Stereoselective Syntheses of the Conjugation-Ready, Downstream Disaccharide and Phosphorylated Upstream, Branched Trisaccharide Fragments of the O-PS of Vibrio cholerae O139

Article abstract of DOI:10.1021/acs.joc.5b00562

N-Bromosuccinimide-mediated 4,6-O-benzylidene ring opening in 8-azido-3,6-dioxaoctyl 4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-β-D-glucopyranoside afforded the corresponding 4-O-benzoyl-6-bromo-6-deoxy analogue, which was coupled with 3,4,6-tri-O-ace

Full text of DOI:10.1021/acs.joc.5b00562

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Stereoselective Syntheses of the Conjugation-Ready, Downstream
Disaccharide and Phosphorylated Upstream, Branched Trisaccharide
Fragments of the O‑PS of Vibrio cholerae O139
Sameh E. Soliman†,‡ and Pavol Kovac*,†
́
̌
†NIDDK, LBC, Section on Carbohydrates, National Institutes of Health, Bethesda, Maryland 20892-0815, United States
‡Department of Chemistry, Faculty of Science, Alexandria University, Alexandria 21321, Egypt
S
* Supporting Information
ABSTRACT: N-Bromosuccinimide-mediated 4,6-O-benzylidene ring opening
in 8-azido-3,6-dioxaoctyl 4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-β-D-
glucopyranoside afforded the corresponding 4-O-benzoyl-6-bromo-6-deoxy
analogue, which was coupled with 3,4,6-tri-O-acetyl-2-O-benzyl-α-D-galactopyr-
anosyl chloride to give the 1,2-cis α-linked disaccharide as the major product.
Conventional hydroxyl group manipulation in the latter and products of further
conversions gave the desired, functionalized disaccharide α-D-GalpA-(1→3)-β-
D-QuipNAc. The rare, foregoing sequence forms the downstream end in the O-
specific polysaccharide of both Vibrio cholerae O22 and O139. Halide-assisted
glycosylation at 4I-OH in 8-azido-3,6-dioxaoctyl 6-O-benzyl-2-deoxy-3-O-
(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2-trichloroacetamido-β-D-gluco-
pyranoside, obtained by regioselective reductive opening of the acetal ring in
the parent 4I,6I-O-benzylidene derivative, with 2,4-di-O-benzyl-α-colitosyl
bromide, gave exclusively the α-linked trisaccharide. The latter was sequentially
deacetylated and selectively benzylated to give 8-azido-3,6-dioxaoctyl 2,4-di-O-benzyl-3,6-dideoxy-α-L-xylo-hexopyranosyl-(1→
4)-[3-O-benzyl-β-D-galactopyranosyl-(1→3)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-D-glucopyranoside. Subsequent selec-
tive phosphorylation of the triol, thus obtained, with 2,2,2-trichloroethyl phosphorodichloridate afforded isomeric (R,S)-(P)-
4II,6II-cyclic phosphates, which were both obtained in crystalline form and fully characterized. Each of the latter was globally
deprotected by catalytic (Pd/C) hydrogenation/hydrogenolysis to give the desired, amino-functionalized, spacer-equipped,
phosphorylated upstream trisaccharide fragment of the O-PS of V. cholerae O139.
INTRODUCTION
■
This laboratory has been engaged in developing conjugate
vaccines from fragments of bacterial lipopolysaccharides for a
number of years.1−4 To evaluate specificity,5,6 we often use in
these studies synthetic oligosaccharide probes that mimic O-
antigens. We intend to perform such a study also in connection
with our ongoing work toward a synthetic vaccine for a disease
caused by Vibrio cholerae O139.7−9 The O-antigen of V. cholerae
O139 is a phosphorylated hexasaccharide [sequence FD(E)-
CBA, Figure 1] consisting of five different monosaccharides.10
Collecting a wide range of fragments of the protective antigen
requires extensive synthetic work. We often synthesize11−13
Figure 1. Structure of the O-antigen of V. cholerae O139
lipopolysaccharide.
such fragments in spacer-equipped form to make them
amenable for conjugation to proteins because use of
glycoconjugates made from such substances often helps in
solved,12,14 but the first synthesis of the sequence α-D-GalpA-
(1→3)-β-D-QuipNAc (sequence BA) is yet to be reported. The
latter forms the downstream end in the O-PS of both V.
cholerae O139 and O22 (Figures 1 and 2, respectively).10,15
Because synthesis of virtually any disaccharide is generally not
solving questions that arise during immunological/immunoge-
nicity studies. Synthesis of every individual fragment of the
hexasaccharide (Figure 1) presents its own problems, the most
formidable challenge being construction of the 1,2-cis glycosidic
linkage, which is present also in target compounds of this
communication. Construction of the α-colitosyl linkage to form
fragments of the hexasaccharide (Figure 1) has been largely
Received: March 12, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.joc.5b00562
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11:1). Unlike in the case of coupling product 3, running the
reaction in solvent containing a large proportion of Et2O had
no effect on stereoselectivity and only made the reaction
considerably slower. The 13C NMR spectrum of disaccharide
5α showed signals characteristic of the presence of both donor
and acceptor moieties, while the α-configuration of the
interglycosidic linkage was established from the 1H NMR
spectrum (δ 4.93, H-1II, J1,2 = 3.6 Hz).
́
Zemplen de-O-acylation of 5α gave tetraol 6 in virtually
Figure 2. Structure of the repeating unit of O-PS of V. cholerae O22,
the highlighted disaccharide showing the sequence BA present also in
O-PS of V. cholerae O139.
quantitative yield, and subsequent isopropylidenation (2,2-
dimethoxypropane and camphorsulfonic acid in DMF at 45
°C) gave selectively the 3II,4II-O-isopropylidene derivative 7.
The location of the isopropylidene group in 7 (Scheme 1) was
evident from the comparison of 13C NMR spectra of 6 and 7,
the latter showing downfield shifts for C-3II and C-4II (from δC
69.5 to 76.5 and from 70.3 to 75.0 ppm, respectively), as a
result of isopropylidenation at those positions.
considered difficult, the absence of synthesis of the sequence
BA underscores the difficulty of the task. Here, we report on
the first syntheses of the linker-equipped downstream
disaccharide 10 and the phosphorylated upstream trisaccharide
20 [sequences BA and D(E)C, respectively, Figure 1]. The
syntheses of the aforementioned, end fragments were important
for choosing and optimizing the synthetic and protecting group
strategy to be used in the planned synthesis of the complete
hexasaccharide, which is a much more involved, yet to be solved
task.
The primary hydroxyl group in 7 was then regioselectively
oxidized with [bis(acetoxy)iodo]benzene (BAIB) in the
presence of 2,2,6,6-tetramethylpiperidinyloxy free radical
(TEMPO).25 For confirmation of structure and easier
purification, the crude product of the oxidation was treated
with benzyl bromide and potassium carbonate26 in anhydrous
DMF to provide benzyl uronate 8 (73% over two steps). The
13C NMR spectrum of 8 showed a signal at δC 167.3 ppm,
which confirms the presence of a carboxyl group in the
oxidation product. It is worth noting that attempts to convert 6
directly into 9 (not described in the Experimental Section) gave
complex mixtures. O-Deisopropylidenation (80% aq AcOH, 80
°C) of 8 was followed by catalytic hydrogenation/hydro-
genolysis (Pd/C, H2, rt), which affected one-pot transformation
of azide to amine, multiple debenzylations, debromination at C-
6I, as well as conversion of the N-trichloroacetyl to N-acetyl
group to afford the desired disaccharide 10. The expected
structure 10 was confirmed by mass and NMR spectra.
Thus far, only very few attempts to synthesize fragments of
the hexasaccharide antigen of V. cholerae O139 have been
reported.11−14 Most notable are the two syntheses of the
upstream, terminal tetrasaccharide sequence FD(E)C (Figure
1),12,14 each having its characteristic advantage. The present
synthesis of trisaccharide 20 (Scheme 2) incorporates the
salient features of both approaches. First, it starts with
compound 11 where the linker/spacer is already in place.12
Thus, chemical manipulation with the fully assembled
sequence,14 other than final deprotection, can be avoided.
Second, the synthesis was designed with the isomers yielding
phosphorylation as the penultimate step,14 but choosing
protecting groups that allow global deprotection in one step,
to afford the final product 20 in increased overall yield.
RESULTS AND DISCUSSION
■
Synthesis of the downstream disaccharide 10 started (Scheme
1) with the spacer-equipped glycosyl acceptor 8-azido-3,6-
dioxaoctyl 4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-β-
D-glucopyranoside (1).16 Construction of the 1,2-cis α-D-
galactopyranosidic linkage, to arrive at sequence BA, required
use of a glycosyl donor bearing at C-2 a group incapable of
anchimeric assistance.17 For that purpose, we chose 2-O-
benzylated galactosyl chloride 218 and treated it in CH2Cl2 with
1 at subzero temperature under silver triflate catalysis. These
conditions are known19 to favor 1,2-cis glycosidation. The
reaction was fast and high yielding (4 h, ∼90%), but the
stereoselectivity of formation of the α-glycosidic linkage was
poor (α/β = 2:1). It is known that stereoselectivity of
glycosylation can vary depending on solvent and that ether-
type solvents, such as 1,2-dimethoxyethane20 and particularly
diethyl ether,21 favor formation of the 1,2-cis glycosidic linkage.
Therefore, in an attempt to enhance formation of the desired α-
linkage by solvent effect, limited by solubility of 1 in Et2O, we
performed condensation of 1 with 2 in 1:1 CH2Cl2−Et2O. The
selectivity improved (α/β = 4:1), but we deemed looking for
ways to further enhance the yield of the α-linked product
warranted. Experimental evidence has shown22 that unlike fast
substitution reactions, the slow SN1-type reactions, such as
glycosylations with donors bearing nonparticipating groups at
C-2, tend to occur without inversion of configuration. In
addition, reactivity of hydroxyl groups in carbohydrates can be
controlled to some extent by substituents at neighboring
positions;23 namely, their reactivity can be increased by vicinal
electron-donating substituents and vice versa. Accordingly,
regioselective ring opening in the 4,6-O-benzylidene acetal in 1
with N-bromosuccinimide,24 to form 4 (85%, Scheme 1),
positioned the electron-withdrawing benzoyl group at C-4 and
simultaneously deoxygenated position 6, which was desirable to
effect later conversion of GlcpNAc into QuipNAc. Subsequent
silver triflate mediated glycosylation of 4 with galactosyl
chloride 2, in CH2Cl2 as solvent, gave predominantly the
desired α-linked disaccharide 5α, together with a small amount
of the β-anomer 5β (overall yield of glycosylation, 84%; α/β =
Accordingly, the 4I,6I-O-benzylidene ring in 8-azido-3,6-
dioxaoctyl 4,6-O-benzylidene-2-deoxy-3-O-(2,3,4,6-tetra-O-ace-
tyl-β-D-galactopyranosyl)-2-trichloroacetamido-β-D-glucopyra-
noside16 (11) was regioselectively opened with NaCNBH3 and
HCl in THF27 to give the crystalline 6I-O-benzyl derivative 12
(92%). Compared to the 13C NMR spectrum of 11,16 the signal
for C-4I in 12 showed significant upfield shift (by ∼ 10 ppm)
because of the absence of the positive shift effect28 of the
1
benzylidene group, and the H−1H COSY cross-peak between
H-4I (δ 3.57 ppm) and the proton of the newly generated
hydroxyl group (δ 3.66 ppm) confirmed that the reductive
opening of the benzylidene acetal produced the 6I- and not 4I-
benzyl ether. Because the shift effect of alkylation and
alkylidination is similar, the difference in chemical shift for C-
B
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Scheme 1. Synthesis of the Spacer-Equipped Disaccharide 10
1
6I in 11 and 12 is small and not diagnostic for the mode of
opening of the benzylidene ring. The disaccharide acceptor 12,
thus formed, was subjected to halide-assisted29 glycosylation
with the α-colitosyl bromide 14 (made by treatment30 of the
corresponding β-ethyl thioglycoside 1331 with bromine) to give
the trisaccharide 15 (95%). Identification of the coupling
product 15 was based on its H and 13C NMR spectra, which
included signals characteristic for presence of both the donor
and the acceptor moieties. As expected, compared to the 13C
NMR spectrum of 12, the signal of C-4I (the site of
glycosylation) was shifted downfield. The coupling constant
(J = 3.0 Hz) shown by the doublet for the anomeric proton of
C
DOI: 10.1021/acs.joc.5b00562
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Scheme 2. Synthesis of the Spacer-Equipped, Phosphorylated Trisaccharide 20
the colitose residue (δ 5.07 ppm) confirmed formation of the
α-glycosidic linkage, which was also supported32 by the 1JC‑1,H‑1
= 170.2 Hz.
elevated pressure and temperature were not required to achieve
smooth, complete deprotection by catalytic hydrogenation/
hydrogenolysis. Because the same protecting groups were
transformed in both situations, the likely explanation for the
harsher conditions required previously was different activity of
the Pd/C catalyst used.
De-O-acetylation of 15 through transesterification with
methanolic sodium methoxide afforded 8-azido-3,6-dioxaoctyl
2,4-di-O-benzyl-3,6-dideoxy-α-L-xylo-hexopyranosyl-(1→4)-[β-
D-galactopyranosyl-(1→3)]-6-O-benzyl-2-deoxy-2-trichloroace-
tamido-β-D-glucopyranoside (16) in virtually theoretical yield.
Subsequent selective benzylation at the 3II-OH via stannylation
gave the 3II-O-benzyl derivative 17 (93%). Formation of the 4II-
O-benzyl isomer was not observed (TLC). Identification of
compound 17 was based on its 13C NMR spectrum, in which
the signal for C-3II was shifted downfield (δ 79.9 ppm)
compared to δ 73.6 ppm in the spectrum of 16.
Treatment of trisaccharide 17 with the phosphorylating
reagent, 2,2,2-trichloroethyl phosphorodichloridate,33,34 gave
isomeric (S,R)-(P)-4II,6II-cyclic 2,2,2-trichloroethyl phosphates
18 and 19 (∼3:1, 31P NMR). The isomers 18 and 19 were
separated by chromatography and isolated in excellent
combined yield. Both substances were obtained crystalline
and fully characterized.
Similar global deprotection of the other phosphate isomer 19
gave the same product 20 (1H NMR, 13C NMR, 31P NMR,
TLC, MS) in 90% yield. In addition to MS, the presence of the
cyclic phosphate in 20 followed from the 31P NMR spectrum,
showing JP,H = 22.2 Hz.35
3
Many features in the present, successful synthetic approach
will be incorporated in the future synthesis of the full O-
antigen, the phosphorylated hexasaccharide [sequence FD(E)-
CBA, Figure 1], which will be a much more involved project,
not realized to date.
CONCLUSIONS
■
We have developed an efficient approach to synthesize the rare
disaccharide sequence 10, which forms the downstream end in
the O-PS of both V. cholerae O22 and O139 and the
phosphorylated upstream, branched trisaccharide 20, which is
one of the terminal determinants of the O-specific poly-
saccharide of V. cholerae O139. The final, amino-functionalized
spacer-equipped fragments 10 and 20 are ready for conjugation
to proteins.
Global deprotection of the foregoing phosphate 18 was
achieved in one step by catalytic hydrogenation/hydrogenolysis
in the presence of Pd/C catalyst and pH = 7, 0.1 M potassium
phosphate buffer, to protect the acid-labile9 α-colitosyl group.
In this way, the desired phosphorylated trisaccharide 20 was
obtained in 93% yield. Contrary to our previous observation,12
D
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Continued elution gave the desired α-linked disaccharide 3α (2.49
g, 60%, 90% overall, α/β ∼ 2:1).
EXPERIMENTAL SECTION
■
General Methods. Optical rotations were measured at ambient
temperature for solution in CHCl3 unless stated otherwise. All
reactions were monitored by thin-layer chromatography (TLC) on
silica gel 60 coated glass slides. Column chromatography was
performed by elution from prepacked columns of silica gel and
monitored with the evaporative light scattering detector. Nuclear
magnetic resonance (NMR) spectra were measured at 500 or 600
20
1
Data for 3α. [α]D = +47.6 (c 0.7, CHCl3). H NMR (600 MHz,
CDCl3): δ = 7.38−7.04 (m, 10 H, 2 Ph), 7.21 (d, 1 H, J2,NH = 7.6 Hz,
NH), 5.69 (d, 1 H, J1,2 = 3.6 Hz, H-1II), 5.38 (dd, 1 H, J3,4 = 3.2 Hz, J4,5
= 1.1 Hz, H-4II), 5.34 (s, 1H, PhCH), 5.27 (dd, 1 H, J2,3 = 10.7 Hz, J3,4
= 3.2 Hz, H-3II), 5.12 (d, 1 H, J1,2 = 8.3 Hz, H-1I), 4.62 (t, 1 H, J = 9.4
Hz, H-3I), 4.50 (d, 1 H, 2J = 12.4 Hz, PhCHH), 4.37 (d, 1 H, 2J = 12.4
Hz, PhCHH), 4.33 (dd, 1 H, J = 5.0, 10.5 Hz, H-6Ia), 4.28−4.25 (m, 1
1
MHz for H, 125 or 150 MHz for 13C, and 162 MHz for 31P. Solvent
H, H-5II), 4.07 (dd, 1 H, J = 8.1, 10.9 Hz, H-6II ), 3.98−3.93 (m, 2 H,
a
peaks were used as internal reference relative to TMS for H and 13C
1
H-1′a, H-6II ), 3.80−3.78 (m, 1 H, H-1′b), 3.77−3.73 (m, 3 H, H-6Ib,
b
chemical shifts (ppm); 31P chemical shifts (ppm) were reported
relative to 85% H3PO4 in D2O external reference. Assignments of
NMR signals were made by homonuclear and heteronuclear two-
dimensional correlation spectroscopy, run with the software supplied
with the spectrometers. When reporting assignments of NMR signals,
nuclei associated with the spacer are denoted with a prime; sugar
residues are serially numbered, beginning with the one bearing the
aglycon, and are identified by a Roman numeral superscript in listings
of signal assignments, with nuclei of the colitose residue as III (see
Scheme 2, 15). The density of 2,2,2-trichloroethyl phosphorodi-
chloridate (Aldrich/Sigma, d ≈ 1.7 g/mL at 20 °C) was determined by
weighing of 1 mL of the reagent. Palladium-on-charcoal catalyst (5%,
Escat 103) was purchased from Engelhard Industries. Solutions in
organic solvents were dried with anhydrous Na2SO4 and concentrated
at 40 °C/2 kPa.
H-4I, H-2II), 3.70−3.64 (m, 8 H, H-2′, H-3′, H-4′, H-5′), 3.63−3.61
(m, 1 H, H-2I), 3.60−3.56 (m, 1 H, H-5I), 3.41 (t, 2 H, J = 5.0 Hz, H-
6′), 2.03, 2.02, 1.95 (3 s, 9 H, 3 COCH3). 13C NMR (150 MHz,
CDCl3): δ = 170.4−169.9 (3 OCOCH3), 162.0 (NCOCCl3), 137.8,
139.8 (2 ipso Ph), 129.4−126.2 (Ar), 101.8 (PhCH), 99.5 (C-1I), 96.3
(C-1II), 92.4 (CCl3), 82.8 (C-4I), 72.8 (C-3I), 72.5 (C-2II), 71.7
(PhCH2), 70.6, 70.5, 70.4, 70.0 (C-2′, C-3′, C-4′, C-5′), 68.9 (C-1′),
68.8 (C-3II), 68.7 (C-6I), 67.9 (C-4II), 66.2 (C-5II), 65.5 (C-5I), 61.0
(C-6II), 58.2 (C-2I), 50.6 (C-6′), 20.7−20.5 (3 COCH3). HRMS (ESI-
TOF): m/z [M + NH4]+ calcd for C40H53Cl3N5O16 964.2553, found
964.2552. Anal. Calcd for C40H49Cl3N4O16: C, 50.67; H, 5.21; N, 5.91.
Found: C, 50.55; H, 5.30; N, 6.07.
(b) A suspension of glycosyl acceptor 1 (833 mg, 1.46 mmol),
powdered AgOTf (633 mg, 2.49 mmol), and activated molecular
sieves (4 Å, 2 g) in anhydrous CH2Cl2 (15 mL) and anhydrous Et2O
(30 mL) was stirred under nitrogen for 1 h. A solution of galactosyl
donor 2 (933 mg, 2.25 mmol) and sym-collidine (0.4 mL, 2.5 mmol)
in anhydrous CH2Cl2 (15 mL) was added at room temperature. The
reaction mixture was stirred for 2 days until TLC (6:1 chloroform−
acetone) indicated that all acceptor was consumed. The mixture was
worked up as described above and chromatographed. The combined
yield of 3α and 3β was 88% (α/β ∼ 4:1).
8-Azido-3,6-dioxaoctyl 4,6-O-Benzylidene-2-deoxy-2-tri-
chloroacetamido-3-O-(3,4,6-tri-O-acetyl-2-O-benzyl-α-D-galac-
topyranosyl)-β-D-glucopyranoside (3α) and 8-Azido-3,6-diox-
aoctyl 4,6-O-Benzylidene-2-deoxy-2-trichloroacetamido-3-O-
(3,4,6-tri-O-acetyl-2-O-benzyl-β-D-galactopyranosyl)-β-D-glu-
copyranoside (3β). (a) A mixture of 8-azido-3,6-dioxaoctyl 4,6-O-
benzylidene-2-deoxy-2-trichloroacetamido-β-D-glucopyranoside (1)16
(2.5 g, 4.39 mmol), powdered AgOTf (1.90 g, 7.46 mmol), and
activated molecular sieves (4 Å, 7 g) in anhydrous CH2Cl2 (150 mL)
was stirred under nitrogen for 1 h. The suspension was cooled to −30
°C, and a solution of 3,4,6-tri-O-acetyl-2-O-benzyl-α-D-galactopyrano-
syl chloride (2)18 (2.80 g, 6.76 mmol) in anhydrous CH2Cl2 (50 mL)
containing sym-collidine (1.20 mL, 7.5 mmol) was added dropwise.
The cooling was removed, and with continued stirring, the mixture
was allowed to warm to room temperature. After 4 h, analysis by TLC
(6:1 chloroform−acetone) indicated that all acceptor was consumed.
The mixture was diluted with CH2Cl2 (100 mL) and filtered through a
Celite pad. The filtrate was washed successively with 0.5 M aq HCl, aq
NaHCO3, and brine. The organic phase was coevaporated with water
to remove excess sym-collidine, and chromatography (9:1 chloro-
form−acetone) gave first the β-linked disaccharide 3β (1.25 g, 30%).
8-Azido-3,6-dioxaoctyl 4-O-Benzoyl-6-bromo-2,6-dideoxy-
2-trichloroacetamido-β-D-glucopyranoside (4). N-Bromosuccini-
mide (812 mg, 4.6 mmol) was added at 100 °C, under argon, to a
stirred mixture of 8-azido-3,6-dioxaoctyl 4,6-O-benzylidene-2-deoxy-2-
trichloroacetamido-β-D-glucopyranoside (1)16 (2.0 g, 3.5 mmol) and
barium carbonate (3.47 g, 17.6 mmol) in 4:1 carbon tetrachloride−
tetrachloroethane (75 mL). The mixture was kept at reflux for 2.5 h,
cooled to room temperature, and filtered. The filtrate was
concentrated, and the residue was chromatographed (12:1 → 6:1
chloroform−acetone) to afford 4 (1.93 g, 85%). [α]D20 = −18.2 (c 0.8,
1
CHCl3). H NMR (600 MHz, CDCl3): δ = 8.05−7.45 (m, 5 H, Ph),
7.57 (d, 1 H, J2,NH = 7.6 Hz, NH), 5.11 (t, 1 H, J3,4 = J4,5 = 9.3 Hz, H-
4), 5.04 (d, 1 H, J1,2 = 8.3 Hz, H-1), 4.30−4.25 (m, 1 H, H-3), 4.04−
4.00 (m, 1 H, H-1′a), 3.92−3.88 (m, 1 H, H-1′b), 3.87−3.84 (m, 1 H,
H-5), 3.75−3.72 (m, 1 H, H-2), 3.78−3.65 (m, 8 H, H-2′, H-3′, H-4′,
H-5′), 3.58 (d, 1 H, J3,OH = 6.2 Hz, 3-OH), 3.55 (dd, 1 H, J = 2.4, 11.3
Hz, H-6a), 3.48 (dd, 1 H, J = 7.7, 11.3 Hz, H-6b), 3.45−3.42 (m, 2 H,
H-6′). 13C NMR (150 MHz, CDCl3): δ = 166.2 (CO), 163.0 (CO),
133.7−128.5 (Ar), 128.7 (ipso Ph), 99.8 (C-1), 92.2 (CCl3), 74.3 (C-
4), 73.5 (C-5), 71.8 (C-3), 70.7, 70.5, 70.2, 69.9 (C-2′, C-3′, C-4′, C-
5′), 68.7 (C-1′), 59.1 (C-2), 50.4 (C-6′), 31.3 (C-6). HRMS (ESI-
TOF): m/z [M + Na]+ calcd for C21H26Cl3BrN4O8Na 668.9897,
found 668.9884. Anal. Calcd for C21H26Cl3BrN4O8: C, 38.88; H, 4.04;
N, 8.64. Found: C, 39.02; H, 4.09; N, 8.64.
20
1
Data for 3β. [α]D = +13.2 (c 0.7, CHCl3). H NMR (600 MHz,
CDCl3): δ = 7.49−7.19 (m, 10 H, 2 Ph), 7.10 (d, 1 H, J2,NH = 7.2 Hz,
NH), 5.59 (s, 1H, PhCH), 5.29 (dd, 1 H, J3,4 = 3.5 Hz, J4,5 = 1.0 Hz,
H-4II), 5.22 (d, 1 H, J1,2 = 8.3 Hz, H-1I), 4.88 (dd, 1 H, J2,3 = 10.1 Hz,
2
J3,4 = 3.5 Hz, H-3II), 4.75 (d, 1 H, J = 11.8 Hz, PhCHH), 4.65 (t
partially overlapped, 1 H, J = 9.5 Hz, H-3I), 4.63 (d, 1 H, J1,2 = 7.7 Hz,
2
H-1II), 4.58 (d, 1 H, J = 11.8 Hz, PhCHH), 4.37 (dd, 1 H, J = 4.8,
10.4 Hz, H-6Ia), 4.13 (dd, 1 H, J = 6.8, 11.2 Hz, H-6II ), 3.99−3.96 (m,
a
1 H, H-6II ), 3.95−3.91 (m, 1 H, H-1′a), 3.84−3.81 (m, 1 H, H-6Ib),
b
3.80−3.75 (m, 2 H, H-4I, H-1′b), 3.73 (dt, 1 H, J = 1.0, 6.8, 7.6 Hz, H-
5II), 3.69−3.67 (m, 1 H, H-2II), 3.67−3.61 (m, 8 H, H-2′, H-3′, H-4′,
H-5′), 3.58−3.54 (m, 1 H, H-5I), 3.49−3.44 (m, 1 H, H-2I), 3.39 (t, 2
H, J = 5.1 Hz, H-6′), 2.07, 2.01, 1.88 (3 s, 9 H, 3 COCH3). 13C NMR
(150 MHz, CDCl3): δ = 170.4−170.0 (3 OCOCH3), 162.0
(NCOCCl3), 138.2, 136.9 (2 ipso Ph), 129.1−126.0 (Ar), 102.4 (C-
1II), 101.2 (PhCH), 99.4 (C-1I), 92.2 (CCl3), 78.9 (C-4I), 76.8 (2C, C-
2II, C-3I), 75.2 (PhCH2), 72.4 (C-3II), 70.7, 70.6, 70.5, 70.1 (C-2′, C-
3′, C-4′, C-5′), 70.6 (C-5II), 69.1 (C-1′), 68.6 (C-6I), 67.3 (C-4II),
66.2 (C-5I), 61.4 (C-6II), 59.6 (C-2I), 50.6 (C-6′), 20.65, 20.64, 20.61
(3 COCH3). HRMS (ESI-TOF): m/z [M + NH4]+ calcd for
C40H53Cl3N5O16 964.2553, found 964.2549. Anal. Calcd for
C40H49Cl3N4O16: C, 50.67; H, 5.21; N, 5.91. Found: C, 50.76; H,
5.01; N, 5.65.
8-Azido-3,6-dioxaoctyl 4-O-Benzoyl-6-bromo-2,6-dideoxy-
2-trichloroacetamido-3-O-(3,4,6-tri-O-acetyl-2-O-benzyl-α-D-
galactopyranosyl)-β-D-glucopyranoside (5α) and 8-Azido-3,6-
dioxaoctyl 4-O-Benzoyl-6-bromo-2,6-dideoxy-2-trichloroace-
tamido-3-O-(3,4,6-tri-O-acetyl-2-O-benzyl-β-D-galactopyrano-
syl)-β-D-glucopyranoside (5β). A suspension of the glycosyl
acceptor 4 (2.49 g, 3.84 mmol), powdered AgOTf (218.4 mg, 0.85
mmol), and activated molecular sieves (4 Å, 10 g) in anhydrous
CH2Cl2 (150 mL) was stirred under nitrogen for 1 h. A solution of
3,4,6-tri-O-acetyl-2-O-benzyl-α-D-galactopyranosyl chloride (2)18 (2.47
g, 5.76 mmol) and sym-collidine (1.1 mL, 7.7 mmol) in anhydrous
CH2Cl2 (50 mL) was added portionwise at room temperature. Stirring
was continued for 2 days, when analysis by TLC (6:1 toluene−
E
DOI: 10.1021/acs.joc.5b00562
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Featured Article
acetone) indicated that all acceptor was consumed. The mixture was
diluted with CH2Cl2 (100 mL) and filtered through a Celite pad. The
filtrate was washed successively with 0.5 M aq HCl, aq NaHCO3, and
brine. The organic phase was coevaporated with water to remove
excess sym-collidine, and chromatography (9:1 toluene−acetone) gave
first the desired α-linked disaccharide 5α (3.03 g, 77%).
(C-2II), 74.4 (C-5I), 74.3 (PhCH2), 73.1 (C-4I), 70.7, 70.5, 70.2 (C-2′,
C-3′, C-4′), 70.3 (C-4II), 70.2 (C-5II), 69.8 (C-5′), 69.5 (C-3II), 68.6
(C-1′), 62.0 (C-6II), 57.3 (C-2I), 50.5 (C-6′), 32.6 (C-6I). HRMS
(ESI-TOF): m/z [M + NH4]+ calcd for C27H42Cl3BrN5O12 812.1073,
found 812.1075.
To a solution of the deacetylated product 6 in anhydrous DMF (6
mL) were added 2,2-dimethoxypropane (95 μL, 0.75 mmol) followed
by dry camphorsulfonic acid (12 mg, 0.05 mmol). The mixture was
stirred at 45 °C for 16 h, neutralized with Et3N (50 μL), concentrated
to dryness, and coevaporated with toluene (twice) to remove traces of
Et3N. A solution of the crude product in 10:1 MeOH−H2O (22 mL)
was boiled under reflux for 3 h. The solvents were evaporated and the
residue was purified by flash chromatography (5:1 toluene−acetone)
to afford the isopropylidene derivative 7 (345 mg, 82% over two
steps). [α]D20 = +46.2 (c 0.4, CHCl3). 1H NMR (600 MHz, CDCl3): δ
= 7.36−7.30 (m, 5 H, Ph), 7.18 (d, 1 H, J2,NH = 8.4 Hz, NH), 4.99 (d,
1 H, J1,2 = 3.4 Hz, H-1II), 4.92 (d, 1 H, 2J = 12.1 Hz, PhCHH), 4.86 (s,
1 H, 4I-OH), 4.83 (d, 1 H, J1,2 = 8.3 Hz, H-1I), 4.73 (d, 1 H, 2J = 12.1
Hz, PhCHH), 4.44 (dd, 1 H, J2,3 = 7.6 Hz, J3,4 = 5.8 Hz, H-3II), 4.27
(dd, 1 H, J3,4 = 5.5 Hz, J4,5 = 4.3 Hz, H-4II), 4.17 (dd, 1 H, J4,5 = 4.4
Hz, J5,6 = 7.4 Hz, H-5II), 3.96−3.93 (m, 1 H, H-1′a), 3.88−3.84 (m, 2
H, H-6II), 3.83−3.81 (m, 2 H, H-1′b, H-3I), 3.76−3.72 (m, 2 H, H-6Ia,
20
1
Data for 5α. [α]D = +28.0 (c 0.7, CHCl3). H NMR (600 MHz,
CDCl3): δ = 8.04−7.04 (m, 11 H, 2 Ph, NH), 5.35 (br s, 1 H, H-4II),
5.34 (d, 1 H, J1,2 = 7.4 Hz, H-1I), 5.31 (dd, 1 H, J = 8.1 Hz, J = 8.9 Hz,
H-4I), 5.18 (dd, 1 H, J2,3 = 10.6 Hz, J3,4 = 3.3 Hz, H-3II), 4.93 (d, 1 H,
J1,2 = 3.6 Hz, H-1II), 4.56 (dd, 1 H, J = 8.1 Hz, J = 8.7 Hz, H-3I), 5.31
2
(dd, 1 H, J = 1.3 Hz, J = 7.1 Hz, H-5II), 4.23 (d, 1 H, J = 12.3 Hz,
PhCHH), 4.10 (d, 1 H, 2J = 12.3 Hz, PhCHH), 4.07−4.04 (m, 2 H, H-
1′a, H-6II ), 3.95 (ddd, 1 H, J = 3.2, 8.5, 11.8 Hz, H-5I), 3.85−3.81 (m,
a
2 H, H-1′b, H-6II ), 3.71−3.65 (m, 8 H, H-2′, H-3′, H-4′, H-5′), 3.63
b
(dd, 1 H, J1,2 = 3.5 Hz, J2,3 = 10.5 Hz, H-2II), 3.58 (dd, 1 H, J = 3.3,
11.2 Hz, H-6Ia), 3.53−3.49 (m, 2 H, H-6Ib, H-2I), 3.41 (t, 2 H, J = 5.1
Hz, H-6′), 2.04, 2.03, 1.87 (3 s, 9 H, 3 COCH3). 13C NMR (150 MHz,
CDCl3): δ = 170.5−169.7 (3 OCOCH3), 165.1 (CO), 162.0 (CO),
137.6, 129.2 (2 ipso Ph), 133.5−127.7 (Ar), 98.3 (C-1I), 97.8 (C-1II),
92.2 (CCl3), 76.9 (C-3I), 73.8 (C-5I), 73.1 (PhCH2), 73.0 (C-2II), 71.9
(C-4II), 70.7, 70.6, 70.4, 70.1 (C-2′, C-3′, C-4′, C-5′), 69.4 (C-3II),
69.2 (C-1′), 68.2 (C-4I), 67.5 (C-5II), 62.2 (C-6II), 58.6 (C-2I), 50.7
(C-6′), 31.5 (C-6I), 20.7−20.6 (3 COCH3). HRMS (ESI-TOF): m/z
[M + NH4]+ calcd for C40H52Cl3BrN5O16 1042.1658, found
1042.1652. Anal. Calcd for C40H48Cl3BrN4O16: C, 46.78; H, 4.71;
N, 5.46. Found: C, 47.03; H, 4.71; N, 5.45.
H-2I), 3.70−3.60 (m, 8 H, H-2′, H-3′, H-4′, H-5′), 3.58 (dd, 1 H, J1,2
=
3.4 Hz, J2,3 = 7.8 Hz, H-3II), 3.50−3.46 (m, 3 H, H-4I, H-5I, H-6Ib),
3.42−3.40 (m, 2 H, H-6′), 2.41 (d, 1 H, J6,OH = 6.2 Hz, 6II-OH), 1.41,
1.36 (2 s, 6 H, C(CH3)2). 13C NMR (150 MHz, CDCl3): δ = 162.4
(NCOCCl3), 136.7 (ipso Ph), 128.6−128.3 (Ar), 109.7 (C(CH3)2),
101.0 (C-1II), 100.4 (C-1I), 92.5 (CCl3), 82.3 (C-3I), 76.6 (C-2II), 76.5
(C-3II), 75.0 (C-4II), 74.4 (C-5I), 73.1 (C-4I), 72.7 (PhCH2), 71.0,
70.5, 70.3 (C-2′, C-3′, C-4′), 69.9 (C-5′), 68.3 (C-1′), 67.8 (C-5II),
62.9 (C-6II), 57.1 (C-2I), 50.4 (C-6′), 32.4 (C-6I), 28.2, 26.4
(C(CH3)2). HRMS (ESI-TOF): m/z [M + NH4]+ calcd for
C30H46Cl3BrN5O12 852.1392, found 852.1395. Anal. Calcd for
C30H42Cl3BrN4O12: C, 43.05; H, 5.06; N, 6.69. Found: C, 43.34; H,
5.32; N, 6.60.
Continued elution gave the β-linked disaccharide 5β (0.28 g, 7%,
84% overall, α/β ∼ 11:1).
20
1
Data for 5β. [α]D = +3.4 (c 0.8, CHCl3). H NMR (600 MHz,
CDCl3): δ = 8.06−7.25 (m, 10 H, 2 Ph), 6.92 (d, 1 H, J2,NH = 6.9 Hz,
NH), 5.27 (d, 1 H, J1,2 = 8.2 Hz, H-1I), 5.20 (t, 1 H, J = 9.3 Hz, H-4I),
5.14 (br d, 1 H, J = 3.3 Hz, H-4II), 4.76 (dd, 1 H, J2,3 = 10.2 Hz, J3,4
=
3.3 Hz, H-3II), 4.69 (t partially overlapped, 1 H, J = 9.4 Hz, H-3I), 4.68
(d, 1 H, 2J = 11.7 Hz, PhCHH), 4.61 (d, 1 H, 2J = 11.7 Hz, PhCHH),
4.44 (d, 1 H, J1,2 = 7.6 Hz, H-1II), 4.02−3.98 (m, 1 H, H-1′a), 3.90−
3.87 (m, 1 H, H-5I), 3.85−3.81 (m, 1 H, H-1′b), 3.71−3.63 (m, 8 H,
H-2′, H-3′, H-4′, H-5′), 3.61 (t, 1 H, J = 6.8 Hz, H-5II), 3.57−3.53 (m,
8-Azido-3,6-dioxaoctyl 6-Bromo-2,6-dideoxy-2-trichloroa-
cetamido-3-O-(benzyl 2-O-benzyl-3,4-O-isopropylidene-α-D-
galactopyranosyluronate)-β-D-glucopyranoside (8). To a flask
charged with compound 7 (200 mg, 0.24 mmol), TEMPO (15 mg,
0.10 mmol), and BAIB (232 mg, 0.72 mmol) was added CH2Cl2−H2O
(2:1, v/v, 15 mL), and the two-phase reaction mixture was stirred
vigorously at room temperature until analysis by TLC (3:1 toluene−
acetone) showed complete conversion of the starting material to a
slower moving product (∼24 h). The mixture was diluted with EtOAc
(100 mL) and washed with 1:1 (v/v) aq Na2S2O3 and aq NaH2PO4 (2
× 50 mL). The combined aqueous washes were extracted with EtOAc
(2 × 100 mL), and the organic phases were combined, dried, and
concentrated.
2 H, H-6Ia, H-6II ), 3.49−3.46 (m, 2 H, H-6Ib, H-2II), 3.44 (dd, 1 H, J =
a
7.7, 11.1 Hz, H-6II ), 3.40 (t, 2 H, J = 5.1 Hz, H-6′), 3.37−3.33 (m, 1
b
H, H-2I), 2.00, 1.87, 1.85 (3 s, 9 H, 3 COCH3). 13C NMR (150 MHz,
CDCl3): δ = 170.2−169.9 (3 OCOCH3), 165.3 (CO), 162.1 (CO),
138.4, 129.5 (2 ipso Ph), 133.6−127.8 (Ar), 102.8 (C-1II), 98.4 (C-1I),
92.0 (CCl3), 77.1 (C-2II), 75.6 (C-3I), 75.4 (PhCH2), 73.5 (C-5I), 72.4
(C-3II), 71.8 (C-4I), 70.7, 70.6, 70.5, 70.0 (C-2′, C-3′, C-4′, C-5′), 70.3
(C-5II), 69.2 (C-1′), 66.9 (C-4II), 60.5 (C-6II), 59.9 (C-2I), 50.7 (C-
6′), 31.2 (C-6I), 20.6−20.3 (3 COCH3). HRMS (ESI-TOF): m/z [M
+ NH4]+ calcd for C40H52Cl3BrN5O16 1042.1658, found 1042.1652.
Anal. Calcd for C40H48Cl3BrN4O16: C, 46.78; H, 4.71; N, 5.46. Found:
C, 47.07; H, 4.84; N, 5.15.
Anhydrous K2CO3 (57.16 mg, 0.31 mmol) followed by BnBr (40
μL, 0.32 mmol) was added at 0 °C under argon to a solution of the
foregoing material in DMF (15 mL). After being stirred for 15 min at
0 °C, the mixture was allowed to warm to room temperature (∼2 h),
diluted with CH2Cl2 (150 mL), and washed with water (2 × 50 mL).
The organic layer was dried, concentrated, and coevaporated with
toluene (twice). Chromatography (6:1 toluene−acetone) afforded the
benzyl uronate 8 (164 mg, 73% over two steps) as a colorless foam.
8-Azido-3,6-dioxaoctyl 6-Bromo-2,6-dideoxy-2-trichloroa-
cetamido-3-O-(2-O-benzyl-3,4-O-isopropylidene-α-D-galacto-
pyranosyl)-β-D-glucopyranoside (7). A solution of sodium
methoxide in methanol (1 M, 0.7 mL) was added under argon to a
solution of 5α (520 mg, 0.5 mmol) in 1:9 CH2Cl2−MeOH (20 mL),
and the reaction mixture was stirred at room temperature for 6 h. The
mixture was neutralized with Amberlite IR-120 (H+) resin and filtered,
and the solids were washed with MeOH (25 mL). The filtrate was
concentrated to give compound 6 as an amorphous solid in almost
theoretical yield. For identification and spectral analysis, a portion was
20
1
[α]D = +17.2 (c 0.5, CHCl3). H NMR (600 MHz, CDCl3): δ =
7.36−7.30 (m, 10 H, 2Ph), 6.98 (d, 1 H, J2,NH = 8.8 Hz, NH), 5.36 (d,
1 H, 2J = 12.3 Hz, PhCHH), 5.13 (d, 1 H, J1,2 = 3.5 Hz, H-1II), 5.01 (d,
1 H, 2J = 12.3 Hz, PhCHH), 4.94 (d, 1 H, 2J = 12.1 Hz, PhCHH), 4.91
(d, 1 H, J4,OH = 1.8 Hz, 4I-OH), 4.75 (d, 1 H, J1,2 = 8.2 Hz, H-1I),
1
purified by chromatography (12:1 CH2Cl2−MeOH). H NMR (600
MHz, CDCl3 + D2O): δ = 7.36−7.30 (m, 5 H, Ph), 5.08 (d, 1 H, J1,2
=
4.74−4.72 (m, 2 H, PhCHH, H-5II), 4.49 (dd, 1 H, J3,4 = 5.5 Hz, J4,5
=
3.6 Hz, H-1II), 4.86 (d, 1 H, J1,2 = 8.4 Hz, H-1I), 4.83 (d, 1 H, 2J = 11.7
3.1 Hz, H-4II), 4.46 (dd, 1 H, J2,3 = 7.5 Hz, J3,4 = 5.5 Hz, H-3II), 3.91−
3.87 (m, 1 H, H-1′a), 3.85−3.83 (m, 1 H, H-1′b), 3.79 (t, 1 H, J1,2 = J2,3
= 8.5 Hz, H-2I), 3.74−3.69 (m, 2 H, H-3I, H-6Ia), 3.68−3.59 (m, 9 H,
H-2II, H-2′, H-3′, H-4′, H-5′), 3.52−3.45 (m, 3 H, H-4I, H-5I, H-6Ib),
3.44−3.37 (m, 2 H, H-6′), 1.37, 1.27 (2 s, 6 H, C(CH3)2). 13C NMR
(150 MHz, CDCl3): δ = 167.3 (COOBn), 162.4 (NCOCCl3), 136.6,
135.2 (2 ipso Ph), 128.7−128.2 (Ar), 109.6 (C(CH3)2), 101.3 (C-1II),
100.8 (C-1I), 92.4 (CCl3), 85.9 (C-3I), 76.1 (C-3II), 75.9 (C-2II), 74.4
2
Hz, PhCHH), 4.70 (d, 1 H, J = 11.7 Hz, PhCHH), 4.03−3.99 (m, 2
H, H-3II, H-4II), 3.94−3.88 (m, 3 H, H-3I, H-5II, H-1′a), 3.80−3.75 (m,
4 H, H-2II, H-6II, H-1′b), 3.74−3.69 (m, 1 H, H-6Ia), 3.68−3.58 (m, 9
H, H-2I, H-2′, H-3′, H-4′, H-5′), 3.52−3.49 (m, 2 H, H-5I, H-4I),
3.48−3.43 (m, 1 H, H-6Ib), 3.42−3.39 (m, 2 H, H-6′). 13C NMR (150
MHz, CDCl3 + D2O): δ = 162.7 (NCOCCl3), 137.0 (ipso Ph), 128.7−
128.4 (Ar), 100.0 (C-1II), 99.9 (C-1I), 92.4 (CCl3), 82.7 (C-3I), 77.0
F
DOI: 10.1021/acs.joc.5b00562
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Featured Article
(C-5I), 73.5 (C-4II), 73.3 (C-4I), 72.9 (PhCH2), 71.0, 70.5, 70.3 (C-2′,
C-3′, C-4′), 69.9 (C-5′), 68.3 (C-1′), 68.2 (C-5II), 66.8 (PhCH2), 56.4
(C-2I), 50.5 (C-6′), 32.3 (C-6I), 28.0, 26.1 (C(CH3)2). HRMS (ESI-
TOF): m/z [M + NH4]+ calcd for C37H50Cl3BrN5O13 956.1654, found
956.1638. Anal. Calcd for C37H46Cl3BrN4O13: C, 47.22; H, 4.93; N,
5.95. Found: C, 47.45; H, 5.08; N, 5.80.
and brine (20 mL), and the organic extract was dried and
concentrated. Chromatography (6:1 toluene−acetone) afforded 12
(919 mg, 92%). Mp: 85−86 °C (EtOAc−Et2O). [α]D20 = −1.7 (c 0.5,
1
CHCl3). H NMR (600 MHz, CDCl3): δ = 7.36−7.26 (m, 5 H, Ph),
6.94 (d, 1 H, J2,NH = 7.5 Hz, NH), 5.38 (d, 1 H, J3,4 = 3.3 Hz, H-4II),
5.24 (dd, 1 H, J1,2 = 8.1 Hz, J2,3 = 10.4 Hz, H-2II), 4.92 (dd, 1 H, J2,3
=
10.5 Hz, J3,4 = 3.4 Hz, H-3II), 4.88 (d, 1 H, J1,2 = 8.3 Hz, H-1I), 4.63 (d,
1 H, 2J = 12.3 Hz, PhCHH), 4.60 (d, 1 H, 2J = 12.3 Hz, PhCHH), 4.56
(d, 1 H, J1,2 = 8.0 Hz, H-1II), 4.55 (dd, 1 H,, J2,3 = 10.4 Hz, J3,4 = 8.1
Hz, H-3I), 4.16−4.10 (m, 2 H, H-6II), 3.99 (t, 1 H, J = 6.6 Hz, H-5II),
3.96−3.93 (m, 1 H, H-1′a), 3.87−3.85 (dd, 1 H, J = 1.2, J = 10.7 Hz,
H-6Ib), 3.80−3.76 (m, 1 H, H-1′b), 3.71−3.68 (m, 1 H, H-6Ia), 3.68−
3.65 (m, 3 H, H-5′, 4I-OH), 3.64−3.59 (m, 6 H, H-2′, H-3′, H-4′),
3.57 (t, 1 H, J = 8.5 Hz, H-4I), 3.53−3.48 (m, 2 H, H-2I, H-5I), 3.39 (t,
2 H, J = 5.0 Hz, H-6′), 2.16, 2.08, 2.03, 1.97 (4 s, 12 H, 4 COCH3).
13C NMR (150 MHz, CDCl3): δ = 170.4, 170.1, 170.0, 169.5 (4
8-Azido-3,6-dioxaoctyl 6-Bromo-2,6-dideoxy-2-trichloroa-
cetamido-3-O-(benzyl 2-O-benzyl-α-D-galactopyranosyluro-
nate)-β-D-glucopyranoside (9). Compound 8 (360 mg, 0.38
mmol) was dissolved in 80% aq AcOH (5 mL) and stirred at 80 °C
for 3 h, when TLC (3:1 toluene−acetone) showed complete
conversion of the starting material into a product with lower mobility.
The solvents were evaporated, the residue was coevaporated with
toluene (twice) to remove AcOH, and chromatography (4:1 toluene−
20
1
acetone) gave 9 (307 mg, 89%). [α]D = +25.7 (c 0.3, CHCl3). H
NMR (600 MHz, CDCl3): δ = 7.38−7.30 (m, 10 H, 2Ph), 7.17 (d, 1
H, J2,NH = 8.5 Hz, NH), 5.26 (d, 1 H, 2J = 12.2 Hz, PhCHH), 5.16 (d,
1 H, J1,2 = 3.5 Hz, H-1II), 5.08 (d, 1 H, 2J = 12.3 Hz, PhCHH), 4.86 (d,
1 H, 2J = 11.8 Hz, PhCHH), 4.80 (d, 1 H, J1,2 = 8.4 Hz, H-1I), 4.75 (d,
1 H, 2J = 11.8 Hz, PhCHH), 4.69 (d, 1 H, J4,OH = 1.2 Hz, 4I-OH), 4.53
(d, 1 H, J4,5 = 1.5 Hz, H-5II), 4.28 (br s, 1 H, H-4II), 4.22−4.19 (m, 1
H, H-3II), 3.89−3.86 (m, 1 H, H-1′a), 3.84−3.79 (m, 3 H, H-2II, H-3I,
H-1′b), 3.75−3.68 (m, 2 H, H-2I, H-6Ia), 3.67−3.56 (m, 8 H, H-2′, H-
3′, H-4′, H-5′), 3.52−3.49 (m, 2 H, H-5I, H-4I), 3.48−3.44 (m, 1 H,
H-6Ib), 3.43−3.36 (m, 2 H, H-6′), 2.47 (d, 1 H, J3,OH = 5.8 Hz, 3II-
OH), 2.42 (br s, 1 H, 4II-OH). 13C NMR (150 MHz, CDCl3): δ =
168.1 (COOBn), 162.6 (NCOCCl3), 136.6, 135.1 (2 ipso Ph), 128.9−
128.4 (Ar), 101.3 (C-1II), 100.6 (C-1I), 92.4 (CCl3), 85.0 (C-3I), 75.9
(C-2II), 74.7 (PhCH2), 74.3 (C-5I), 73.6 (C-4I), 71.1 (C-5II), 70.8 (C-
4II), 70.9, 70.5, 70.3 (C-2′, C-3′, C-4′), 69.9 (C-5′), 69.4 (C-3II), 68.5
(C-1′), 67.1 (PhCH2), 56.8 (C-2I), 50.5 (C-6′), 32.4 (C-6I). HRMS
(ESI-TOF): m/z [M + NH4]+ calcd for C34H46Cl3BrN5O13 916.1341,
found 916.1337. Anal. Calcd for C34H42Cl3BrN4O13: C, 45.33; H, 4.70;
N, 6.22. Found: C, 45.57; H, 4.49; N, 5.86.
OCOCH3), 162.0 (NCOCCl3), 138.2 (ipso Ph), 128.3−127.6 (Ar),
100.9 (C-1I), 99.1 (C-1II); 92.4 (CCl3), 82.3 (C-3I), 75.3 (C-5I), 73.5
(PhCH2), 71.1 (C-5II), 70.8 (C-3II), 70.7, 70.6, 70.5 (C-2′, C-3′, C-4′),
69.9 (C-5′), 69.5 (C-6I), 69.2 (C-4I), 68.4 (C-1′), 68.3 (C-2II), 66.8
(C-4II), 61.2 (C-6II), 58.0 (C-2I), 50.6 (C-6′), 20.9, 20.6, 20,5, 20.4 (4
OCOCH3). HRMS (ESI-TOF): m/z [M + Na]+ calcd for
C35H47Cl3N4O17Na 923.1899, found 923.1896. Anal. Calcd for
C35H47Cl3N4O17: C, 46.60; H, 5.25; N, 6.21. Found: C, 46.45; H,
5.24; N, 6.16.
8-Azido-3,6-dioxaoctyl 2,4-Di-O-benzyl-3,6-dideoxy-α-L-
xylo-hexopyranosyl-(1→4)-[3,4,6-tri-O-acetyl-β-D-galactopyra-
nosyl-(1→3)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-D-
glucopyranoside (15). Bromine (113 μL, 2.2 mmol) was added to a
solution of ethyl 2,4-di-O-benzyl-3,6-dideoxy-1-thio-β-L-xylo-hexopyr-
anoside (13)31 (410 mg, 1.1 mmol) in CCl4 (6 mL). The mixture was
shaken gently for 10 min, and then hex-1-ene (555 μL, 4.4 mmol) was
added. After concentration and coevaporation with CCl4 (3 × 6 mL), a
solution of the crude bromide 14 in dry CH2Cl2 (2 mL) was added to
a stirred mixture of 12 (330 mg, 0.366 mmol), Bu4NBr (354 mg, 1.1
mmol), and powdered molecular sieves (4 Å, 750 mg) in 2.5:1
CH2Cl2−DMF (2.8 mL). After being stirred under argon atmosphere
for 48 h at room temperature, when TLC (4:1 toluene−acetone)
showed that all glycosyl acceptor 12 had been consumed, the mixture
was diluted with CH2Cl2 (10 mL) and filtered through a Celite pad,
and the solids were washed with CH2Cl2 (2 × 5 mL). The combined
filtrate and washings were successively washed with satd aq NaHCO3
(15 mL) and H2O (15 mL), dried, and concentrated. Chromatography
(6:1 toluene−acetone) gave trisaccharide 15 (422 mg, 95%) as a
8-Amino-3,6-dioxaoctyl 2,6-Dideoxy-2-acetamido-3-O-(α-D-
galactopyranosyluronate)-β-D-glucopyranoside (10). A mixture
of 9 (250 mg, 0.28 mmol) and Pd/C (250 mg) in a mixture of MeOH
(10 mL) and 0.1 M potassium phosphate buffer (10 mL; pH = 7) was
stirred under H2 (1 atm) at room temperature. After 2 days, when
TLC (25:1 MeOH−25% NH4OH) showed complete conversion of
the starting material into a more polar product, the mixture was
filtered through a Celite pad, the catalyst was washed with H2O (15
mL), and the filtrate was concentrated. Chromatography (30:1
MeOH−25% NH4OH) followed by lyophilization afforded the desired
disaccharide 10 (122 mg, 86%) as a white solid. 1H NMR (600 MHz,
D2O): δ = 5.27 (d, 1 H, J1,2 = 3.1 Hz, H-1II), 4.47 (d, 1 H, J1,2 = 8.5 Hz,
H-1I), 4.17 (br s, 1 H, H-4II), 4.08 (br s, 1 H, H-5II), 3.91−3.88 (m, 1
H, H-1′a), 3.78−3.74 (m, 2 H, H-2II, H-3II), 3.71−3.66 (m, 2 H, H-2I,
H-1′b), 3.64−3.61 (m, 8 H, H-2′, H-3′, H-4′, H-5′), 3.60−3.57 (m, 1
H, H-3I), 3.46−3.42 (m, 1 H, H-5I), 3.35 (t, 1 H, J3,4 = J4,5 = 9.2 Hz,
H-4I), 3.10 (t, 2 H, J = 5.2 Hz, H-6′), 1.91 (s, 3H, COCH3), 1.23 (d, 3
H, J = 6.1 Hz, H-6I). 13C NMR (150 MHz, D2O): δ = 175.6 (CO),
174.7 (CO), 101.3 (C-1I), 100.5 (C-1II), 81.1 (C-3I), 76.2 (C-4I), 72.4
(C-5II), 71.8 (C-5I), 71.0 (C-4II), 69.9, 69.9, 69.8 (C-2′, C-3′, C-4′),
69.7 (C-2II or C-3II), 69.3 (C-5′), 68.4 (C-2II or C-3II), 66.8 (C-1′),
54.4 (C-2I), 39.3 (C-6′), 22.4 (COCH3), 16.7 (C-6I). HRMS (ESI-
TOF): m/z [M − H]− calcd for C20H35N2O13 511.2145, found
511.2149.
8-Azido-3,6-dioxaoctyl 6-O-Benzyl-2-deoxy-3-O-(2,3,4,6-
tetra-O-acetyl-β-D-galactopyranosyl)-2-trichloroacetamido-β-
D-glucopyranoside (12). A mixture of the benzylidene acetal 1116
(1.0 g, 1.11 mmol) and freshly activated powdered molecular sieves (3
Å, 1.5 g) in dry THF (20 mL) was stirred under argon for 2 h at room
temperature. The solution was cooled to 0 °C (ice−water bath), and
NaCNBH3 (0.84 g, 13.32 mmol) was added portionwise. After the
mixture was stirred for 20 min at 0 °C, 2 M HCl−Et2O was added
dropwise at 0 °C until the effervescence ceased and the pH remained
acidic. The mixture was stirred for an additional 15 min at room
temperature, diluted with CH2Cl2 (25 mL), and filtered through
Celite. The filtrate was washed with cold satd aq NaHCO3 (20 mL)
20
1
colorless solid. [α]D = −29.5 (c 0.9, CHCl3). H NMR (600 MHz,
CDCl3): δ = 7.34−7.26 (m, 15 H, Ph), 7.04 (d, 1 H, J2,NH = 8.5 Hz,
NH), 5.34 (d, 1 H, J3,4 = 3.1 Hz, H-4II), 5.14 (dd, 1 H, J1,2 = 8.3, J2,3
10.4 Hz, H-2II), 5.07 (d, 1 H, J1,2 = 3.0 Hz, H-1III), 4.90 (dd, 1 H, J2,3
=
=
10.5, J3,4 = 3.4 Hz, H-3II), 4.82 (d, 1 H, J1,2 = 8.2 Hz, H-1II), 4.75 (d, 1
H, J1,2 = 7.2 Hz, H-1I), 4.59 (d, 1 H, 2J = 12.4, PhCHH), 4.54 (s, 2 H,
PhCH2), 4.46 (s, 2 H, PhCH2), 4.44 (m, 1 H, H-5III), 4.33 (d, 1 H, 2J
= 12.4, PhCHH), 4.15 (t, 1 H, J = 7.2 Hz, H-3I), 4.11−4.02 (m, 3 H,
H-4I, H-6II), 3.92−3.84 (m, 4 H, H-6Ia, H-1′a, H-2III, H-5II), 3.81 (m, 1
H, H-2II), 3.73−3.66 (m, 3 H, H-1′b, H-6Ib, H-5I), 3.65−3.55 (m, 8 H,
H-2′, H-3′, H-4′, H-5′), 3.38 (m, 3 H, H-4III, H-6′), 2.15 (dt, 1 H, J2,3
= J3,4 = 3.6, 2J = 12.8 Hz, 1 H, H-3IIIeq), 2.08, 2.01, 1.94, 1.84 (4 s, 12
2
H, 4 COCH3), 1.80 (dt, 1 H, J3,4 = 1.9, J2,3 = J = 12.2 Hz, 1 H, H-
3IIIax), 1.25 (d, 3 H, J5,6 = 6.2 Hz, H-6III). 13C NMR (150 MHz,
CDCl3): δ = 170.3, 170.0, 169.9, 169.5 (4 OCOCH3), 161.6
(NCOCCl3), 138.3, 138.1, 138.0 (3 ipso Ph), 128.4−127.5 (Ar),
100.0 (JC,H = 161.4 Hz, C-1II), 99.5 (JC,H = 164.3 Hz, C-1I), 95.7 (JC,H
= 170.2 Hz, C-1III), 92.5 (CCl3), 76.6 (C-3I), 75.5 (C-4III), 75.2 (C-5I),
73.3 (PhCH2), 71.5 (C-4I), 71.3 (PhCH2), 70.7, 70.6, 70.5 (PhCH2, C-
2′, C-3′, C-4′), 70.9 (C-3II), 70.8 (C-2III), 70.6 (C-5II), 70.0 (C-5′),
68.3 (C-1′), 68.1 (C-2II), 68.1 (C-6I), 66.9 (C-4II), 66.3 (C-5III), 60.5
(C-6II), 57.2 (C-2I), 50.6 (C-6′), 26.8 (C-3III), 20.7, 20.6, 20.5, 20.4 (4
OCOCH3), 16.5 (C-6III). HRMS (ESI-TOF): m/z [M + NH4]+ calcd
for C55H73Cl3N5O20 1228.3914, found 1228.3962. Anal. Calcd for
C55H69Cl3N4O20: C, 54.48; H, 5.74; N, 4.62. Found: C, 54.26; H, 5.79;
N, 4.45.
G
DOI: 10.1021/acs.joc.5b00562
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Featured Article
8-Azido-3,6-dioxaoctyl 2,4-Di-O-benzyl-3,6-dideoxy-α-L-
xylo-hexopyranosyl-(1→4)-[β-D-galactopyranosyl-(1→3)]-6-O-
benzyl-2-deoxy-2-trichloroacetamido-β-D-glucopyranoside
(16). Compound 15 (903 mg, 0.76 mmol) was dissolved in 6:1
CH2Cl2−MeOH (35 mL), a solution of sodium methoxide in
methanol (1 M, 1.0 mL) was added, and the reaction mixture was
stirred at room temperature for 3 h. The mixture was processed as
described above for preparation of 6, and chromatography (9:1
CH2Cl2−MeOH) gave 16 in virtually theoretical yield. [α]D20 = −13.4
for C54H67Cl3N4O16: C, 57.17; H, 5.95; N, 4.94. Found: C, 57.43; H,
5.93; N, 4.91.
8-Azido-3,6-dioxaoctyl 2,4-Di-O-benzyl-3,6-dideoxy-α-L-
xylo-hexopyranosyl-(1→4)-[3-O-benzyl-β-D-galactopyranosyl-
(S)-(P)-4,6-cyclic 2,2,2-trichloroethyl phosphate-(1→3)]-6-O-
benzyl-2-deoxy-2-trichloroacetamido-β-D-glucopyranoside
(18) and 8-Azido-3,6-dioxaoctyl 2,4-Di-O-benzyl-3,6-dideoxy-
α-L-xylo-hexopyranosyl-(1→4)-[3-O-benzyl-β-D-galactopyrano-
syl-(R)-(P)-4,6-cyclic 2,2,2-trichloroethyl phosphate-(1→3)]-6-
O-benzyl-2-deoxy-2-trichloroacetamido-β-D-glucopyranoside
(19). 2,2,2-Trichloroethyl phosphorodichloridate (95 μL, 0.593 mmol)
was added dropwise at −20 °C to a solution of 17 (560 mg, 0.494
mmol) and pyridine (400 μL, 4.94 mmol) in CH2Cl2 (7 mL). After 30
min, when TLC (3:1 toluene−acetone) indicated incomplete
conversion of 17, an additional portion of 2,2,2-trichloroethyl
phosphorodichloridate (95 μL, 0.593 mmol) was added, and stirring
was continued for 30 min at −20 °C. Excess reagent was destroyed by
addition of MeOH (0.5 mL), the mixture was concentrated, and
EtOAc (5 mL) was added to the residue. The precipitate was filtered
off and washed with EtOAc (2 × 3 mL). The combined filtrates were
concentrated, and chromatography (3:1 toluene−acetone) gave 18
(464 mg, 71%) and 19 (160 mg, 24%), in that order, as colorless
solids.
(c 0.5, CHCl3). 1H NMR (600 MHz, CDCl3): δ = 8.02 (d, 1 H, J2,NH
=
7.2 Hz, NH), 7.34−7.26 (m, 15 H, Ph), 5.12 (d, 1 H, J1,2 = 7.1 Hz, H-
2
1I), 4.99 (d, 1 H, J1,2 = 3.1 Hz, H-1III), 4.52 (d, 1 H, J = 12.1,
PhCHH), 4.44 (s, 2 H, PhCH2), 4.43−4.39 (m, 4 H, H-1II, PhCH2, H-
3I), 4.38 (d, 1 H, 2J = 12.1, PhCHH), 4.20 (dd, 1 H, J4,5 = 12.7, J5,6
=
6.3 Hz, H-5III), 3.98 (m, 1 H, H-1′b), 3.94 (m, 1 H, H-4I), 3.91 (dd, 1
H, J = 4.6, J = 10.7 Hz, H-6Ia), 3.88 (d, 1 H, J3,4 = 2.7 Hz, H-4II), 3.83
(m, 1 H, H-6II ), 3.82 (m, 1 H, H-2III), 3.77 (m, 1 H, H-6II ), 3.74 (m,
a
b
1 H, H-6Ib), 3.71 (m, 1 H, H-1′a), 3.67 (m, 1 H, H-5I), 3.64−3.61 (m,
9 H, H-2I, H-2′, H-3′, H-4′, H-5′), 3.58 (m, 1 H, H-2II), 3.56 (dd, 1 H,
J2,3 = 9.7, J3,4 = 2.9 Hz, H-3II), 3.49 (m, 1 H, H-5II), 3.43 (br s, 1 H, H-
4III), 3.38 (t, 2 H, J = 5.1 Hz, H-6′), 2.11 (dt, 1 H, J2,3 = J3,4 = 3.7, 2J =
12.9 Hz, 1 H, H-3IIIeq), 1.81 (dt, 1 H, J3,4 = 2.1, J2,3 = 2J = 12.8 Hz, 1 H,
H-3IIIax), 1.17 (d, 3 H, J5,6 = 6.5 Hz, H-6III). 13C NMR (150 MHz,
CDCl3): δ = 162.1 (NCOCCl3), 138.2, 138.1, 137.9 (3 ipso Ph),
128.4−127.6 (Ar), 100.0 (C-1II), 98.9 (C-1I), 97.5 (C-1III), 92.5
(CCl3), 77.1 (C-3I), 75.7 (C-4III), 75.7 (C-5II), 74.6 (C-4I), 74.5 (C-
5I), 73.6 (C-3II), 73.1 (PhCH2), 71.4 (PhCH2), 71.2 (C-2III), 71.0
(PhCH2), 70.8 (C-2II), 70.5, 70.4, 70.3, 69.8 (C-2′, C-3′, C-4′, C-5′),
68.7 (C-1′), 68.6 (C-4II), 68.5 (C-6I), 67.3 (C-5III), 62.2 (C-6II), 56.1
(C-2I), 50.6 (C-6′), 27.1 (C-3III), 16.4 (C-6III). HRMS (ESI-TOF):
m/z [M + NH4]+ calcd for C47H65Cl3N5O16 1060.3486, found
1060.3487. Anal. Calcd for C47H61Cl3N4O16: C, 54.05; H, 5.89; N,
5.36. Found: C, 53.92; H, 6.01; N, 5.17.
20
Data for 18. Mp: 152−153 °C (EtOH). [α]D = −17.4 (c 0.6,
1
CHCl3). H NMR (500 MHz, CDCl3): δ = 7.57 (d, 1 H, J2,NH = 7.4
Hz, NH), 7.36−7.23 (m, 20 H, Ph), 5.04 (d, 1 H, J1,2 = 3.3 Hz, H-1III),
5.01 (d, 1 H, J1,2 = 6.7 Hz, H-1I), 4.74 (m, 3 H, H-4II, PhCH2), 4.68−
4.59 (m, 2 H, CH2CCl3), 4.55−4.47 (m, 7 H, H-6II, H-1II, 2 PhCH2),
4.46−4.36 (m, 4 H, H-3I, H-5III, PhCH2), 4.04 (t, 1 H, J3,4 = J4,5 = 7.9
Hz, H-4I), 4.03−3.96 (m, 3 H, H-4I, H-6II , H-1′a), 3.90−3.84 (m, 2 H,
a
H-2III, H-2II), 3.79 (br s, 1 H, 2II-OH), 3.74−3.65 (m, 5 H, H-6II , H-
b
1′b, H-4III, H-2I, H-5I), 3.64−3.57 (m, 8 H, H-2′, H-3′, H-4′, H-5′),
3.46 (br s, 1 H, H-5II), 3.41 (dt, 1 H, J2,3 = 9.4, J3,4 = 3.8 Hz, H-3II),
3.34 (t, 2 H, J = 5.0 Hz, H-6′), 2.09 (dt, 1 H, J2,3 = J3,4 = 3.5, 2J = 12.8
Hz, 1 H, H-3IIIeq), 1.82 (dt, 1 H, J3,4 = 2.2, J2,3 = 2J = 12.7 Hz, 1 H, H-
3IIIax), 1.24 (d, 3 H, J5,6 = 6.5 Hz, H-6III). 13C NMR (125 MHz,
CDCl3): δ = 162.0 (NCOCCl3), 138.8, 138.2, 138.0, 137.2 (4 ipso Ph),
128.7−127.3 (Ar), 101.4 (C-1II), 98.6 (C-1I), 96.9 (C-1III), 95.0 (d,
8-Azido-3,6-dioxaoctyl 2,4-Di-O-benzyl-3,6-dideoxy-α-L-
xylo-hexopyranosyl-(1→4)-[3-O-benzyl-β-D-galactopyranosyl-
(1→3)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-D-gluco-
pyranoside (17). A suspension of compound 16 (773 mg, 0.74
mmol) and dibutyltin oxide (204 mg, 0.82 mmol) in dry MeOH (25
mL) was boiled under reflux until the solution became clear (∼2 h).
The mixture was cooled to room temperature, the solvent was
evaporated, a solution of the residue was concentrated with toluene (3
× 5 mL), and the white residue was dried under vacuum for 3 h. A
mixture of the stannylene derivative, thus obtained, CsF (225 mg, 1.48
mmol), and benzyl bromide (176 μL, 1.48 mmol) in anhyd DMF (15
mL) was stirred at room temperature overnight and concentrated
(∼13 kPa). A solution of the residue in toluene (5 mL) was
concentrated, and chromatography (first toluene, to remove the tin
derivatives, followed by 4:1 toluene−acetone) afforded 17 (781 mg,
JC,P = 10.3 Hz, CH2CCl3), 92.3 (COCCl3), 77.0 (C-3II), 76.9 (d, JC,P
=
3.9 Hz, CH2CCl3), 76.3 (C-4III), 76.2 (C-4II), 76.21 (C-3I), 74.7 (C-
5I), 73.1 (PhCH2), 72.8 (C-4I), 72.1 (PhCH2), 72.0 (PhCH2), 71.0
(PhCH2), 70.9 (C-2III), 70.7 (d, JC,P = 7.5 Hz, C-6II), 70.6, 70.5, 69.9
(C-2′, C-3′, C-4′, C-5′), 69.6 (C-2II), 68.2 (C-6I), 68.1 (C-1′), 67.0
(C-5III), 66.3 (d, JC,P = 6.2 Hz, C-5II), 56.7 (C-2I), 50.6 (C-6′), 27.4
(C-3III), 16.4 (C-6III). 31P NMR (162 MHz, CDCl3): δ = −10.95.
HRMS (ESI-TOF): m/z [M + NH4]+ calcd for C56H71Cl6N5O18P:
1342.2657, found 1342.2660. Anal. Calcd for C56H67Cl6N4O18P: C,
50.66; H, 5.09; N, 4.22. Found: C, 50.96; H, 5.09; N, 4.24.
20
Data for 19. Mp: 75−76 °C (EtOH). [α]D = −15.0 (c 0.5,
20
1
1
93%) as a colorless syrup. [α]D = −12.8 (c 0.7, CHCl3). H NMR
(600 MHz, CDCl3): δ = 7.87 (d, 1 H, J2,NH = 7.3 Hz, NH), 7.38−7.22
(m, 20 H, Ph), 5.09 (d, 1 H, J1,2 = 6.5 Hz, H-1I), 5.03 (d, 1 H, J1,2 = 3.0
CHCl3). H NMR (500 MHz, CDCl3): δ = 7.51 (d, 1 H, J2,NH = 8.0
Hz, NH), 7.40−7.27 (m, 20 H, Ph), 5.00 (br d, 2 H, H-1III, H-1I), 4.88
(d, 1 H, J3,4 = 3.0 Hz, H-4II), 4.81 (d, 1 H, 2J = 12.1, PhCHH), 4.76 (d,
2
2
Hz, H-1III), 4.85 (d, 1 H, J = 12.0, PhCHH), 4.74 (d, 1 H, J = 12.0,
1 H, 2J = 12.2, PhCHH), 4.65−4.57 (m, 3 H, H-6II , CH2CCl3), 4.56−
b
2
4.41 (m, 7 H, H-1II, H-6II , 2 PhCH2, PhCHH), 4.39 (d, 1 H, 2J = 12.0,
PhCHH), 4.53 (d, 1 H, J = 12.0, PhCHH), 4.54−4.41 (m, 4 H, 2
a
PhCH2), 4.40−4.35 (m, 3 H, H-1II, H-3I, PhCHH), 4.14 (dd, 1 H, J4,5
= 12.9 Hz, J5,6 = 6.3 Hz, H-5III), 3.99−3.95 (m, 2 H, H-1′b, H-4I), 3.92
(dd, 1 H, J = 5.0, J = 10.7 Hz, H-6Ia), 3.89 (br s, 1 H, H-4II), 3.87−3.82
(m, 2 H, H-6II , H-2III), 3.78−3.73 (m, 4 H, H-6II , H-6Ib, H-2II, H-5I),
PhCHH), 4.24 (t, 1 H, J3,4 = J4,5 = 7.2 Hz, H-3I), 4.11 (br. q, 1 H, J5,6
=
6.5 Hz, H-5III), 4.04 (t, 1 H, J3,4 = J4,5 = 7.0 Hz, H-4I), 4.01 (m, 1 H, H-
1′a), 3.95 (dd, 1 H, J = 5.1, J = 10.4 Hz, H-6Ia), 3.87−3.81 (m, 3 H, H-
2II, H-2III, H-2I), 3.78 (m, 1 H, H-5I), 3.72 (dd, 1 H, J = 3.3, J = 10.5
Hz, H-6Ib), 3.67 (m, 2 H, H-1′b), 3.65−3.59 (m, 8 H, H-2′, H-3′, H-4′,
H-5′), 3.56 (br s, 1 H, H-5II), 3.51 (dt, 1 H, J2,3 = 9.4, J3,4 = 3.5 Hz, H-
3II), 3.45 (br s, 1 H, H-4III), 3.35 (t, 2 H, J = 5.0 Hz, H-6′), 2.13 (dt, 1
H, J2,3 = J3,4 = 3.6, 2J = 12.6 Hz, 1 H, H-3IIIeq), 1.79 (dt, 1 H, J3,4 = 1.9,
a
b
3.72−3.65 (m, 2 H, H-1′a, H-2I), 3.63−3.58 (m, 8 H, H-2′, H-3′, H-4′,
H-5′), 3.43−3.39 (m, 3 H, H-4III, H-5II, H-3II), 3.34 (t, 2 H, J = 5.0 Hz,
H-6′), 2.10 (dt, 1 H, J2,3 = J3,4 = 3.5, 2J = 12.9 Hz, 1 H, H-3IIIeq), 1.83
(dt, 1 H, J3,4 = 2.3, J2,3 = 2J = 13.0 Hz, 1 H, H-3IIIax), 1.19 (d, 3 H, J5,6
=
6.4 Hz, H-6III). 13C NMR (150 MHz, CDCl3): δ = 163.0 (NCOCCl3),
138.3, 138.3, 138.04, 138.01 (4 ipso Ph), 128.5−127.5 (Ar), 100.5 (C-
1II), 99.0 (C-1I), 97.2 (C-1III), 92.5 (CCl3), 79.9 (C-3II), 76.9 (C-3I),
75.8 (C-4III), 74.6 (C-5II), 74.5 (C-5I), 74.2 (C-4I), 73.1 (PhCH2),
72.7 (PhCH2), 71.4 (PhCH2), 71.2 (C-2III), 71.0 (PhCH2), 70.8 (C-
2II), 70.6, 70.5, 70.4, 69.9 (C-2′, C-3′, C-4′, C-5′), 68.9 (C-6I), 68.7
(C-1′), 67.6 (C-4II), 67.4 (C-5III), 62.3 (C-6II), 55.4 (C-2I), 50.6 (C-
6′), 27.3 (C-3III), 16.4 (C-6III). HRMS (ESI-TOF): m/z [M + NH4]+
calcd for C54H71Cl3N5O16 1150.3956, found 1150.3959. Anal. Calcd
J2,3 = J = 12.8 Hz, 1 H, H-3IIIax), 1.21 (d, 3 H, J5,6 = 6.6 Hz, H-6III).
2
13C NMR (125 MHz, CDCl3): δ = 161.2 (NCOCCl3), 138.2, 138.1,
137.9, 137.3 (4 ipso Ph), 128.6−127.8 (Ar), 100.5 (C-1II), 99.2 (C-
1III), 96.7 (C-1I), 94.4 (d, JC,P = 9.6 Hz, CH2CCl3), 92.2 (COCCl3),
77.7 (d, JC,P = 4.6 Hz, CH2CCl3), 77.4 (C-3I), 76.9 (C-3II), 75.6 (C-
4III), 75.2 (d, JC,P = 4.5 Hz, C-4II), 74.2 (C-5I), 73.2 (C-4I), 73.1
(PhCH2), 72.2 (PhCH2), 71.3 (PhCH2), 71.1 (PhCH2), 70.8 (C-2III),
70.6, 70.5, 70.4, 69.9 (C-2′, C-3′, C-4′, C-5′), 70.0 (d, JC,P = 2.4 Hz, C-
6II), 69.7 (C-2II), 68.8 (C-6I), 68.4 (C-1′), 67.5 (C-5III), 66.8 (d, JC,P
=
H
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6.3 Hz, C-5II), 54.1 (C-2I), 50.6 (C-6′), 27.3 (C-3III), 16.5 (C-6III). 31
P
Bhuiyan, M. S.; Saksena, R.; Clements, J. D.; Calderwood, S. B.; Qadri,
NMR (162 MHz, CDCl3): δ = −7.06. HRMS (ESI-TOF): m/z [M +
NH4]+ calcd for C56H71Cl6N5O18P 1342.2657, found 1342.2669. Anal.
Calcd for C56H67Cl6N4O18P: C, 50.66; H, 5.09; N, 4.22. Found: C,
50.54; H, 4.94; N, 4.16.
́ ̌
F.; Kovac, P.; Ryan, E. T. Clin. Vaccine Immunol. 2012, 19, 594−602.
(3) Alam, M. M.; Bufano, M. K.; Xu, P.; Kalsy, A.; Yu, Y.; Freeman, Y.
W.; Sultana, T.; Rashu, M. R.; Desai, I.; Eckhoff, G.; Leung, D. T.;
Charles, R. C.; LaRocque, R. C.; Harris, J. B.; Clements, J. D.;
8-Amino-3,6-dioxaoctyl 3,6-Dideoxy-α-L-xylo-hexopyrano-
syl-(1→4)-[β-D-galactopyranosyl-4,6-cyclic phosphate-(1→3)]-
2-deoxy-2-acetamido-β-D-glucopyranoside (20). (a) A mixture
of 18 (214 mg, 0.16 mmol) and Pd/C (214 mg) in a mixture of
MeOH (8 mL) and 0.1 M potassium phosphate buffer (8 mL; pH = 7)
was stirred under H2 (1 atm) at room temperature. After 3 days, when
TLC (2:1 i-PrOH−30% NH4OH) showed complete conversion of the
starting material into a more polar product, the mixture was filtered
through a Celite pad, the catalyst was washed with H2O (10 mL), and
the filtrate was concentrated. Chromatography (2:1:0.1 i-PrOH−
H2O−30% NH4OH) followed by lyophilization afforded the desired
trisaccharide 20 (106 mg, 93%) as a white powder. [α]D20 = −36.4 (c
0.3, H2O). 1H NMR (600 MHz, D2O): δ = 4.85 (d, 1 H, J1,2 = 3.8 Hz,
H-1III), 4.73 (q, 1 H, J5,6 = 6.7 Hz, H-5III), 4.50 (d, 1 H, J3,4 = 3.5 Hz,
H-4II), 4.48 (d, 1 H, J1,2 = 8.8 Hz, H-1I), 4.46 (d, 1 H, J1,2 = 8.1 Hz, H-
Calderwood, S. B.; Firdausi, Qadri; Vann, W. F.; Kovac
PLOS Neglected Trop. Dis. 2014, e2683.
(4) Xu, P.; Alam, M. M.; Kalsy, A.; Charles, R. C.; Calderwood, S. B.;
Qadri, F.; Ryan, E. T.; Kovac P. Bioconjugate Chem. 2011, 21, 2179−
2185.
(5) Wang, J.; Villeneuve, S.; Zhang, J.; Lei, P. s.; Miller, C. E.; Lafaye,
P.; Nato, F.; Szu, S. C.; Karpas, A.; Bystricky, S.; Robbins, J. B.; Kovac
́ ̌
, P.; Ryan, E. T.
́ ̌
,
́ ̌
,
P.; Fournier, J. M.; Glaudemans, C. P. J. J. Biol. Chem. 1998, 273,
2777−2783.
́ ̌
(6) Pavliak, V.; Pozsgay, V.; Kovac, P.; Karpas, A.; Chu, C.;
Schneerson, R.; Robbins, J.; Glaudemans, C. P. J. J. Biol. Chem. 1993,
268, 25797−25802.
(7) Albert, M. J.; Siddique, A. K.; Islam, M. S.; Faruque, A. S. G.;
Ansaruzzaman, M.; Faruqe, S. M.; Sack, R. B. Lancet 1993, 341, 704−
704.
1II), 4.33 (br d, 1 H, J = 12.5 Hz, H-6II ), 4.23 (m, 1 H, H-6II ), 4.05
b
(br s, 1 H, H-4III), 3.87−3.88 (m, 4 H, H-3I, H-2III, H-6Ia, H-1′aa), 3.84
(t, 1 H, J1,2 = J2,3 = 9.3 Hz, H-2I), 3.80 (dd, 1 H, J = 4.3, J = 12.3 Hz,
H-6Ib), 3.71−3.66 (m, 4 H, H-4I, H-1′b, H-5′), 3.65−3.59 (m, 7 H, H-
3II, H-2′, H-3′, H-4′), 3.55 (br s, 1 H, H-5II), 3.49−3.43 (m, 2 H, H-5I,
(8) Manning, P. A.; Stroeher, U. H.; Morona, R. In Vibrio cholerae
and Cholera: Molecular to Global Perspectives; Wachsmuth, I. K., Blake,
P. A., Olsvik, O., Eds.; American Society for Microbiology:
Washington, D.C., 1994; pp 77−94.
(9) Kaper, J. B.; Morris, J. G.; Levine, M. M. Clin. Microbiol. Rev.
1995, 8, 48−86.
(10) (a) Knirel, Y. A.; Widmalm, G.; Senchenkova, S. N.; Jansson, P.-
E.; Weintraub, A. Eur. J. Biochem. 1997, 247, 402−410. (b)
Previously,12,13 we erroneously showed the structure of the O-antigen
of V. cholerae O139 as containing the β-D-galactopyranosyluronic acid
linkage..
H-2II), 3.13 (t, 2 H, J = 4.9 Hz, H-6′), 1.95 (s, 3 H, COCH3), 1.92 (dt,
2
1 H, J2,3 = J3,4 = 2.5, J = 12.9 Hz, 1 H, H-3IIIeq), 1.81 (dt, 1 H, J3,4
=
3.9, J2,3 = J = 12.9 Hz, 1 H, H-3IIIax), 1.06 (d, 3 H, J5,6 = 6.8 Hz, H-
6III). 13C NMR (150 MHz, D2O): δ = 174.5 (COCH3), 103.2 (C-1II),
101.1 (C-1I), 98.1 (C-1III), 77.1 (C-3I), 76.0 (d, JC,P = 4.3 Hz, C-4II),
75.7 (C-5I), 72.9 (C-4I), 71.3 (d, JC,P = 7.3 Hz, C-3II), 69.8, 69.8, 69.6
(C-2′, C-3′, C-4′), 69.7 (C-2II), 69.2 (C-1′), 68.8 (d, JC,P = 5.4 Hz, C-
6II), 68.3 (C-4III), 67.5 (d, JC,P = 4.9 Hz, C-5II), 66.7 (C-5III), 66.6 (C-
5′), 63.3 (C-2III), 59.8 (C-6I), 55.9 (C-2I), 39.3 (C-6′), 32.8 (C-3III),
22.5 (COCH3), 15.6 (C-6III). 31P NMR (162 MHz, D2O): δ = −3.88
(3JP,H 22.2 Hz). HRMS (ESI-TOF): m/z [M − H]− calcd for
C26H46N2O18P 705.2489, found 705.2488.
2
(11) Ruttens, B.; Kovac
(12) Ruttens, B.; Saksena, R.; Kovac
4366−4375.
́ ̌
(13) Hou, S.-j.; Kovac, P. Carbohydr. Res. 2011, 346, 1394−1397.
́
,
̌
P. Helv. Chim. Acta 2006, 89, 320−332.
́ ̌
,
P. Eur. J. Org. Chem. 2007,
(14) Turek, D.; Sundgren, A.; Lahmann, M.; Oscarson, S. Org.
Biomol. Chem. 2006, 4, 1236−1241.
(b) Compound 19 (150 mg, 0.11 mmol) was treated with Pd/C
(150 mg) and worked up, as described for 18, to afford 20 (72 mg,
90%), which was in all aspects identical with the compound described
above.
(15) Cox, A. D.; Brisson, J. R.; Thibault, P.; Perry, M. B. Carbohydr.
Res. 1997, 304, 191−208.
́ ̌
(16) Soliman, S. E.; Kovac, P. Synthesis 2014, 46, 748−751.
(17) Paulsen, H.; Lockhoff, O. Chem. Ber. 1981, 114, 3079−3101.
(18) Kovac P. J. Carbohydr. Chem. 1992, 11, 999−1014.
(19) Demchenko, A. V. Curr. Org. Chem. 2003, 7, 35−79.
(20) Adinolfi, M.; Iadonisi, A.; Ravida, A.; Schiattarella, M.
Tetrahedron Lett. 2004, 45, 4485−4488.
ASSOCIATED CONTENT
■
́ ̌
,
S
* Supporting Information
1
Copies of H and 13C NMR spectra for all new compounds.
̀
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.joc.5b00562.
(21) Igarashi, K.; Irisawa, J.; Honma, T. Carbohydr. Res. 1975, 39,
213−225.
(22) Demchenko, A. V. In General Aspects of the Glycosidic Bond
Formation, in Handbook of Chemical Glycosylation: Advances in
Stereoselectivity and Therapeutic Relevance; Demchenko, A. V., Ed.;
Wiley−VCH Verlag: Weinheim, 2008; DOI: 10.1002/
9783527621644.ch1.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: kpn@helix.nih.gov.
Notes
(23) Ernst, B.; Hart, G. W.; Sinay, P. Protecting Groups: Effects on
́
The authors declare no competing financial interest.
Reactivity, Glycosylation Stereoselectivity, and Coupling Efficiency, in
Carbohydrates in Chemistry and Biology; Wiley−VCH Verlag:
Weinheim, 2000; DOI: 10.1002/9783527618255.ch17.
ACKNOWLEDGMENTS
■
(24) Hanessian, S.; Plessas, N. R. J. Org. Chem. 1969, 34, 1045−1053.
(25) Epp, J. B.; Widlanski, T. S. J. Org. Chem. 1999, 64, 293−295.
(26) Walvoort, M. T. C.; Sail, D.; van der Marell, G. A.; Codee, J. D.
This research was supported by the Intramural Research
Program of the National Institutes of Health (NIH) and
National Institute of Diabetes and Digestive and Kidney
Diseases (NIDDK).
In Carbohydrate Chemistry: Proven Synthetic Methods; Kovac
́ ̌
, P., Ed.;
CRC/Taylor and Francis: Boca Raton, 2011.
(27) Garegg, P. J.; Hultberg, H.; Wallin, S. Carbohydr. Res. 1982, 108,
97−101.
REFERENCES
■
(28) Shashkov, A. S.; Chizhov, O. S. Bioorg. Khim. 1976, 2, 437−496.
(29) Lemieux, R. U.; Hendriks, K. B.; Stick, R. V.; James, K. J. Am.
Chem. Soc. 1975, 97, 4056−4062.
(1) Chernyak, A.; Kondo, S.; Wade, T. K.; Meeks, M. D.; Alzari, P.
M.; Fournier, J. M.; Taylor, R. K.; Kovac
2002, 185, 950−962.
(2) Tarique, A. A.; Kalsy, A.; Arifuzzaman, M.; Rollins, S. M.;
Charles, R. C.; Leung, D. T.; Harris, J. B.; LaRocque, R. C.; Sheikh, A.;
́
,
̌
P.; Wade, W. F. J. Infect. Dis.
(30) Weygand, F.; Ziemann, H. Ann. 1962, 657, 179−198.
(31) Ruttens, B.; Kovac, P. Synthesis 2004, 2505−2508.
́ ̌
I
DOI: 10.1021/acs.joc.5b00562
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Featured Article
(32) Bock, K.; Pedersen, C. J. Chem. Soc., Perkin Trans. 2 1974, 293−
297.
(33) Grzeskowiak, K. Synthesis 1980, 831−833.
́
(34) Greene, T. W.; Wutts, P. G. M. Protecting Groups in Organic
Synthesis; John Wiley & Sons, Inc.: New York, 1999.
(35) Knirel, Y. A.; Paredes, L.; Jansson, P.-E.; Weintraub, A.;
Widmalm, G.; Albert, M. J. Eur. J. Biochem. 1995, 232, 391−396.
J
DOI: 10.1021/acs.joc.5b00562
J. Org. Chem. XXXX, XXX, XXX−XXX