 Hz, 1A-H) in the H NMR spectrum, and
Reaction of 1,2,3,4-tetra-O-acetyl-2-deoxy-2-N-phthal- δ = 98.1 (C-1A), 96.2 (C-1C), 95.4 (C-1B) in the 13C NMR
imido-β-d-glucopyranose (2)[22] with 2-N-carboxybenz- spectrum]. The JC-1,1-H coupling constants of 154.0, 165.0,
1
ylamino ethanol in the presence of boron trifluoride–diethyl and 171.0 Hz in the H-coupled 13C NMR spectrum con-
ether (BF3·OEt2) at elevated temperature gave compound 3 firmed the presence of two α glycosidic linkages and one β
in 76% yield. Removal of the O-acetyl groups using sodium linkage[30,31] in compound 12 (Scheme 2).
methoxide,[23] followed by benzylidene acetal formation[24]
In another experiment, stereoselective 1,2-cis glycosyl-
using benzaldehyde dimethyl acetal in the presence of ation of l-rhamnosyl thioglycoside acceptor 6 with d-gluc-
pTsOH gave compound 4 in 77% yield. Regioselective ring ose-derived trichloroacetimidate derivative 9 in the presence
[19]
opening[25] of the benzylidene acetal in compound 4 using of NOBF4 in CH2Cl2/Et2O (2:1) gave disaccharide thio-
a combination of triethylsilane and molecular iodine gave glycoside derivative 13 in 74% yield. This reaction relies on
compound 5 in 72% yield. In another experiment, ethyl 2,4- the orthogonal properties[21] of the anomeric thioethyl
di-O-benzyl-1-thio-α-l-rhamnopyranoside
(6)[15]
was group in compound 6. Spectral analysis of compound 13
treated with p-methoxybenzyl chloride in the presence of confirmed its formation [signals at δ = 5.25 (d, J = 1.5 Hz,
sodium hydroxide[26] to give thioglycoside derivative 7 in 1E-H), 5.16 (d, J = 3.5 Hz, 1F-H) in the 1H NMR spectrum,
88% yield (Scheme 1).
and δ = 94.5 (C-1F), 82.2 (C-1E) in the 13C NMR spectrum].
Stereo- and regioselective glycosylation of d-glucosamine The anomeric thioethyl group in compound 13 was re-
diol acceptor 5 with d-glucose trichloroacetimidate deriva- moved by treatment with N-bromosuccinimide (NBS)[32] to
[19]
tive 8 in the presence of NOBF4 in a CH2Cl2/Et2O (1:2) give the disaccharide hemiacetal derivative, which, on treat-
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Hexasaccharide from Shigella flexneri Serotype 1d
Spectral analysis confirmed the exclusive formation of com-
pound 16 under these reaction conditions. Signals in the 1H
and 13C NMR spectra [δ = 5.72 (br. s, 1F-H), 5.21 (d, J =
8.0 Hz, 1A-H), 5.20–5.18 (1B-H, 1C-H, 1E-H), 5.04 (br. s,
1D-H) in the 1H NMR spectrum, and δ = 99.9 (C-1D), 97.9
(2 C, C-1A, C-1C), 97.2 (C-1E), 94.6 (C-1B), 94.5 (C-1F) in
the 13C NMR spectrum] and the JC-1,1-H coupling con-
stants[30,31] in the gated 1H-coupled 13C NMR spectrum
(154.0, 165.0, 171.0, 165.0, 171.5, and 171.5 Hz) confirmed
the presence of five α glycosidic linkages and one β linkage.
Compound 16 was subjected to a sequence of deprotection
reactions comprising (a) removal of the N-phthaloyl group
using ethylenediamine,[34] followed by acetylation using ace-
tic anhydride and pyridine; (b) catalytic transfer hydrogena-
tion[35] using a combination of triethylsilane and Pd/C
(10%) for the removal of the benzyl ethers and the benzyl-
oxycarbonyl (Cbz) protecting group, and (c) removal of the
O-acetyl groups using sodium methoxide, to give depro-
tected hexasaccharide 1 as its 2-aminoethyl glycoside in
54% overall yield. The product was passed through a col-
umn of LH-20 Sephadex using CH3OH/H2O (4:1) as eluent
to give pure compound 1. Spectral analysis of compound 1
unambiguously confirmed its formation [δ = 5.23 (d, J =
3.0 Hz, 1B-H), 5.12 (br. s, 1D-H), 5.03 (br. s, 1C-H), 5.00 (d,
J = 3.0 Hz, 1F-H), 4.91 (br. s, 1E-H), 4.49 (d, J = 7.5 Hz,
1A-H) in the 1H NMR spectrum, and δ = 101.9 (C-1E),
100.7 (C-1C), 100.6 (2 C, C-1A, C-1D), 97.7 (C-1B), 95.3 (C-
1F) in the 13C NMR spectrum] (Scheme 4).
Scheme 2. Reagents: (a) NOBF4, CH2Cl2/Et2O (1:2), –10 °C,
30 min, 73 %; (b) NIS, TfOH, CH2Cl2/Et2O (2:1), MS (4 Å),
–25 °C, 30 min, then 0–5 °C, 30 min, 71%.
ment with trichloroacetonitrile[33] in the presence of DBU
(1,8-diazabicycloundec-7-ene), gave compound 14 in 72%
yield. This compound was used immediately in the next
glycosylation reaction. NOBF4-catalyzed stereoselective α-
glycosylation[19] of disaccharide trichloroacetimidate donor
14 with thioglycoside acceptor 10 in a CH2Cl2/Et2O (1:2)
solvent mixture gave trisaccharide thioglycoside derivative
15 in 70% yield. The presence of characteristic signals of
compound 15 in the 1H and 13C NMR spectra, and
the JC-1,1-H coupling constants (165.0, 171.0, and
171.5 Hz)[30,31] in the gated 1H-coupled 13C NMR spectrum
confirmed its formation [δ = 5.25 (d, J = 3.0 Hz, 1F-H),
1
5.21 (br. s, 1E-H), 5.13 (br. s, 1D-H) in the H NMR spec-
trum, and δ = 99.8 (C-1D), 94.6 (C-1F), 83.7 (C-1E) in the
13C NMR spectrum] (Scheme 3).
Scheme 4. Reagents: (a) NIS, TfOH, CH2Cl2/Et2O (1:1), MS (4 Å),
–25 °C, 30 min, 69 %; (b) ethylenediamine, nBuOH, 90 °C, 8 h;
(c) acetic anhydride, pyridine, room temperature, 2 h; (d) Et3SiH,
Pd/C (10 %), CH3OH/CH2Cl2 (9:1), room temperature, 10 h;
(e) CH3ONa (0.1 m), CH3OH, room temperature, 3 h, 54% over
four steps.
Scheme 3. Reagents: (a) NOBF4, CH2Cl2/Et2O (1:2), –10 °C,
30 min, 74% for compound 13, and 70% for compound 15; (b) N-
bromosuccinimide (NBS) acetone/H2O (9:1), room temperature, Conformational Analysis
40 min; (c) CCl3CN, DBU, CH2Cl2, –10 °C, 1 h, 72 % over two
steps.
Molecular dynamics (MD) simulation in combination
with NOE-based NOESY or ROESY NMR experiments
Finally, iodonium-ion-catalyzed stereoselective conver- can provide the conformations of a molecule in solution.
gent [3+3] α-glycosylation of trisaccharide thioglycoside do- One- and two-dimensional homonuclear NMR spectra,
nor 15 with trisaccharide acceptor 12 in the presence of a such as the 1H NMR and 1H–1H NOESY/TOCSY spectra,
combination of NIS and TfOH[28,29] in CH2Cl2/Et2O (1:1) of hexasaccharide 1 showed severe signal overlap, due to
gave branched hexasaccharide derivative 16 in 69% yield. the similar chemical and electronic environments of the
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D. Dhara, R. K. Kar, A. Bhunia, A. K. Misra
FULL PAPER
carbohydrate protons (see Supporting Information, Fig- ergy state was also found for the vdW contribution, where
ure S1). In order to explore the conformational behavior the solvent energy is energetically stable in comparison to
of hexasaccharide 1 in solution, an all-atoms explicit MD the solute itself. These results indicate that the solution-
simulation was performed for a time period of 30 ns. The state conformations of hexasaccharide 1 are energetically
initial conformational states for hexasaccharide 1 were favorable, and hence the solvent-perturbation-induced con-
evaluated by examining proton–proton distances at the formations are very stable. A simple overview of the com-
inter-glycosidic linkages. The proton–proton distance as an- puted conformational states of hexasaccharide 1 are shown
alyzed from the trajectory was found to be very rigid for in Figure 3 as an ensemble structure. The rigidity of
3A-H/1C-H, 2C-H/1D-H, and 2D-H/1F-H which were all the backbone for the α-l-rhamnose moiety (C), the α-l-
within a range of 2–4 Å (see Supporting Information, Fig- rhamnose moiety (D), and the α-l-rhamnose moiety (E)
ure S2). The fact that such a confined distance was ob- can be observed, and the relative flexibility of the β-d-
served throughout the time of the simulation reveals that glucosamine moiety (A), the α-d-glucose moiety (B), and
the compound has a rigid conformation with respect to the α-d-glucose moiety (F) can also be observed. Interest-
these glycosidic linkages. The distance for 2C-H/1D-H was ingly the flexibility of the glycosidic linkage between the α-l-
found to be dynamic (see Supporting Information, Fig- rhamnose moiety (C) and the α-l-rhamnose moiety
ure S2A), which suggests a greater degree of freedom with (D) is well correlated with the weak NOE cross-peak
respect to the linkage between the β-d-glucosamine moiety in the NOESY spectrum (see Supporting Information, Fig-
(A) and the α-d-glucose moiety (B). On the other hand, a ure S1).
very small deviation for the proton–proton distance
of 2E-H/1F-H was found, which cannot be accounted for
by a conformationally flexible linkage between the α-l-
rhamnose moiety (E) and the α-d-glucose moiety (F) (see
Supporting Information, Figure S2B). This result is in good
agreement with the NOESY spectrum of hexasaccharide 1,
where no significant NOE was observed between the α-d-
glucose moiety (B) and the α-l-rhamnose moiety (C) (see
Supporting Information, Figure S1). In another approach,
the torsional angles were measured using the definition of
the oligosaccharide dihedral angles as φn (Hn–Cn–On–Cn+1
)
and ψn (Cn–On–Cn+1–Hn+1). The torsional distributions for
the respective moieties were found to be well conserved and
within the conventional range of oligosaccharides, except
for the α-d-glucose moiety (B) (see Supporting Information,
Figure S3). The φ angle distribution (HA–CA–OB–CB) was
found to be more flexible than the ψ angle (CA–OB–CB–
HB), which seems to be in the range –50° to +50°. In a
precise search of the conformational forms that are possible
in the solution state, cluster analysis based on root-mean-
square deviation (RMSD) of trajectory frames was at-
tempted. The cluster analysis plot (Figure 2) is based on the
average method of hierarchical cluster linkage, using 2 Å as
the merging distance cut-off, and it is presented as a heat
map with respect to hexasaccharide 1 conformations in the
time of the MD simulation. The maximum numbers of pop-
ulations were found to be in the range of 2–3.5 Å in com-
parison to the initial frame of reference. This suggests that
the oligosaccharide conformation is not very flexible, that
it remains rigid with respect to the glycosidic linkages be-
tween the sugar moieties. The energetics of hexasaccharide
1 in the solution state were also theoretically evaluated to
get an account of the Coulombic and van der Waals (vdW)
contributions, and the perturbations in these contributions
over the different conformations. The Coulomb energy of
hexasaccharide 1 (solute molecule) was found to be approx-
imately 400 kcalmol–1, whereas the Coulombic energy of
hexasaccharide 1 owing to interaction with solvent was
found to be energetically favorable (–200 kcalmol–1) (see
Figure 2. Hierarchical cluster analysis of conformations of hexasac-
charide 1, based on the MD simulation trajectory.
Figure 3. Ensemble conformational structures of hexasaccharide 1
Supporting Information, Figure S4). A similar favorable en- sampled over a 3 ns time interval from MD simulation.
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Eur. J. Org. Chem. 2014, 4577–4584

Hexasaccharide from Shigella flexneri Serotype 1d
COCH3) ppm. ESI-MS: m/z = 635.1 [M + Na]+. C30H32N2O12
(612.19): calcd. C 58.82, H 5.27; found C 58.65, H 5.50.
Conclusions
In summary, a concise chemical synthetic strategy has
been developed for the synthesis of the hexasaccharide re-
peating unit of the cell-wall O-antigen of Shigella flexneri
type 1d using a [3+3] block glycosylation and a minimum
number of steps. The yields of both the glycosylations and
the functional group manipulations were excellent. Confor-
mational analysis of the synthesized hexasaccharide was
carried out using 2D NOESY NMR spectroscopy and MD
simulation. The conformational analysis showed that the
molecule has a partly flexible and partly rigid structure in
solution.
2-(Benzyloxycarbonylamino)ethyl 4,6-O-Benzylidene-2-deoxy-2-N-
phthalimido-β-D-glucopyranoside (4): A solution of compound 3
(4 g, 6.53 mmol) in CH3ONa (0.05 m solution in MeOH; 100 mL)
was allowed to stir at room temperature for 1 h. The reaction mix-
ture was neutralized with Dowex 50W X8 (H+) resin, then it was
filtered, and the filtrate was concentrated under reduced pressure
to give the de-O-acetylated product.
The de-O-acetylated product (3.1 g) was dissolved in anhydrous
DMF (5 mL), and benzaldehyde dimethyl acetal (1.5 mL,
9.99 mmol) and pTsOH (200 mg) were added. The reaction mixture
was stirred at room temperature for 12 h. The reaction mixture was
neutralized with Et3N (1 mL), and the solvents were removed un-
der reduced pressure. The crude product was diluted with CH2Cl2
(100 mL), and the organic layer was washed with water, dried
(Na2SO4) and concentrated. The crude product was crystallized
from EtOH to give pure compound 4 (2.9 g, 77%) as a white solid,
m.p. 114–115 °C (EtOH). [α]D = –32 (c = 1.0, CHCl3). IR (KBr):
Experimental Section
General Methods: All reactions were monitored by thin-layer
chromatography on silica-gel-coated TLC plates. The spots on TLC
plates were visualized by spraying the plates with ceric sulfate [2%
Ce(SO4)2 in 2 n H2SO4], and warming with a hot plate. Silica gel
230–400 mesh was used for column chromatography. NMR spectra
were recorded with a Bruker Avance 500 MHz instrument using
CDCl3 as solvent and tetramethylsilane as an internal reference
unless otherwise stated. Chemical shifts are expressed in ppm on
the δ scale. Complete assignments of proton and carbon spectra
were carried out using a standard set of NMR experiments, e.g.,
1H NMR, 13C NMR, 13C DEPT 135, 2D COSY, 2D HSQC, etc.
In addition, 2D NOESY (300 ms mixing time) experiments were
carried out to assist in the conformational analysis. The NOESY
experiments were performed with 456 increments in t1, and 2K
data points in t2. The spectral width was normally 10 ppm in both
dimensions. After 16 dummy scans, 80 scans were recorded per t1
increment. After zero-filling in t1, 4K (t2) ϫ 1K (t1) data matrices
were obtained. The two-dimensional NMR spectroscopic data were
processed using the TopSpin software suite (Bruker, Switzerland).
ESI-MS spectra were recorded with a Micromass mass spectrome-
ter. Optical rotations were recorded with a Jasco P-2000 spectrome-
ter. Commercially available grades of organic solvents of adequate
purity were used in all reactions.
ν = 3448, 3019, 2883, 1773, 1715, 1388, 1233, 1113, 1086, 759,
˜
697 cm–1. 1H NMR (500 MHz, CDCl3): δ = 7.86–7.23 (m, 14 H,
Ar-H), 5.50 (s, 1 H, PhCH), 5.20 (d, J = 8.5 Hz, 1 H, 1-H), 5.08–
5.06 (m, 1 H, NH), 4.95–4.85 (dd, J = 12.5 Hz, 2 H, PhCH2), 4.55
(t, J = 9.5 Hz, 1 H, 4-H), 4.31 (dd, J = 10.0, 4.0 Hz, 1 H, 6-Ha),
4.16 (dd, J = 8.5 Hz, 1 H, 6-Hb), 3.75–3.71 (m, 2 H, OCH2), 3.57–
3.51 (m, 3 H, 2-H, 3-H, 5-H), 3.21–3.19 (m, 2 H, NCH2) ppm. 13
C
NMR (125 MHz, CDCl3): δ = 167.1 (2 C, PhthCO), 156.2
(CbzCO), 137.0–123.5 (Ar-C), 101.8 (PhCH), 99.1 (C-1), 82.0 (C-
3), 69.1 (C-6), 68.5 (OCH2), 68.4 (C-4), 66.5 (PhCH2), 66.2 (C-5),
56.7 (C-2), 40.8 (NCH2) ppm. ESI-MS: m/z = 597.1 [M + Na]+.
C31H30N2O9 (574.19): calcd. C 64.80, H 5.26; found C 64.64, H
5.44.
2-(Benzyloxycarbonylamino)ethyl 6-O-Benzyl-2-deoxy-2-N-phthal-
imido-β-D-glucopyranoside (5): Et3SiH (1.5 mL, 9.39 mmol) and io-
dine (500 mg, 1.97 mmol) were added to a solution of compound
4 (2.5 g, 4.35 mmol) in CH3CN (15 mL) at 5 °C, and the reaction
mixture was stirred at the same temperature for 30 min. The reac-
tion mixture was diluted with CH2Cl2 (50 mL), and the organic
layer was successively washed with Na2S2O3 (5% aq.) and
NaHCO3 (satd.), then it was dried (Na2SO4) and concentrated. The
crude product was purified over SiO2 using hexane/EtOAc (3:1) as
eluent to give pure compound 5 (1.8 g, 72%) as a yellow oil. [α]D
2-(Benzyloxycarbonylamino)ethyl 3,4,6-Tri-O-acetyl-2-deoxy-2-N-
phthalimido-β-D-glucopyranoside (3): Compound 2 (5 g, 10.47 mmol)
= –14 (c = 1.0, CHCl ). IR (neat): ν = 3390, 3031, 2924, 1713,
˜
3
and 2-(benzyloxycarbonylamino)ethanol (4 g, 20.5 mmol) were dis-
solved in (CH2Cl)2 (50 mL), and BF3·OEt2 (1.5 mL, 12.15 mmol)
was added. The reaction mixture was allowed to stir at 55 °C for
16 h. The reaction mixture was poured into satd. NaHCO3 solu-
tion, and extracted with CH2Cl2 (100 mL). The organic layer was
washed with water, dried (Na2SO4), and concentrated under re-
duced pressure. The crude product was purified over SiO2 using
hexane/EtOAc (3:1) as eluent to give pure compound 3 (4.9 g, 76%)
as a white solid, m.p. 109–110 °C (EtOH). [α]D = +22 (c = 1.0,
1520, 1391, 1069, 754, 721, 697 cm–1. 1H NMR (500 MHz, CDCl3):
δ = 7.75–7.23 (m, 14 H, Ar-H), 5.36–5.34 (m, 1 H, NH), 5.16 (d,
J = 8.0 Hz, 1 H, 1-H), 4.97–4.89 (dd, J = 12.5 Hz each, 2 H,
PhCH2), 4.54–4.46 (dd, J = 12.5 Hz, 2 H, PhCH2), 4.25 (t, J =
9.0 Hz, 1 H, 3-H), 4.07 (t, J = 9.0 Hz, 1 H, 2-H), 3.75–3.70 (m, 3
H, 5-H, 6-Hab), 3.66–3.53 (m, 3 H, 4-H, OCH2), 3.35–3.20 (m, 2
H, NCH2) ppm. 13C NMR (125 MHz, CDCl3): δ = 168.4 (2 C,
PhthCO), 156.4 (CbzCO), 137.6–123.5 (Ar-C), 98.6 (C-1), 74.2 (C-
4), 73.6 (PhCH2), 73.2 (C-5), 71.7 (C-3), 69.9 (C-6), 69.5 (OCH2),
66.5 (PhCH2), 56.3 (C-2), 41.1 (NCH2) ppm. ESI-MS: m/z = 599.2
[M + Na]+. C31H32N2O9 (576.21): calcd. C 64.57, H 5.59; found C
64.40, H 5.78.
CHCl ). IR (KBr): ν = 3362, 3031, 2932, 1757, 1452, 1388, 1233,
˜
3
1113, 1086, 759, 697 cm–1. 1H NMR (500 MHz, CDCl3): δ = 7.77–
7.24 (m, 9 H, Ar-H), 5.69 (dd, J = 10.5, 9.0 Hz, 1 H, NH), 5.35
(d, J = 8.0 Hz, 1 H, 1-H), 5.14–5.07 (m, 2 H, 3-H, 4-H), 5.03–4.87
(m, 2 H, PhCH2), 4.27–4.23 (m, 2 H, 6-Hab), 4.13–4.10 (m, 1 H, Ethyl 2,4-Di-O-benzyl-3-O-(p-methoxybenzyl)-1-thio-α-
L-rhamno-
5-H), 3.81–3.74 (m, 2 H, 2-H, OCH2), 3.69–3.62 (m, 1 H, OCH2), pyranoside (7): Powdered NaOH (500 mg, 12.5 mmol) and p-meth-
3.35–3.18 (m, 2 H, NCH2), 2.07, 2.05, 1.82 (3 s, 9 H, 3 COCH3)
oxybenzyl chloride (1.4 mL, 10.32 mmol) were added to a solution
ppm. 13C NMR (125 MHz, CDCl3): δ = 170.5, 170.0, 169.3 (3 of compound 6 (2 g, 5.15 mmol) in DMF (10 mL), and the reaction
COCH3), 167.1 (2 C, PhthCO), 156.1 (CbzCO), 136.4–123.7 (Ar- mixture was stirred at room temperature for 3 h. The reaction mix-
C), 98.3 (C-1), 71.9 (C-3), 70.6 (C-4), 69.5 (C-6), 68.7 (OCH2), 66.5
ture was poured into water, and extracted with CH2Cl2 (100 mL).
(PhCH2), 61.8 (C-5), 54.5 (C-2), 40.8 (NCH2), 20.7, 20.6, 20.4 (3 The organic layer was washed with water, dried (Na2SO4), and con-
Eur. J. Org. Chem. 2014, 4577–4584
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4581

D. Dhara, R. K. Kar, A. Bhunia, A. K. Misra
FULL PAPER
centrated. The crude product was purified over SiO2 using hexane/
3400, 3019, 2927, 1736, 1717, 1388, 1216, 1046, 758 cm–1. 1H NMR
EtOAc (10:1) as eluent to give pure compound 7 (2.3 g, 88%) as a
(500 MHz, CDCl3): δ = 7.74–7.17 (m, 34 H, Ar-C), 5.43 (d, J =
yellow oil. [α]D = –61 (c = 1.0, CHCl ). IR (neat): ν = 3446, 3094, 3.0 Hz, 1 H, 1B-H), 5.35–5.31 (m, 1 H, NH), 5.25 (br. s, 1 H,
˜
3
2930, 1723, 1531, 1454, 1371, 1237, 1095, 752, 698 cm–1. H NMR
(500 MHz, CDCl3): δ = 7.40–7.23 (m, 12 H, Ar-H), 6.85 (d, J =
9.0 Hz, 2 H, Ar-H), 5.24 (br. s, 1 H, 1-H), 4.97 (d, J = 11.0 Hz, 1
H, PhCH2), 4.74, 4.70 (2 d, J = 12.5 Hz, 2 H, PhCH2), 4.65 (d, J
= 11.0 Hz, 1 H, PhCH2), 4.51 (br. s, 2 H, PhCH2), 4.04–4.00 (m, 1
1C-H), 5.20 (d, J = 8.5 Hz, 1 H, 1A-H), 5.05–4.94 (dd, J = 11.0 Hz,
2 H, PhCH2), 4.85–4.60 (m, 8 H, 4B-H, PhCH2), 4.54–4.48 (m, 3
H, 3A-H, PhCH2), 4.34–4.30 (m, 2 H, 2A-H, PhCH2), 3.97 (t, J =
8.5 Hz, 1 H, 4A-H), 3.85 (dd, J = 10.0, 3.0 Hz, 1 H, 3C-H), 3.80–
3.55 (m, 11 H, 2C-H, 3B-H, 5A-H, 5B-H, 5C-H, 6A-Hab, 6B-Hab,
1
H, 5-H), 3.81 (s, 3 H, OCH3), 3.79–3.76 (m, 2 H, 2-H, 3-H), 3.61 OCH2), 3.42 (dd, J = 10.0, 3.5 Hz, 1 H, 2B-H), 3.38–3.25 (m, 3 H,
(t, J = 9.0 Hz, 1 H, 4-H), 2.62–2.52 (m, 2 H, SCH2CH3), 1.34 (d, 4C-H, NCH2), 1.92, 1.49 (2 s, 6 H, 2 COCH3), 1.27 (d, J = 6.0 Hz,
J = 6.0 Hz, 3 H, CCH3), 1.27 (t, J = 7.0 Hz, 3 H, SCH2CH3) ppm.
3 H, CCH3) ppm. 13C NMR (125 MHz, CDCl3): δ = 170.4, 169.0
(2 COCH3), 167.8 (2 C, PhthCO), 156.2 (CbzCO), 138.5–123.5 (Ar-
13C NMR (125 MHz, CDCl3): δ = 159.2–113.7 (Ar-C), 81.9 (C-1),
80.5 (C-4), 79.9 (C-2), 76.6 (PhCH2), 75.3 (PhCH2), 72.2 (PhCH2), C), 98.1 (C-1A), 96.2 (C-1C), 95.4 (C-1B), 81.6 (C-4C), 79.0 (C-2B),
71.7 (C-3), 68.4 (C-5), 55.1 (OCH3), 25.4 (SCH2CH3), 18.0 (CCH3), 78.3 (C-3B), 77.9 (C-2C), 77.8 (C-3A), 74.9 (PhCH2), 74.8 (PhCH2),
15.2 (SCH2CH3) ppm. ESI-MS: m/z
C30H36O5S (508.23): calcd. C 70.84, H 7.13; found C 70.67, H 7.28.
=
531.2 [M
+
Na]+.
74.4 (C-4A), 73.4 (PhCH2), 73.3 (PhCH2), 73.2 (C-3C), 72.8
(PhCH2), 71.2 (C-4B), 69.5 (C-6B), 68.9 (2 C, C-5C, C-6A), 68.8
(C-5A), 68.7 (C-5B), 66.6 (PhCH2), 61.3 (OCH2), 55.4 (C-2A), 41.2
(NCH2), 20.6, 20.4 (2 COCH3), 18.0 (CCH3) ppm. MALDI-MS:
m/z = 1351.5 [M + Na]+. C75H80N2O20 (1328.53): calcd. C 67.76,
H 6.07; found C 67.57, H 6.24.
2-(Benzyloxycarbonylamino)ethyl (4,6-Di-O-acetyl-2,3-di-O-benzyl-
α-
D-glucopyranosyl)-(1Ǟ4)-6-O-benzyl-2-deoxy-2-N-phthalimido-β-
D-glucopyranoside (11):
A
solution of compound (1.5 g,
5
2.60 mmol) and compound 8 (2.3 g, 3.90 mmol) in anhydrous
CH2Cl2/Et2O (1:2 v/v; 10 mL) was cooled to –10 °C under argon.
NOBF4 (250 mg, 2.14 mmol) was added, and the reaction mixture
was stirred at the same temperature for 30 min. The reaction mix-
ture was diluted with CH2Cl2 (100 mL), and the organic layer was
successively washed with satd. NaHCO3 and water, then it was
dried (Na2SO4) and concentrated. The crude product was purified
over SiO2 using hexane/EtOAc (4:1) as eluent to give pure com-
pound 11 (1.9 g, 73%) as a colorless oil. [α]D = +16 (c = 1.0,
Ethyl (2,3,4,6-Tetra-O-benzyl-α-
D-glucopyranosyl)-(1Ǟ3)-2,4-di-O-
benzyl-1-thio-α- -rhamnopyranoside (13): A solution of compound
L
6 (1.5 g, 3.86 mmol) and compound 9 (3 g, 4.38 mmol) in CH2Cl2/
Et2O (1:2 v/v; 15 mL) was cooled to –10 °C under argon. NOBF4
(350 mg, 2.99 mmol) was added, and the reaction mixture was
stirred at the same temperature for 30 min. The reaction mixture
was diluted with CH2Cl2 (100 mL), and the organic layer was suc-
cessively washed with satd. NaHCO3 and water, then it was dried
(Na2SO4) and concentrated. The crude product was purified over
SiO2 using hexane/EtOAc (10:1) as eluent to give pure compound
CHCl ). IR (neat): ν = 3410, 3019, 2927, 1710, 1518, 1389, 1210,
˜
3
1047, 756 cm–1. 1H NMR (500 MHz, CDCl3): δ = 7.78–7.22 (m, 24
H, Ar-H), 5.47–5.45 (m, 1 H, NH), 5.10 (d, J = 8.0 Hz, 1 H, 1A-
H), 5.00–4.92 (dd, J = 12.5 Hz, 2 H, PhCH2), 4.89 (t, J = 9.5 Hz,
1 H, 4B-H), 4.79–4.72 (m, 3 H, PhCH2), 4.67 (d, J = 11.5 Hz, 1 H,
PhCH2), 4.62 (br. s, 1 H, 1B-H), 4.61–4.50 (m, 2 H, PhCH2), 4.31–
4.29 (m, 1 H, 3A-H), 4.17 (dd, J = 8.5 Hz, 1 H, 2A-H), 3.89–3.85
(m, 2 H, 3B-H, 5A-H), 3.76–3.66 (m, 7 H, 4A-H, 6A-Hab, 6B-Hab,
OCH2), 3.62–3.59 (m, 1 H, 5B-H), 3.44 (dd, J = 10.0, 3.5 Hz, 1 H,
2B-H), 3.40–3.24 (m, 2 H, NCH2), 1.87, 1.86 (2 s, 6 H, 2 COCH3)
ppm. 13C NMR (125 MHz, CDCl3): δ = 170.3, 169.3 (2 C, 2
COCH3), 156.4 (CbzCO), 137.9–123.4 (Ar-C), 101.7 (C-1B), 98.7
(C-1A), 84.6 (C-3A), 79.2 (C-3B), 78.9 (C-2B), 75.5 (PhCH2), 75.1
(C-4A), 74.5 (PhCH2), 73.5 (PhCH2), 71.4 (C-4B), 70.0 (OCH2),
69.3 (C-6B), 68.9 (C-5A), 68.7 (C-5B), 66.5 (C-6A), 60.8 (PhCH2),
55.5 (C-2A), 41.3 (NCH2), 20.7, 20.5 (2 COCH3) ppm. MALDI-
MS: m/z = 1025.3 [M + Na]+. C55H58N2O16 (1002.38): calcd. C
65.86, H 5.83; found C 65.70, H 6.02.
13 (2.6 g, 74%) as a yellow oil. [α]2D5 = +57 (c = 1.0, CHCl3). IR
1
(neat): ν = 3017, 2921, 1730, 1497, 1216, 1072, 756, 668 cm–1. H
˜
NMR (500 MHz, CDCl3): δ = 7.39–7.11 (m, 30 H, Ar-H), 5.25 (d,
J = 1.5 Hz, 1 H, 1E-H), 5.16 (d, J = 3.5 Hz, 1 H, 1F-H), 4.97–4.57
(m, 10 H, PhCH2), 4.47 (d, J = 11.5 Hz, 1 H, PhCH2), 4.34 (d, J
= 11.5 Hz, 1 H, PhCH2), 4.13 (t, J = 9.5 Hz, 1 H, 3F-H), 4.07–4.00
(m, 4 H, 2E-H, 4E-H, 5E-H, 5F-H), 3.77–3.65 (m, 3 H, 2F-H, 3E-H,
4F-H), 3.61 (dd, J = 12.5, 3.0 Hz, 1 H, 6F-Ha), 3.48 (dd, J = 12.5,
1.5 Hz, 1 H, 6F-Hb), 2.70–2.50 (m, 2 H, SCH2CH3), 1.37 (d, J =
6.0 Hz, 3 H, CCH3), 1.25 (t, J = 7.4 Hz, 3 H, SCH2CH3) ppm. 13
C
NMR (500 MHz, CDCl3): δ = 138.8–127.4 (Ar-C), 94.5 (C-1F),
82.2 (C-1E), 82.1 (C-3F), 79.4 (C-2F), 77.8 (C-3E), 77.3 (C-4F), 76.9
(C-2E), 76.8 (C-4E), 75.6 (2 C, 2 PhCH2), 74.9 (PhCH2), 73.4
(PhCH2), 73.2 (PhCH2), 72.8 (PhCH2), 70.5 (C-5F), 68.6 (C-5E),
68.2 (C-6F), 25.4 (SCH2CH3), 17.9 (CCH3), 15.1 (SCH2CH3) ppm.
MALDI-MS: m/z = 933.4 [M + Na]+. C56H62O9S (910.41): calcd.
C 73.82, H 6.86; found C 73.64, H 7.00.
2-(Benzyloxycarbonylamino)ethyl
pyranosyl)-(1Ǟ3)-[(4,6-di-O-acetyl-2,3-di-O-benzyl-α-
pyranosyl)-(1Ǟ4)]-6-O-benzyl-2-deoxy-2-N-phthalimido-β-
(2,4-Di-O-benzyl-α-
L
-rhamno-
-gluco-
-gluco-
D
(2,3,4,6-Tetra-O-benzyl-α-
D-glucopyranosyl)-(1Ǟ3)-2,4-di-O-
D
benzyl-α- -rhamnopyranosyl Trichloroacetimidate (14): NBS
L
pyranoside (12): Molecular seives (4 Å; 2 g) were added to a solu-
tion of compound 11 (1.5 g, 1.49 mmol) and compound 7 (900 mg,
1.77 mmol) in CH2Cl2/Et2O (2:1 v/v; 10 mL), and the reaction mix-
ture was stirred at room temperature under argon for 30 min. The
reaction mixture was cooled to –25 °C, and NIS (440 mg,
1.95 mmol) and TfOH (15 μL) were added. The reaction mixture
was stirred at the same temperature for 30 min, then the tempera-
ture was raised to 0 °C, and it was stirred at 0 °C for another
30 min. The reaction mixture was diluted with CH2Cl2 (100 mL),
and the organic layer was successively washed with Na2S2O3 (5%),
(470 mg, 2.64 mmol) was added to a solution of compound 13 (2 g,
2.19 mmol) in acetone/H2O (9:1 v/v; 20 mL), and the reaction mix-
ture was stirred at room temperature for 40 min. The reaction mix-
ture was diluted with CH2Cl2 (100 mL), and the organic layer was
successively washed with Na2S2O3 (5% aq.) and water, then it was
dried (Na2SO4), and concentrated under reduced pressure to give
the hemiacetal derivative, which was passed through a short plug
of SiO2.
The hemiacetal derivative (1.4 g) was dissolved in anhydrous
NaHCO3 (satd. aq.), and water, then it was dried (Na2SO4) and CH2Cl2 (10 mL), and CCl3CN (1 mL, 9.97 mmol) was added. The
concentrated. The crude product was purified over SiO2 using hex-
ane/EtOAc (8:1) as eluent to give pure compound 12 (1.4 g, 71%)
mixture was cooled to –10 °C, and DBU (150 μL, 1 mmol) was
added. The reaction mixture was stirred at the same temperature
for 1 h. The solvents were removed under reduced pressure, and the
as a colorless oil. [α]D = +18 (c = 1.0, CHCl ). IR (neat): ν =
˜
3
4582
www.eurjoc.org
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 4577–4584

Hexasaccharide from Shigella flexneri Serotype 1d
crude product was purified over SiO2 using hexane/EtOAc (15:1) as
1.19 (3 d, J = 6.0 Hz, 9 H, 3 CCH3) ppm. 13C NMR (125 MHz,
eluent to give pure compound 14 (1.6 g, 72%), which was used CDCl3): δ = 170.6, 169.1 (2 COCH3), 167.8 (2 C, PhthCO), 156.5
immediately in the next step without spectral characterization.
Ethyl (2,3,4,6-Tetra-O-benzyl-α- -glucopyranosyl)-(1Ǟ3)-(2,4-di-O-
benzyl-α- -rhamnopyranosyl)-(1Ǟ2)-3,4-di-O-benzyl-1-thio-α-
(CbzCO), 138.8–123.6 (Ar-C), 99.9 (C-1D), 97.9 (2 C, C-1A, C-1C),
97.2 (C-1E), 94.6 (C-1B), 94.5 (C-1F), 82.2 (2 C, C-3C, C-3F), 80.7
(C-4C), 80.5 (C-4F), 79.8 (C-4D), 79.5 (C-2B), 79.2 (3 C, C-2C, C-
2E, C-3D), 77.9 (2 C, C-2F, C-4A), 77.7 (2 C, C-2D, C-4E), 77.3 (C-
3A), 75.6 (2 C, PhCH2), 75.4 (C-3E), 75.1 (PhCH2), 75.0 (2 C, 2
PhCH2), 74.9 (PhCH2), 73.7 (PhCH2), 73.4 (PhCH2), 73.1
(PhCH2), 72.7 (PhCH2), 72.6 (PhCH2), 72.5 (PhCH2), 72.3
(PhCH2), 70.4 (2 C, C-4B, C-5D), 69.5 (C-5F), 69.2 (C-6F), 68.9 (C-
6B), 68.8 (C-5E), 68.7 (C-5C), 68.5 (C-5B), 68.3 (C-5A), 68.2 (C-6A),
66.6 (OCH2), 61.4 (PhCH2), 55.5 (C-2A), 41.0 (NCH2), 20.6, 20.5
(2 COCH3), 18.3, 18.2, 18.0 (3 CCH3) ppm. MALDI-MS: 2526.0
[M + Na]+. C149H158N2O33 (2503.07): calcd. C 71.45, H 6.36; found
C 71.28, H 6.50.
D
L
L-
rhamnopyranoside (15): A solution of compound 10 (500 mg,
1.28 mmol) and compound 14 (1.5 g, 1.48 mmol) in CH2Cl2/Et2O
(1:2 v/v; 10 mL) was cooled to –10 °C under argon. NOBF4 (80 mg,
0.68 mmol) was added, and the mixture was stirred at the same
temperature for 30 min. The reaction mixture was diluted with
CH2Cl2 (100 mL), and the organic layer was successively washed
with satd. NaHCO3 and water, then it was dried (Na2SO4) and
concentrated. The crude product was purified over SiO2 using hex-
ane/EtOAc (8:1) as eluent to give pure compound 15 (1.1 g, 70%)
as a yellow oil. [α]D = +1 (c = 1.0, CHCl ). IR (neat): ν = 3017,
˜
3
2928, 1496, 1454, 1216, 1067, 757, 668 cm–1. H NMR (500 MHz,
1
2-Aminoethyl (α-
(1Ǟ2)-(α- -rhamnopyranosyl)-(1Ǟ3)-(α-
(1Ǟ3)-[(α- -glucopyranosyl)-(1Ǟ4)]-2-acetamido-2-deoxy-β-D-
D-Glucopyranosyl)-(1Ǟ3)-(α-
L-rhamnopyranosyl)-
L
L
-rhamnopyranosyl)-
CDCl3): δ = 7.39–7.13 (m, 40 H, Ar-H), 5.25 (d, J = 3.0 Hz, 1 H,
1F-H), 5.21 (br. s, 1 H, 1E-H), 5.13 (br. s, 1 H, 1D-H), 5.00–4.34
(m, 16 H, PhCH2), 4.20 (dd, J = 9.5, 3.0 Hz, 1 H, 3E-H), 4.16 (t,
J = 9.0 Hz, 1 H, 3F-H), 4.12 (br. s, 1 H, 2E-H), 4.09–4.01 (m, 3 H,
2D-H, 5D-H, 5F-H), 3.87–3.84 (m, 1 H, 5E-H), 3.80 (dd, J = 9.0,
3.0 Hz, 1 H, 3D-H), 3.77 (t, J = 9.5 Hz, 1 H, 4F-H), 3.72–3.64 (m,
3 H, 2F-H, 4D-H, 6F-Ha), 3.54 (dd, J = 10.5, 1.5 Hz, 1 H, 6F-Hb),
3.49 (t, J = 9.0 Hz, 1 H, 4E-H), 2.60–2.45 (m, 2 H, SCH2CH3),
1.41, 1.32 (2 d, J = 6.0 Hz, 6 H, 2 CCH3) ppm. 13C NMR
(125 MHz, CDCl3): δ = 138.8–127.4 (Ar-C), 99.8 (C-1D), 94.6 (C-
1F), 83.7 (C-1E), 82.2 (C-3F), 80.9 (C-4E), 80.3 (2 C, C-4D, C-4F),
79.3 (C-2F), 77.8 (C-3D), 76.2 (C-3E), 75.6 (2 C, 2 PhCH2), 75.4 (2
C, C-2D, PhCH2), 75.2 (C-2E), 74.9 (PhCH2), 73.5 (PhCH2), 72.9
(PhCH2), 72.8 (PhCH2), 72.4 (PhCH2), 70.5 (C-5F), 68.8 (C-5D),
68.5 (C-5E), 68.3 (C-6F), 25.5 (SCH2CH3), 18.2, 18.1 (2 CCH3),
15.2 (SCH2CH3) ppm. MALDI-MS: m/z = 1259.5 [M + Na]+.
C76H84O13S (1236.56): calcd. C 73.76, H 6.84; found C 73.60, H
7.05.
D
glucopyranoside (1): Ethylene diamine (0.3 mL, 4.48 mmol) was
added to a solution of compound 16 (1 g, 0.40 mmol) in nBuOH
(20 mL), and the reaction mixture was allowed to stir at 90 °C for
8 h. The solvents were removed under reduced pressure.
The crude product was dissolved in acetic anhydride (3 mL) and
pyridine (3 mL), and the solution was kept at room temperature
for 2 h. The solvents were removed under reduced pressure and the
crude product was passed through a short pad of SiO2 to remove
by-products.
The acetylated product was dissolved in CH3OH/CH2Cl2 (9:1, v/v;
10 mL), and Pd/C (10%; 150 mg) and Et3SiH (3 mL, 18.78 mmol)
were added. The reaction mixture was stirred at room temperature
for 10 h. The reaction mixture was filtered through a Celite® pad,
and the filter pad was washed with CH3OH (50 mL). The com-
bined filtrate was concentrated under reduced pressure.
The hydrogenated product was dissolved in CH3ONa (0.1 m in
CH3OH; 10 mL), and the mixture was stirred at room temperature
for 3 h. The reaction mixture was neutralized with Dowex 50W X8
(H+) resin, filtered, and concentrated to give compound 1. This
material was passed through an LH-20 Sephadex column using
CH3OH/H2O (3:1) as eluent to give pure compound 1 (220 mg,
54%) as a white solid, [α]2D5 = +5 (c = 1.0, H2O). 1H NMR
(500 MHz, D2O): δ = 5.23 (d, J = 3.0 Hz, 1 H, 1B-H), 5.12 (br. s,
1 H, 1D-H), 5.03 (br. s, 1 H, 1C-H), 5.00 (d, J = 3.0 Hz, 1 H, 1F-
H), 4.91 (br. s, 1 H, 1E-H), 4.49 (d, J = 7.5 Hz, 1 H, 1A-H), 4.18
(br. s, 1 H, 2E-H), 4.00–3.92 (m, 4 H, 2D-H, 4A-H, 5F-H, OCH2),
3.90–3.76 (m, 8 H, 2A-H, 3A-H, 3C-H, 3D-H, 3E-H, 4D-H, 5A-H,
OCH2), 3.75–3.57 (m, 11 H, 4E-H, 4F-H, 5C-H, 5D-H, 5E-H, 6A-
Hab, 6B-Hab, 6F-Hab), 3.55–3.44 (m, 4 H, 2C-H, 3B-H, 3F-H, 5B-H),
3.42–3.32 (m, 4 H, 2B-H, 2F-H, 4B-H, 4C-H), 3.18–3.05 (m, 2 H,
NCH2), 1.94 (s, 3 H, COCH3), 1.24–1.17 (m, 9 H, 3 CCH3) ppm.
13C NMR (125 MHz, D2O): δ = 174.7 (COCH3), 101.9 (C-1E),
100.7 (C-1C), 100.6 (2 C, C-1A, C-1D), 97.7 (C-1B), 95.3 (C-1F),
78.3 (C-3A), 78.1 (C-2D), 76.9 (C-5A), 76.6 (C-3C), 75.2 (C-2E), 74.3
(C-3E), 72.9 (C-3F), 72.8 (C-4D), 72.6 (C-4C), 72.1 (C-5B), 71.7 (C-
2F), 71.6 (C-4E), 71.5 (C-3B), 71.4 (C-2C), 70.2 (C-2B), 70.1 (C-4A),
69.8 (2 C, C-3D, C-5F), 69.4 (C-5C), 69.2 (C-4B), 69.1 (C-4F), 69.0
(C-5D), 66.6 (C-5E), 65.7 (OCH2), 60.7 (C-6F), 60.3 (C-6A), 60.2
(C-6B), 54.4 (C-2A), 39.4 (NCH2), 22.8 (COCH3), 16.9, 16.8, 16.5
(3 CCH3) ppm. ESI-MS: m/z = 1049.4 [M + Na]+. C40H70N2O28
(1026.41): calcd. C 46.78, H 6.87; found C 46.56, H 7.10.
2-(Benzyloxycarbonylamino)ethyl (2,3,4,6-Tetra-O-benzyl-α-
glucopyranosyl)-(1Ǟ3)-(2,4-di-O-benzyl-α- -rhamnopyranosyl)-
(1Ǟ2)-(3,4-di-O-benzyl-α- -rhamnopyranosyl)-(1Ǟ3)-(2,4-di-O-
benzyl-α- -rhamnopyranosyl)-(1Ǟ3)-[(4,6-di-O-acetyl-2,3-di-O-
benzyl-α-
imido-β-
added to a solution of compound 12 (1 g, 0.75 mmol) and com-
pound 15 (1 g, 0.81 mmol) in CH2Cl2/Et2O (1:1 v/v; 10 mL). The
reaction mixture was stirred at room temperature under argon for
30 min. The reaction mixture was cooled to –25 °C, and NIS
(200 mg, 0.89 mmol) and TfOH (5 μL) were added. The reaction
mixture was stirred at the same temperature for 30 min, then it was
diluted with CH2Cl2 (100 mL), and the organic layer was success-
ively washed with Na2S2O3 (5%), NaHCO3 (satd. aq.), and water,
then it was dried (Na2SO4) and concentrated. The crude product
was purified over SiO2 using hexane/EtOAc (8:1) as eluent to give
pure compound 16 (1.3 g, 69%) as a colorless oil. [α]D = +7 (c =
D-
L
L
L
D
-glucopyranosyl)-(1Ǟ4)-]-6-O-benzyl-2-deoxy-2-N-phthal-
-glucopyranoside (16): Molecular sieves (4 Å; 2 g) were
D
1.0, CHCl ). IR (neat): ν = 3033, 2928, 1737, 1726, 1495, 1456,
˜
3
1338, 1223, 1049, 751, 667 cm–1. 1H NMR (500 MHz, CDCl3): δ =
7.32–7.08 (m, 74 H, Ar-H), 5.72 (br. s, 1 H, 1F-H), 5.21 (d, J =
8.0 Hz, 1 H, 1A-H), 5.20–5.18 (m, 4 H, 1B-H, 1C-H, 1E-H, NH),
5.04 (br. s, 1 H, 1D-H), 5.00–4.40 (m, 27 H, 4B-H, PhCH2), 4.36 (t,
J = 8.0 Hz, 1 H, 2A-H), 4.25 (d, J = 11.0 Hz, 1 H, PhCH2), 4.16–
4.10 (m, 3 H, 3F-H, 5A-H, PhCH2), 4.08–4.02 (m, 2 H, 5E-H, 5F-
H), 3.99 (br. s, 1 H, 2D-H), 3.95 (br. s, 1 H, 2E-H), 3.84–3.70 (m,
10 H, 2C-H, 3C-H, 3D-H, 3E-H, 4A-H, 5D-H, 6A-Hab, 6B-Hab), 3.69–
3.58 (m, 5 H, 2B-H, 2F-H, 3B-H, 5C-H, 6F-Ha), 3.56–3.40 (m, 5 H, Molecular Dynamics Simulation: The structure of hexasaccharide 1
3A-H, 4C-H, 4D-H, 4F-H, 6F-Hb), 3.38–3.25 (m, 3 H, 4E-H, NCH2), was built in the Maestro panel, and the structure was cleaned for
3.22–3.19 (m, 1 H, 5B-H), 1.86, 1.47 (2 s, 6 H, 2 COCH3), 1.26–
optimal bond parameters, keeping in mind the requisite α/β config-
Eur. J. Org. Chem. 2014, 4577–4584
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4583

D. Dhara, R. K. Kar, A. Bhunia, A. K. Misra
FULL PAPER
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Published Online: June 12, 2014
4584
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Eur. J. Org. Chem. 2014, 4577–4584