Chemical Synthesis of Residue-Selectively 13C and 2H Double-Isotope-Labeled Oligosaccharides as Chemical Probes for the NMR-Based Conformational Analysis of Oligosaccharides

Article abstract of DOI:10.1021/acs.joc.0c01939

The conformational analysis of oligosaccharide is a fundamental issue in glycobiology. NMR measurements of atom-selectively 13C-labeled oligosaccharides have provided valuable information concerning their conformation, which would not be possible using nonlabeled oligosaccharides. The amount of accessible information from an atom-selectively labeled molecule, however, is limited. In this work, we report on the chemical synthesis of residue-selectively 13C- and 2H-labeled oligosaccharides and their use in conformational analysis. 1H NMR measurements of such double isotope-labeled compounds can provide a great deal of information on the dihedral angles across glycosidic linkages. We demonstrated this method in the conformational analyses of some linear and branched β(1,3)-glucan oligosaccharides.

Full text of DOI:10.1021/acs.joc.0c01939

pubs.acs.org/joc
Article
13
2
Chemical Synthesis of Residue-Selectively C and H Double-
Isotope-Labeled Oligosaccharides as Chemical Probes for the NMR-
Based Conformational Analysis of Oligosaccharides
Hiroki Hamagami, Yoshiki Yamaguchi, and Hiroshi Tanaka*
Cite This: https://dx.doi.org/10.1021/acs.joc.0c01939
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: The conformational analysis of oligosaccharide is a
fundamental issue in glycobiology. NMR measurements of atom-
selectively 13C-labeled oligosaccharides have provided valuable
information concerning their conformation, which would not be
possible using nonlabeled oligosaccharides. The amount of
accessible information from an atom-selectively labeled molecule,
however, is limited. In this work, we report on the chemical
2
synthesis of residue-selectively 13C- and H-labeled oligosacchar-
ides and their use in conformational analysis. 1H NMR
measurements of such double isotope-labeled compounds can provide a great deal of information on the dihedral angles across
glycosidic linkages. We demonstrated this method in the conformational analyses of some linear and branched β(1,3)-glucan
oligosaccharides.
isotopically labeled compounds.6 We hypothesized that
INTRODUCTION
■
replacing the [1H] hydrogens on the uniformly 13C-labeled
Cell-surface oligosaccharides play important roles in biological
1
sugar unit with H NMR-silent [2H] hydrogens would be
events such as protein trafficking and cellular recognition.1 To
1
effective in simplifying the H NMR spectrum. Herein we
understand these types of structure−activity relationships,
conformational analysis of oligosaccharides is highly important.
NMR spectroscopy of oligosaccharides is a valuable tool for
elucidating their solution conformation by providing structure-
report on the synthesis and NMR analysis of some linear and
2
branched oligosaccharides containing uniformly 13C and H
double-isotope-labeled monosaccharide units.
related parameters such as homo- and heteronuclear couplings
RESULTS AND DISCUSSION
(J-couplings) and nuclear Overhauser effects (NOEs).2
■
1
However, severe overlaps of 1H signals and the low abundance
Our strategy was to analyze H NMR spectra of oligosacchar-
ides containing a uniformly 13C- and 2H-labeled mono-
of NMR-active [13C] carbon frequently hamper obtaining
these parameters. To overcome these difficulties, selectively
13C-labeled oligosaccharides have been used as chemical
probes.3 The enriched molecules can provide specific J-
couplings in a complex structure. Serianni et al. reported that a
set of dihedral angles φ and Ψ across a glycosidic linkage can
be estimated by using two different oligosaccharides containing
a [13C] carbon at an anomeric or C2 position, which provide
inter-residue 3JCH and 3JCC coupling constants.4 Seeberger et al.
recently reported that the glucose-ring conformation of β(1,6)-
hexaglucosides can be determined by using hexasaccharides
containing a uniformly 13C-labeled glucose unit at different
3
saccharide unit in order to obtain multiple JCH coupling
constants across glycosidic linkages (Figure 1). Double isotope
Figure 1. 13C-Labeled oligosaccharides for conformational studies.
positions, which provides JCH at the anomeric center.5
1
Uniformly 13C-labeled sugar units have the potential for
3
Special Issue: A New Era of Discovery in Carbohy-
drate Chemistry
providing multiple inter-residue JCH coupling constants.
However, these [13C] carbons complicate the 1H NMR
spectrum by coupling with all protons one, two, or three
Received: August 10, 2020
2
bonds apart. On the other hand, a uniformly 13C and H
double-isotope-labeled glucose is commercially available and
has been used in studies related to the biosynthesis of
https://dx.doi.org/10.1021/acs.joc.0c01939
© XXXX American Chemical Society
J. Org. Chem. XXXX, XXX, XXX−XXX
A

The Journal of Organic Chemistry
pubs.acs.org/joc
Article
3
labeling would allow one to assign JCH coupling constants
between [1H] hydrogens on the attached glycosides and the
enriched [13C] carbons. The enriched [13C] carbon at the
3
anomeric position has a JCH coupling related to dihedral
angles Ψ. The other enriched [13C] carbons have 3JCH coupling
related to dihedral angles φ.
To demonstrate the utility of this method, we compared
3
inter-residue JCH coupling constants of the branched and
linear β(1,3)-oligosaccharides 5 and 6, which are partial
structures of β(1,3)-glucans (Scheme 1). β(1,3)-Glucans are
Scheme 1. Strategy for the Synthesis of the Isotope-Labeled
Branched Tetrasaccharide 5 and Linear Trisaccharide 6
Figure 2. Naturally occurring β-glucans 1 and 2 and the synthetic
derivatives 3 and 4.
structurally defined oligosaccharides as model compounds for
β(1,3)-glucans would be highly desirable for evaluating the
biological activities of such polysaccharides.9,10 Our recent
studies on oligosaccharides 3 and 4 showed that branching
β(1,6)-glucosides on a β(1,3)-undecasaccharides did not have
positive effects on the binding to dectin-1.9g Therefore, a
comparison of the 3JCH coupling constants of 5 and 6 would be
expected to provide information on the effects of the branching
β(1,6)-glucoside on the conformation of the β(1,3)-backbone.
The retrosynthesis of the branched tetrasaccharide 5 and the
linear trisaccharide 6 containing a uniformly 13C and 2H
double-isotope-labeled glucoside was designed to proceed via
the same synthetic intermediate 8 (Scheme 1). The
tetrasaccharide 5 would be synthesized by the regioselective
glycosylation of the diol 8 with the superarmed glycosyl
imidate 7.11 The diol 8 can be prepared by the gold-catalyzed
glycosylation of the alcohol 1012 with the glycosyl o-
alkynylbenzoate (ABz) 9 followed by the chemoselective
hydrolysis of the 4,6-O-tert-butylsilylene acetal. The benzyli-
dene acetal of 10 would reduce the steric hindrance of the
alcohol at the 3 position. The glycosyl donor 9 can be prepared
by the chemoselective glycosylation of the glucal 12 with
glycosyl imidate 11,13 followed by anomeric transformation via
the stereoselective epoxidation of the vinyl ether.14 The
unhindered alcohol of the glucal 12 would show a high
reactivity with respect to its glycosylation. In addition, O-tert-
butylsilylene protection would improve the stability of glycals
under acidic conditions.15 The glucal can be synthesized from
the commercially available uniformly 13C and 2H double-
isotope-labeled glucose (13) in 5 steps.14
major components of fungal cell walls and certain bacteria and
function to stimulate innate immunity. Branched β-glucans
such as Schizophyllan (1) containing β(1,6)-glucosidic link-
ages on every third glucose unit on the backbone are known to
exhibit a higher affinity for dectin-1 compared to linear β(1,3)-
glucans such as Curdlan (2) (Figure 2).7 The positive effects of
the branching glucosides on affinity for Dectin-1 can be
attributed to a conformational change in the linear β(1,3)-
backbone caused by the attachment of β(1,6)-branching
glycosides.8 However, structure−activity relationships remain
unclear due to the lack of availability of pure, naturally
occurring β(1,3)-glucosides. Therefore, chemical synthesis of
We first examined the preparation of the branched and linear
oligosaccharides by using the nonlabeled glucoside 1414
(Scheme 2). Treatment of the glucal 14 (1.00 equiv) and
the glycosyl imidate 11 (1.00 equiv) with 10 mol % of
TMSOTf at temperatures between −78 to −60 °C gave the
disaccharide 15 in a quantitative yield. Increasing the
temperature from −78 to −50 °C reduced the yield to 85%.
This result can be attributed to the instability of the double
bond of glucals under acidic conditions.16 We also examined
the glycosylation of the 4,6-O-tert-butylsilylene-protected
thioglycoside 1617 with the same donor 11, to provide the
B
https://dx.doi.org/10.1021/acs.joc.0c01939
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
pubs.acs.org/joc
Article
The epoxide 19 was used in the following reaction without
further purification due to its high susceptibility to acids.
Treatment of the epoxide 19 with the o-hexylbenzoate
(ABzOH) in the presence of 4 Å MS provided the alcohol
20. The subsequent acylation of the resulting alcohol of 20
with benzoyl chloride under basic conditions provided the
glycosyl (2-alkynyl)benzoate 21 as an anomeric mixture (α/β
= 17:83) in 65% yield based on the glucal 15. We also
examined the preparation of the thioglycoside 23 from the
epoxide 19. Treatment of the epoxide 19 with ethanethiol in
the presence of a catalytic amount of trifluoroacetic acid
(TFA) provided the β-thioglycoside 22 in 45% yield based on
the glucal 15. The relative stereochemistry at the C1 and C2
positions was confirmed by a coupling constant between H-1
and H-2 (9.8 Hz) indicative of a trans configuration. The
production of the β-thioglycoside 22 clearly indicated that the
stereoselective epoxidation of the glucal 15 had proceeded at
the α-face. Acylation of the β-thioglycoside 22 with benzoyl
chloride under basic conditions was unsuccessful, even at high
temperatures with the substrate being recovered. The steric
hindrance of the equatorially oriented ethanesulfenyl group
and the O-tert-butylsilylene protection likely inhibited the
acylation. Finally, we attempted the direct glycosidation of the
epoxide 19. The epoxide 19 was treated with the alcohol 10 in
the presence of ZnCl2 at temperatures between −78 °C and
room temperature,18 only to give a complex mixture of
products. On the other hand, the use of PPh3·AuOTf as a
catalyst gave the α-product 24 in 25% yield based on the glucal
15.19 The stereochemistry of the anomeric center was
determined by the coupling constant between H-1 and H-2,
3J1,2 = 3.4 Hz to be α. The formation of the α-product 24
implied that an oxocarbenium ion was produced from the
epoxide and that the secondary alcohol 10 attacked the cation
from the α face probably because of the bulky groups at the
opposite face.
Scheme 2. Glycosylation of the Alcohols 14 and 16
3-O-glycosylated product 17 in 44% yield along with a
significant amount of the 2-O-glycosylated product 18 (25%).
The preparation of glycosyl donor 21 from 15 was examined
next (Scheme 3). Exposure of the glucal 15 to dimethyldiox-
irane (DMDO) at temperatures between 0 °C and room
temperature, followed by the removal of the oxidant and
solvents in vacuo, provided the epoxide 19 as a single isomer.
Scheme 3. Conversion of the Glucal 15 to 2-O-Acyl Glycosyl
Donors 21
Synthesis of the tetrasaccharide 31 and the trisaccharide 28
from 21 is shown (Scheme 4). Treatment of the donor 21 and
1.2 equiv of the alcohol 10 with 20 mol % of PPh3·AuOTf in
CH2Cl2 at temperatures between 0 °C and room temperature
provided the trisaccharide 25 in 82% yield. The stereo-
chemistry of the anomeric center was determined based on the
coupling constant between H-1 and H-2, 3J1,2 = 8.0 Hz to be β.
The subsequent removal of the silylene protecting group by
treatment with HF·pyridine in tetrahydrofuran gave the diol 26
in 80% yield. Treatment of the diol 26 and 1.2 equiv of the
imidate 7 with 20 mol % of TMSOTf from −78 °C to −50 °C
provided the tetrasaccharide 29 in 61% yield as a single isomer.
The position of the remaining hydroxyl group was confirmed
by COSY between the proton at the C4 and 4-OH in COSY
spectra. Global deprotection was achieved by the following
procedures. Solvolysis of the tetrasaccharide 29 under basic
conditions provided the corresponding hydrolysate 30 in 69%
yield. Subsequent hydrogenolysis of the benzyl ethers by
treatment with 10% Pd/C under a hydrogen atmosphere
afforded the tetrasaccharide 31 in 61% yield. The trisaccharide
28 was also prepared from the protected trisaccharide 26 in
33% total yield via solvolysis and hydrogenolysis.
We next examined the labeled tetra- and trisaccharide 5 and
2
6 prepared from uniformly 13C and H double-isotope-labeled
glucose (13) according to the established method (Scheme 5).
We further prepared the labeled disaccharide 44 (Scheme
6). The pentaacetyl glucoside 32 was treated with 30% HBr in
AcOH at 0 °C to provide glycosyl bromide 41 in 61% yield.
C
https://dx.doi.org/10.1021/acs.joc.0c01939
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
pubs.acs.org/joc
Article
and 6, i.e., φC3 = ∼+ 40°, ψC1 = ∼−32°. This consistency
suggests that a side chain via β(1,6)-glycosidic linkage would
have negligible effects on the conformation across β(1,3)-
glycosidic linkages. This small conformational change,
however, could lead to small differences in biological activities
between linear and branched β(1,3)-glucan oligosaccharides, as
was reported in our previous study.9g Additionally, consistent
values between φC3 and φC6, i.e., φ = ∼+40°, suggested that
the φ angles were not affected by the position of the β-
glucosidic linkages due to exoanomeric effects. These results
demonstrate that using residue-specific, double-isotope-labeled
oligosaccharides provide a wide range of information on
conformations across glycosidic linkages, based on NMR data.
Scheme 4. Synthesis of the Tetrasaccharide 31 and
Trisaccharide 28
CONCLUSIONS
■
We report herein on the chemical synthesis of a residue-
selectively 13C-and 2H-labeled tetra- and trisaccharides 5 and 6
using 4,6-O-tert-butylsilylene-D-glucal (12) as a key inter-
mediate. The TMSOTf-catalyzed glycosylation of the 4,6-O-
tert-butylsilylene-D-glucal (12) proceeded at low temperatures
to provide a 3-O-glycosylated glucal, which was further
converted into a glycosyl o-alkynyl benzoate via stereoselective
epoxidation. The subsequent gold-catalyzed glycosidation of
the glycosyl benzoate gave a trisaccharide structure. On the
other hand, a tetrasaccharide structure was constructed by the
regioselective glycosylation of a diol, which was prepared by
the selective removal of the silylene group of a trisaccharide,
with a superarmed glycosyl donor. The partially double-
isotope-labeled oligosaccharides that were synthesized proved
to be a practical probe for obtaining some vital heteronuclear
coupling constants across glycosidic linkages based on 1H
NMR spectra. From these coupling constants, glycosidic
torsional angles were obtained via a Karplus equation, and
the findings suggested that a side chain via a β(1,6)-glucosidic
linkage had little effect on the conformation across β(1,3)-
glycosidic linkages. We expect that the strategy using NMR
measurements of residue-specific, double-isotope-labeled
oligosaccharides will expand the efficiency of conformational
studies in glycobiology.
Glycosylation of 10 with the glycosyl bromide 41 in the
presence of AgOTf at −20 °C provided the disaccharide 42 in
59% yield. Deprotection of the disaccharide 42 was achieved
by solvolysis under basic conditions followed by hydro-
genolysis to provide the disaccharide 44 in 96% total yield.
We next carried out an NMR analysis of the tetra-, tri-, and
2
disaccharides 5, 6, and 44 containing a uniformly H and 13C
1
double-isotope-labeled glucose (Figure 3). Figure 4 shows H
EXPERIMENTAL SECTION
■
NMR spectra of the nonlabeled tetrasaccharide 31 and the
labeled tetrasaccharide 5, respectively (400 MHz, 25 °C,
General Methods. All chemicals used were reagent grade and
used as supplied, except where noted. Prior to use, molecular sieves
were activated by heating at 350 °C. All reactions were performed in
oven-dried glassware under an argon atmosphere. Dichloromethane
(DCM), toluene, and tetrahydrofuran (THF) were dispensed from a
solvent purification system. All reactions were monitored by thin-layer
chromatography carried out on 0.2 mm E. Merck silica gel plates
(60F-254) with UV light, visualized by p-anisaldehyde/H2SO4/
ethanol solution. Column chromatography was carried out using the
forced flow of the indicated solvent on Merck silica gel. Preparative
HPLC purifications were carried out on an SSC-3461 pump with an
SSC-5410 UV detector by using a Senshu Pak column (Silica-3301-N,
8 ϕ × 300 mm). NMR spectra were recorded using a Bruker
AVANCE III HD 400 in CDCl3 or D2O. Chemical shifts are reported
in units of parts per million (ppm) relative to the signal (0.00 ppm)
2
2H2O). In the spectra of the labeled tetrasaccharide 5, H-
1
labeling at the center residue reduced the overlap of the H
signals, and three inter-residue vicinal coupling constants 3JC1H
,
3
3JC3H, and JC6H between the 13C atoms and protons were
assigned (Table 1). The corresponding coupling constants of
trisaccharide 6 and the disaccharide 44 were also obtained by
3
the same method. The JC1H (5.0 Hz) for the disaccharide 44
was close to a previously reported value (4.8 Hz).4 From these
coupling constants, the glycosidic torsion angles for the syn-
and anti-conformers were calculated using the Karplus
equation (3JCH = 7.49cos2θ − 0.96cos θ + 0.15) (Table 1).20
The crystal structure of methyl α-laminarabioside, an unlabeled
version of 44 was reported to be the syn-conformer, which was
stabilized by an inter-residue hydrogen bond between 4-OH
and O5, with φ and ψ angles of +28° and −38°.21 In the
solution state of methyl α-laminarabioside, the φ and ψ angles
were also estimated to be (+40°, −9°).4 Hence, the syn-
conformer would be the predominant conformer in β(1,3)-
glucosidic linkages. Our results showed no significant differ-
ence in dihedral angles between the tetra- and trisaccharides 5
1
for internal tetramethylsilane in CDCl3. H NMR spectrum data is
reported as follows: CDCl3 (7.26 ppm) or HOD (4.79 ppm). 13C
NMR spectrum data is reported as follow: CDCl3 (77.1 ppm) or
acetone-d6 (30.9 or 215.9 ppm) or CD3OD (49.5 ppm) as an internal
standard for D2O. Multiplicities are reported by using the following
abbreviations: s; singlet, d; doublet, t; triplet, m; multiplet, J; coupling
constants in hertz. Structural assignments were made with additional
information from gCOSY experiments. Pure shift NMR (broadband
homonuclear-decoupling) of 5, 6, and 44 was measured using an
D
https://dx.doi.org/10.1021/acs.joc.0c01939
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
pubs.acs.org/joc
Article
Scheme 5. Synthesis of the Tetrasaccharide 5 and Trisaccharide 6
signal to the anomeric signals. The pulse sequence was based on the
previous report,22 using an RSnob selective 180° pulse of 60 ms under
a slice-selection gradient of 0.535 G cm−1. 40 t1 increments were
acquired with a relaxation delay of 2.3 s. NMR data were processed
with TOPSPIN (ver 2.1), and the spectra were displayed using
XWINPLOT (ver 3.5). High-resolution mass spectrometry (HRMS)
was performed by Waters LCT Premier XE. HRMS (ESI-TOF) was
calibrated with leucine enkephalin (SIGMA) as an internal standard.
IR spectra were recorded on a JASCO FT/IR-4200 spectropho-
tometer. Only the strongest and/or structurally important peaks are
reported as the IR data given in cm−1. Optical rotations were
measured on a JASCO model P-1020 polarimeter, with concen-
trations expressed in g per 100 mL.
Scheme 6. Synthesis of the Labeled Disaccharide 44
3-O-(4,6-O-Benzylidene-2,3-di-O-benzoyl-β-D-glucopyranosyl)-
4,6-O-tert-butylsilylidene-D-glucal (15). A solution of 1414 (0.800 g,
2.79 mmol) and 1113 (1.77 g, 2.85 mmol) in DCM (57.0 mL) was
dried over 4 Å MS at room temperature for 1 h. Then, a catalytic
amount of TMSOTf (0.100 equiv, 0.0505 mL, 0.279 mmol) was
added to the solution at −78 °C. After being warmed to −60 °C over
4 h, the solution was quenched with NEt3, filtered through a pad of
Celite. The filtrate was poured into saturated NaHCO3 (aq) and
extracted with two portions of ethyl acetate. The combined organic
layer was washed with brine, dried over MgSO4, filtered, and
concentrated in vacuo. The residue was purified by column
chromatography on silica gel (toluene/ethyl acetate = 4:1) to give
15 (2.04 g, 2.79 mmol, quant.) as a white solid: [α]2D8 +0.57 (c 1.11,
AVANCE 600 spectrometer equipped with a 5 mm TXI gradient
probe (Bruker). The oligosaccharide samples 5, 6, and 44 were
dissolved in D2O (D 99.9%), and the 1H chemical shifts were
calibrated based on an outer standard of the chemical shift of 4,4-
dimethyl-4-silapentane-1-sulfonic acid (DSS) at 0 ppm. The probe
temperature was set to 278 K to avoid the overlapping of the HDO
E
https://dx.doi.org/10.1021/acs.joc.0c01939
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
pubs.acs.org/joc
Article
Figure 3. Labeled oligosaccharides 5, 6, and 44.
NEt3, filtered through a pad of Celite. The filtrate was poured into
saturated NaHCO3 (aq) and extracted with two portions of ethyl
acetate. The combined organic layer was washed with brine, dried
over MgSO4, filtered, and concentrated in vacuo. The residue was
purified by column chromatography on silica gel (hexane:ethyl acetate
= 9:1) to give 17 (0.123 g, 0.141 mmol, 44%.) and 18 (0.0692 g,
0.0794 mmol, 25%) as colorless oils. Compound 17: [α]1D8 −0.86 (c
1
1.06, CHCl3); H NMR (400 MHz, CDCl3); δ 8.00−7.94 (m, 4H),
7.55−7.46 (m, 2H), 7.42−7.21 (m, 14H), 5.78 (t, 1H, J = 9.3, 9.3
Hz), 5.55 (s, 1H), 5.47 (dd, 1H, J = 9.0, 7.5 Hz), 5.26 (d, 1H, J = 7.4
Hz), 4.42 (d, 1H, J = 9.8 Hz), 4.41 (dd, 1H, J = 10.5, 4.8 Hz), 4.17
(dd, 1H, J = 10.3, 5.0 Hz), 3.98 (t, 1H, J = 9.5, 9.5 Hz), 3.90−3.79
(m, 3H), 3.67 (dt, 1H, J = 9.6, 9.6, 4.8 Hz), 3.59 (t, 1H, J = 8.6, 8.6
Hz), 3.38 (dt, 1H, J = 9.9, 9.9, 5.0 Hz), 3.34−3.29 (m, 1H), 2.54 (d,
1H, OH, J = 2.4 Hz), 1.08 (s, 9H), 1.01 (s, 9H);13C{1H} NMR (100
MHz, CDCl3) δ 166.1, 165.7, 136.8, 133.4, 133.2, 131.2, 130.0, 129.9,
129.5, 129.4, 129.1, 129.1, 128.5, 128.4, 128.3, 126.2, 101.9, 101.5,
88.0, 85.9, 78.8, 75.7, 74.9, 73.9, 72.2, 71.5, 68.9, 66.8, 66.2, 27.6,
27.2, 22.8, 20.1; FT-IR (neat) 2933, 2860, 1730, 1273, 1179, 1095,
758, 710 cm−1; HRMS (ESI-TOF) m/z [M + NH4]+ calcd for
C47H58NO12SSi 888.3449, found 888.3438. Compound 18: [α]D18
−14.5 (c 0.810, CHCl3); 1H NMR (400 MHz, CDCl3); δ 8.04−8.02
(m, 2H), 7.99−7.96 (m, 2H), 7.53−7.47 (m, 4H), 7.44−7.29 (m,
12H), 5.77 (dd, 1H, J = 9.4, 8.6 Hz), 5.57 (s, 1H), 5.54 (t, 1H, J = 8.5,
8.5 Hz), 5.48 (d, 1H, J = 7.4 Hz), 4.59 (d, 1H, J = 9.7 Hz), 4.41 (dd,
1H, J = 10.5, 5.0 Hz), 4.14−4.09 (m, 2H), 3.97 (t, 1H, J = 10.2, 10.2
Hz), 3.82 (t, 1H, J = 10.2 Hz, 10.2 Hz), 3.76−3.68 (m, 2H), 3.56 (t,
1H, J = 9.2, 9.2 Hz), 3.41 (t, 1H, J = 8.7, 8.7 Hz), 3.27 (dt, 1H, J =
10.0, 10.0, 5.2 Hz), 2.65 (br, 1H, OH), 1.01 (s, 9H), 0.85 (s,
9H);13C{1H} NMR (100 MHz, CDCl3) δ 165.7, 165.5, 137.0, 133.4,
133.0, 132.7, 130.0, 129.9, 129.7, 129.7, 129.1, 128.9, 128.5, 128.4,
128.3, 128.0, 126.3, 101.6, 100.9, 85.9, 79.1, 78.8, 77.0, 76.5, 74.0,
73.2, 72.5, 68.8, 66.6, 66.0, 27.5, 27.0, 22.8, 20.0; FT-IR (neat) 2933,
2883, 2860, 1732, 1473, 1451, 1274, 1095, 770, 707 cm−1; HRMS
(ESI-TOF) m/z [M + NH4]+ calcd for C47H58NO12SSi 888.3449,
found 888.3472.
2-O-Benzoyl-3-O-(4,6-O-benzylidene-2,3-di-O-benzoyl-β-D-glu-
copyranosyl)-4,6-O-tert-butylsilylidene-D-glucopyranosyl O-1-Hex-
ynylbenzoate (21). To a stirred solution of 15 (0.353 g, 0.484 mmol)
in DCM (2.42 mL) was added a solution of DMDO in acetone (4.84
mL, 0.484 mmol) at −20 °C. After being warmed to room
temperature over 4 h, the solution was concentrated in vacuo to
give 19 as a white solid, which was used for the next reaction without
further purification: [α]2D8 +7.11 (c 1.06, CHCl3), 1H NMR (400
MHz, CDCl3) δ 7.98 (d, 4H, J = 7.2 Hz), 7.56−7.49 (m, 2H), 7.42−
7.31 (m, 9H), 7.56 (t, 1H, J = 8.9, 8.9 Hz), 5.57 (s, 1H), 5.50 (t, 1H, J
= 7.5,7.5 Hz), 5.14 (d, 1H, J = 7.2 Hz), 4.74 (br, 1H), 4.42 (dd, 1H, J
= 10.4, 4.7 Hz), 4.11−4.01 (m, 3H), 3.90−3.81 (m, 3H), 3.76 (dt,
1H, J = 10.2, 10.2, 4.7 Hz), 3.62 (dt, 1H, J = 10.2, 10.2, 4.7 Hz), 2.82
(br, 1H), 1.09 (s, 9H), 1.00 (s, 9H); 13C{1H} NMR (100 MHz,
CDCl3) δ 165.8, 165.3, 136.9, 133.6, 133.3, 130.0, 129.9, 129.6, 129.2,
129.2, 128.7, 128.5, 128.4, 126.2, 101.6, 101.0, 78.8, 78.7, 74.4, 73.4,
72.4, 68.9, 66.8, 66.2, 64.6, 52.9, 31.0, 27.5, 27.1, 22.9, 20.0; FT-IR
(neat) 3063, 2934, 2859, 1731, 1603, 1452, 1387, 1272, 1098 cm−1;
HRMS (ESI-TOF) m/z [M + H]+ calcd for C41H49O12Si 761.2993,
found 761.2989.) A solution of 19 in DCM (9.69 mL) was dried over
Figure 4. 1H NMR spectra of the nonlabeled tetrasaccharide 31
(upper side) and isotope-labeled tetrasaccharide 5 (lower side).
Table 1. Glycosidic Torsion Angles Derived from Coupling
Constants
3JC3H
(φC3(syn/
anti))
3JC6H
(φC6(syn/
anti))
3JC1H (ψC1(syn/
entry compound
anti))
1
2
3
5
4.7 Hz (−32°/ + 3.8 Hz (+40°/− 4.2 Hz (+37°/−
136°)
130°)
132°)
6
4.8 Hz (−31°/
+137°)
3.9 Hz (+39°/−
130°)
44
5.0 Hz (−29°/
+138°)
1
CHCl3); H NMR (400 MHz, CDCl3) δ 8.00−7.97 (m, 4H), 7.55−
7.47 (m, 2H), 7.42−7.30 (m, 9H), 6.16 (d, 1H, J = 6.0 Hz), 5.74 (t,
1H, J = 9.0, 9.0 Hz), 5.56 (s, 1H), 5.46 (t, 1H, J = 7.7, 7.7 Hz), 5.10
(d, 1H, J = 7.2 Hz), 4.49 (d, 1H, J = 6.1 Hz), 4.42 (dd, 1H, J = 10.5,
4.7 Hz), 4.30−4.28 (m, 1H, H-3), 4.16−4.11 (m, 2H), 4.06 (t, 1H, J
= 9.4, 9.4 Hz), 3.96 (t, 1H, J = 10.5, 10.5 Hz), 3.89 (t, 1H, J = 10.2,
10.2 Hz), 3.80−3.70 (m, 2H), 1.10 (s, 9H), 1.03 (s, 9H); 13C{1H}
NMR (100 MHz, CDCl3) δ 165.8, 165.3, 144.7, 138.0, 137.0, 133.4,
133.2, 130.0, 129.9, 129.6, 129.5, 129.2, 128.6, 128.4, 128.4, 128.4,
126.2, 125.4, 101.6, 101.1, 101.1, 79.2, 78.8, 75.5, 73.7, 72.8, 72.4,
69.0, 66.9, 65.8, 27.6, 27.2, 22.9, 21.6, 20.0; FT-IR (neat) 3067, 2860,
1731, 1271, 709 cm−1; HRMS (ESI-TOF) m/z [M + Na]+calcd for
C41H48O11SiNa 767.2864, found 767.2841.
Phenyl 3-O-(4,6-O-Benzylidene-2,3-di-O-benzoyl-D-glucopyra-
nosyl)-4,6-O-tert-butylsilylidene-1-thio-β-D-glucoside (17) and
Phenyl 2-O-(4,6-O-Benzylidene-2,3-di-O-benzoyl-D-glucopyrano-
syl)-4,6-O-tert-butylsilylidene-1-thio-β-D-glucoside (18). A solution
of 1617 (0.140 g, 0.339 mmol) and 11 (0.201 g, 0.324 mmol) in
DCM (3.24 mL) was dried over 4 Å MS (324 mg) at room
temperature for 1 h. Then, a catalytic amount of TMSOTf (0.0058
mL, 0.0324 mmol) was added to the solution at −78 °C. After being
warmed to −50 °C over 45 min, the solution was quenched with
F
https://dx.doi.org/10.1021/acs.joc.0c01939
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
pubs.acs.org/joc
Article
4 Å MS at room temperature for 1 h. Then, o-1-hexynyl benzoic acid
(196 mg, 0.969 mmol) was added to the solution at −40 °C. After
being stirred at room temperature overnight, the solution was filtered
through a pad of Celite. The filtrate was poured into saturated
NaHCO3(aq) and extracted with two portions of ethyl acetate. The
combined organic layer was washed with brine, dried over MgSO4,
filtered, and concentrated in vacuo. The residue was purified by flash
column chromatography on silica gel (toluene/ethyl acetate = 14:1)
to give 20 as a colorless oil (368 mg, 0.382 mmol, 79%, α/β = 17:83),
which was used for the next reaction without further purification. To a
stirred solution of 20 in pyridine (4.84 mL) were sequentially added
BzCl (0.169 mL, 1.45 mmol) and a catalytic amount of DMAP (5.9
mg, 0.0382 mmol) at 0 °C. After being stirred in an oil bath at 60 °C
for 5 h, the solution was poured into 3 M HCl (aq) and extracted with
two portions of ethyl acetate. The combined organic layer was washed
with saturated NaHCO3 (aq) and brine, dried over MgSO4, filtered,
and concentrated in vacuo. The residue was purified by column
chromatography on silica gel (hexane/ethyl acetate = 7:1) to give 21
as a yellow oil (335 mg, 0.315 mmol, 65% in 3 steps, α/β = 17:83).
The major product, the β-isomer, was separated from the mixture of
α- and β-isomers by HPLC chromatography for structure
through a pad of Celite. The filtrate was purified by column
chromatography on silica gel (hexane/ethyl acetate = 1:1) to give 24
(14.0 mg, 0.0124 mmol, 25%) as a colorless oil: [α]2D2 +9.00 (c 0.42,
1
CHCl3); H NMR (400 MHz, CDCl3) δ 8.01−7.98 (m, 4H), 7.52−
7.14 (m, 21H), 5.70 (t, 1H, J = 9.2, 9.2 Hz), 5.55 (s, 1H), 5.50 (t, 1H,
J = 7.9, 7.9 Hz), 5.30 (s, 1H), 5.18 (d, 1H, J = 7.3 Hz), 5.07 (d, 1H, J
= 3.9 Hz), 4.85 (d, 1H, J = 12.6 Hz), 4.50 (d, 1H, J = 12.6 Hz), 4.41
(d, 1H, J = 3.4 Hz), 4.38 (dd, 1H, J = 10.8, 4.9 Hz), 4.21 (dd, 1H, J =
10.5, 4.5 Hz), 4.13−3.46 (m, 12H), 3.35 (dd, 1H, J = 8.9, 3.3 Hz),
3.28 (s, 3H), 3.25 (t, 1H, J = 9.3, 9.3 Hz), 1.07 (s, 9H), 1.00 (s, 9H);
13C{1H} NMR (100 MHz, CDCl3) δ 165.9, 165.5, 138.1, 137.0,
136.8, 133.2, 133.1, 130.1, 130.1, 130.0, 129.7, 129.5, 129.1, 128.8,
128.6, 128.5, 128.4, 128.4, 128.1, 126.2, 126.1, 102.2, 101.6, 101.5,
100.0, 98.9, 82.6, 81.8, 79.0, 78.2, 76.0, 73.8, 73.6, 72.6, 72.1, 69.1,
69.0, 67.3, 66.8, 66.7, 61.9, 55.4, 27.7, 27.3, 22.9, 20.1; FT-IR (neat)
3020, 2934, 1732, 1454, 1373, 1271, 1219, 1091 cm−1; HRMS (ESI-
TOF) m/z [M + NH4]+ calcd for C62H76NO18Si 1150.4832, found
1150.4803.
Methyl 3-O-[2-O-Benzoyl-3-O-(4,6-O-benzylidene-2,3-di-O-ben-
zoyl-β-D-glucopyranosyl)-4,6-O-tert-butylsilylidene-β-D-glucopyra-
nosyl]-2-O-benzyl-4,6-O-benzylidene-α-D-glucopyranoside (25). A
solution of 21 (180 mg, 0.169 mmol) and 10 (75.5 mg, 0.203 mmol)
in DCM (3.37 mL) was dried over 4 Å MS at room temperature for 1
h. Then, a solution of 0.100 M solution of PPh3AuOTf in DCM
(0.337 mL, 0.0337 mmol) was added to the solution at 0 °C. After
being warmed to room temperature overnight, the solution was
filtered through a pad of Celite. The filtrate was poured into saturated
NaHCO3 (aq) and extracted with two portions of ethyl acetate. The
combined organic layer was washed with brine, dried over MgSO4,
filtered, and concentrated in vacuo. The residue was purified by
column chromatography on silica gel (toluene/ethyl acetate = 14:1)
to give 25 (169 mg, 0.137 mmol, 82%) as a colorless oil: [α]2D8 −5.16
(c 0.670, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.83 (d, 2H, J = 7.9
Hz), 7.77 (d, 2H, J = 7.8 Hz), 7.58 (d, 2H, J = 7.9 Hz), 7.53−7.16 (m,
24H), 5.55 (t, 1H, J = 8.0, 8.0 Hz), 5.51 (s, 1H), 5.43 (s, 1H), 5.37 (t,
1H, J = 7.9, 7.9 Hz), 5.27 (t, 1H, J = 8.4, 8.4 Hz), 5.08 (d, 1H, J = 7.2
Hz), 4.88 (d, 1H, J = 8.0 Hz), 4.43−4.40 (m, 2H), 4.15−3.79 (m,
10H), 3.72−3.58 (m, 3H), 3.43 (t, 1H, J = 9.2, 9.2 Hz), 3.33−3.27
(m, 1H), 3.22−3.19 (m, 4H), 1.09 (s, 9H), 0.97 (s, 9H); 13C{1H}
NMR (100 MHz, CDCl3) δ 165.7, 165.2, 164.6, 138.4, 137.5, 136.9,
133.1, 132.8, 129.9, 129.9, 129.8, 129.8, 129.5, 129.4, 128.5, 128.4,
128.4, 128.3, 128.3, 128.2, 127.9, 126.2, 126.2, 125.4, 101.4, 101.2,
101.1, 100.7, 99.0, 81.1, 79.9, 79.7, 78.8, 75.5, 74.1, 73.9, 73.2, 72.6,
70.7, 69.1, 66.5, 66.4, 62.2, 55.3, 27.5, 27.2, 22.8, 20.1; FT-IR (neat)
1736, 1269, 1178 cm−1; HRMS (ESI-TOF) m/z [M + H]+ calcd for
C69H77O19Si 1237.4828, found 1237.4825.
confirmation: [α]2D8 +7.11 (c 1.06, CHCl3); H NMR (400 MHz,
1
CDCl3) δ 7.85 (d, 2H, J = 7.8 Hz), 7.74 (d, 1H, J = 8.0 Hz), 7.70 (d,
2H, J = 7.8 Hz), 7.57 (d, 2H, J = 7.8 Hz), 7.51−7.27 (m, 14H), 7.23−
7.14 (m, 3H), 5.95 (d, 1H, J = 8.1 Hz), 5.61 (t, 1H, J = 9.4, 9.4 Hz),
5.54 (s, 1H), 5.50−5.41 (m, 2H), 5.17 (d, 1H, J = 7.2 Hz), 4.44 (dd,
1H, J = 10.4, 4.4 Hz), 4.26 (dd, 1H, J = 10.0, 4.5 Hz), 4.13−4.11 (m,
2H), 3,94 (t, 2H, J = 9.8, 9.8 Hz), 3.85 (t, 1H, J = 10.2, 10.2 Hz),
3.73−3.68 (m, 2H), 2.42 (t, 2H, J = 7.0, 7.0 Hz), 1.61−1.53 (m, 2H),
1.47−1.42 (m, 2H), 1.13 (s, 9H), 1.07 (s, 9H), 0.92 (t, 3H, J = 7.4,
7.4 Hz); 13C{1H} NMR (100 MHz, CDCl3) δ 165.7, 165.1, 164.8,
163.6, 136.9, 134.6, 133.3, 133.1, 133.0, 132.5, 130.9, 129.8, 129.8,
129.8, 129.4, 129.2, 129.1, 128.5, 128.3, 128.2, 127.3, 101.5, 101.0,
97.3, 92.7, 81.0, 79.1, 78.7, 75.5, 73.2, 72.6, 72.2, 71.7, 69.0, 66.6,
66.3, 30.8, 27.6, 27.2, 22.9, 22.2, 20.1, 19.6, 13.8; FT-IR (neat) 2933,
1736, 1270, 1095 cm−1; HRMS (ESI-TOF) m/z [M + Na]+ calcd for
C61H66O15SiNa 1089.4069, found 1089.4073.
Ethyl 3-O-(4,6-O-Benzylidene-2,3-di-O-benzoyl-β-D-glucopyra-
nosyl)-4,6-O-tert-butylsilylidene-1-thio-β-D-glucoside (22). To a
stirred solution of 19 prepared from 15 (51.7 mg, 0.0723 mmol)
according to the established method, and ethanethiol (0.0261 mL,
0.361 mmol) in DCM (0.600 mL) was added TFAA (0.500 equiv,
0.005 mL, 0.0362 mmol). After being stirred at room temperature
overnight, the solution was quenched with triethylamine and
concentrated in vacuo. The residue was purified by column
chromatography on silica gel (hexane/ethyl acetate = 3:1) to give
22 as a colorless oil (26.5 mg, 0.0322 mmol, 45%): [α]2D4 +1.52 (c
1
Methyl 3-O-[2-O-Benzoyl-3-O-(4,6-O-benzylidene-2,3-di-O-ben-
zoyl-β-D-glucopyranosyl)-β-D-glucopyranosyl]-2-O-benzyl-4,6-O-
benzylidene-α-D-glucopyranoside (26). To a stirred solution of 25
(169 mg, 0.137 mmol) in THF (2.77 mL) was added HF·pyridine
(0.139 mL) at 0 °C. After being stirred at 0 °C for 2 h, the solution
was poured into saturated NaHCO3 (aq) and extracted with two
portions of ethyl acetate. The combined organic layer was washed
with brine, dried over MgSO4, filtered, and concentrated in vacuo. The
residue was purified by column chromatography on silica gel (ethyl
acetate) to give 26 (122 mg, 0.111 mmol, 80%) as a colorless oil:
0.683, CHCl3); H NMR (400 MHz, CDCl3) δ 7.99 (d, 2H, J = 8.0
Hz), 7.96 (d, 2H, J = 8.0 Hz), 7.53−7.47 (m, 2H), 7.42−7.30 (m,
9H), 5.78 (t, 1H, J = 9.3, 9.3 Hz), 5.56 (s, 1H), 5.49 (t, 1H, J = 8.0
Hz, 8.0 Hz), 5.28 (d, 1H, J = 7.4 Hz), 4.41 (dd, 1H, J = 10.6, 4.8 Hz),
4.26 (d, 1H, J = 9.8 Hz), 4.15 (dd, 1H, J = 10.2 Hz, 4.9 Hz), 4.01 (t,
1H, J = 9.5, 9.5 Hz), 3.87 (t, 3H, J = 9.8, 9.8 Hz), 3.69 (dt, 1H, J =
9.6, 9.6, 4.8 Hz), 3.59 (t, 1H, J = 8.6, 8.6 Hz), 3.40−3.34 (m, 2H),
2.67−2.54 (m, 2H), 2.47 (br, −OH), 1.21 (t, 3H, J = 7.4, 7.4 Hz),
1.09 (s, 9H), 1.03 (s, 9H); 13C{1H} NMR (100 MHz, CDCl3) δ
166.2, 165.8, 136.9, 133.4, 133.3, 130.0, 130.0, 129.6, 129.6, 129.2,
128.5, 128.5, 128.4, 126.2, 101.9, 101.6, 86.1, 85.8, 78.8, 75.8, 75.0,
74.0, 72.3, 72.3, 69.0, 66.8, 66.3, 27.6, 27.2, 24.4, 22.9, 20.1, 15.3; FT-
IR (neat) 2920, 1732, 1272, 1220, 1098 cm−1; HRMS (ESI-TOF) m/
z [M + NH4]+ calcd for C43H58NO12SSi 840.3449, found 840.3453.
Methyl 3-O-[3-O-(4,6-O-Benzylidene-2,3-di-O-benzoyl-β-D-glu-
copyranosyl)-4,6-O-tert-butylsilylidene-α-D-glucopyranosyl]-2-O-
benzyl-4,6-O-benzylidene-α-D-glucopyranoside (24). A solution of
19 prepared from 15 (36.7 mg, 0.0494 mmol) according to the
established method and 1012 (18.4 mg, 0.0494 mmol) in DCM (1.48
mL) was dried over 4 Å MS at room temperature for 1 h. Then, a
solution of PPh3AuOTf in DCM (0.200 equiv, 0.0988 mL, 0.00988
mmol) was added to the solution at room temperature. After being
stirred at room temperature for 30 min, the solution was filtered
[α]2D8 +0.451 (c 0.650, CHCl3); H NMR (400 MHz, CDCl3) δ 7.82
1
(d, 2H, J = 8.0 Hz), 7.55 (t, 4H, J = 8.7, 8.7 Hz), 7.48−7.18 (m,
22H), 7.11 (t, 2H, J = 7.5, 7.5 Hz), 6.91−6.89 (m, 2H), 5.67 (t, 1H, J
= 9.6, 9.6 Hz), 5.52 (s, 1H), 5.45 (t, 1H, J = 8.5, 8.5 Hz), 5.44 (s,
1H), 5.24 (t, 1H, J = 8.4, 8.4 Hz), 4.87 (d, 1H, J = 6.7 Hz), 4.85 (d,
1H, J = 7.1 Hz), 4.46 (dd, 1H, J = 10.2, 4.5 Hz), 4.31 (d, 1H, J = 12.6
Hz), 4.18−4.14 (m, 2H), 4.06 (t, 1H, H-3, J = 9.2, 9.2 Hz), 3.94−3.57
(m, 9H), 3.44−3.32 (m, 3H), 3.21 (s, 3H), 3.17 (dd, 1H, J = 9.0, 3.2
Hz); 13C{1H} NMR (100 MHz, CDCl3) δ 165.6, 165.3, 164.5, 138.3,
137.5, 136.6, 133.3, 133.1, 132.8, 129.8, 129.8, 129.6, 129.2, 129.1,
129.0, 128.5, 128.4, 128.3, 128.2, 127.9, 127.6, 126.2, 126.1, 102.0,
101.7, 101.5, 100.8, 99.0, 84.7, 80.0, 79.8, 78.5, 77.4, 76.7, 74.9, 74.0,
73.0, 72.3, 71.8, 69.3, 69.0, 68.3, 66.9, 62.4, 60.5, 55.3, 21.2; FT-IR
G
https://dx.doi.org/10.1021/acs.joc.0c01939
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
pubs.acs.org/joc
Article
(neat) 2925, 1733, 1271, 1220 cm−1; HRMS (ESI-TOF) m/z [M +
Na]+ calcd for C61H60O19Na 1119.3627, found 1119.3618.
= 11.5 Hz), 3.95−3.30 (m, 23H); 13C{1H} NMR (100 MHz, D2O +
CD3OD as an internal standard) δ 103.7, 103.7, 100.1, 85.0, 84.1,
77.0, 76.8, 76.6, 76.5, 75.4, 74.4, 74.1, 72.3, 71.5, 70.6, 70.5, 69.7,
69.1, 69.0, 61.7, 61.5, 55.9, 49.8; FT-IR (neat) 3894, 1575, 1263,
1199 cm−1; HRMS (ESI-TOF) m/z [M + Na]+ calcd for
C25H44O21Na 703.2273, found 703.2246.
Methyl 3-O-[2-O-Benzoyl-6-O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-
D-glucopyranosyl)-3-O-(4,6-O-benzylidene-2,3-di-O-benzoyl-β-D-
glucopyranosyl)-β-D-glucopyranosyl]-2-O-benzyl-4,6-O-benzyli-
dene-α-D-glucopyranoside (29). A solution of 26 (66.5 mg, 0.0606
mmol) and 711 (50.8 mg, 0.0727 mmol) in DCM (1.82 mL) was
dried over 4 Å MS (121 mg) at room temperature for 1 h. Then, a
catalytic amount of TMSOTf (0.0022 mL, 0.00121 mmol) was added
to the solution at −78 °C. After being warmed to −50 °C over 90
min, the solution was quenched with NEt3, filtered through a pad of
Celite. The filtrate was poured into saturated NaHCO3 (aq) and
extracted with two portions of ethyl acetate. The combined organic
layer was washed with brine, dried over MgSO4, filtered, and
concentrated in vacuo. The residue was purified by column
chromatography on silica gel (toluene/ethyl acetate = 10:1) to give
29 (60.9 mg, 0.0373 mmol, 61%) as a colorless oil: [α]2D6 +3.53 (c
Methyl 2-O-Benzyl-4,6-O-benzylidene-3-O-[3-O-(4,6-O-benzyli-
dene-β-D-glucopyranosyl)-β-D-glucopyranosyl]-α-D-glucopyrano-
side (27). To a stirred solution of 26 (54.3 mg, 0.0495 mmol) in
MeOH (1.98 mL) was added NaOMe (10.7 mg, 0.198 mmol) at
room temperature. After being stirred in an oil bath at 70 °C for 3 h,
the solution was quenched with Dowex 50W-4X, filtered through a
pad of Celite, and concentrated in vacuo to give 27 (38.5 mg, 0.0491
mmol, 99%) as a colorless oil: [α]2D7 −5.92 (c 0.995, CHCl3); H
1
NMR (400 MHz, CDCl3) δ 7.48−7.44 (m, 4H), 7.37−7.34 (m,
11H), 5.50 (s, 1H), 5.48 (s, 1H, benzylidene), 4.74 (d, 1H, J = 11.9
Hz), 4.63 (d, 1H, J = 11.9 Hz), 4.58−4.56 (m, 2H), 4,49 (d, 1H, J =
7.7 Hz), 4.30−4.16 (m, 3H), 3.81−3.41 (m, 15H), 3.33 (s, 3H),
3.28−3.24 (m, 1H); 13C{1H} NMR (100 MHz, CDCl3) δ 137.2,
137.0, 129.4, 129.4, 128.8, 128.6, 128.6, 128.5, 126.4, 126.1, 105.3,
104.3, 102.0, 101.6, 98.4, 88.3, 80.2, 80.2, 79.1, 78.6, 75.8, 74.9, 73.5,
73.4, 73.3, 68.9, 68.8, 68.4, 66.8, 62.6, 62.2, 55.5, 50.9; FT-IR (neat)
3329, 2912, 1078 cm−1; HRMS (ESI-TOF) m/z [M + Na]+ calcd for
C40H48O16Na 807.2840, found 807.2821.
1
0.760, CHCl3); H NMR (400 MHz, CDCl3) δ 8.14 (d, 2H, J = 7.7
Hz), 7.80 (d, 2H, J = 7.8 Hz), 7.53−7.08 (m, 46H), 5.61 (t, 1H, J =
9.4, 9.4 Hz), 5.49 (s, 2H), 5.39 (t, 1H, J = 8.5, 8.5 Hz), 5.30 (t, 1H, J
= 7.9, 7.9 Hz), 5.16 (t, 1H, J = 8.6, 8.6 Hz), 4.84 (d, 1H, J = 8.1 Hz),
4.79−4.75 (m, 3H,), 4,70 (d, 1H, J = 10.9 Hz), 4.64 (d, 1H, J = 11.0
Hz), 4.58 (d, 1H, J = 12.1 Hz), 4.54 (d, 1H, J = 11.1 Hz), 4.44−4.35
(m, 3H), 4.22 (d, 1H, J = 3.4 Hz), 4.15−4.02 (m, 4H), 3.85 (t, 1H, J
= 9.3, 9.3 Hz), 3.80−3.63 (m, 8H), 3.58 (t, 1H, J = 9.3, 9.3 Hz),
3.48−3.44 (m, 3H), 3.32−3.20 (m, 3H), 3.14 (dd, 1H, J = 9.1, 3.4
Hz), 3.12 (s, 3H); 13C{1H} NMR (100 MHz, CDCl3) δ 165.6, 165.4,
165.2, 164.4, 138.7, 138.5, 138.4, 138.1, 138.0, 137.7, 136.6, 133.2,
133.0, 132.7, 130.2, 130.2, 129.8, 129.8, 129.6, 129.4, 129.2, 129.2,
129.2, 129.0, 128.9, 128.6, 128.4, 128.4, 128.3, 128.3, 128.1, 128.0,
127.9, 127.8, 127.7, 127.6, 127.6, 126.3, 126.2, 125.4, 101.7, 101.7,
101.1, 100.9, 100.3, 99.4, 84.1, 83.5, 80.0, 79.7, 78.5, 77.6, 77.4, 75.9,
75.0, 75.0, 74.5, 74.1, 73.8, 73.5, 72.9, 72.2, 71.9, 69.1, 68.9, 68.3,
68.2, 66.7, 62.6, 55.6, 21.6; FT-IR (neat) 2869, 1733, 1269, 1092
cm−1; HRMS (ESI-TOF) m/z [M + NH4]+ calcd for C95H96NO25
1650.6271, found 1650.6265.
Methyl 3-O-[3-O-(β-D-Glucopyranosyl)-β-D-glucopyranosyl]-α-D-
glucopyranoside (28). To a stirred solution of 27 (38.5 mg, 0.0491
mmol) in MeOH (0.981 mL) was added 10% Pd/C (98.1 mg) at
room temperature. After being stirred at room temperature under
hydrogen, the solution was filtered through a pad of Celite and
concentrated in vacuo. The residue was purified by reverse-phase
column chromatography on Bond Elut-C18 (methyl alcohol) and
diluted with water. Freeze-drying of the aqueous solution gave 28 (8.5
mg, 0.0164 mmol, 33%) as a white solid: [α]2D4 +1.85 (c 0.610, H2O);
1H NMR (400 MHz, D2O) δ 4.83 (d, 1H, J = 3.7 Hz), 4.76 (d, 1H, J
= 7.9 Hz), 4.75 (d, 1H, J = 8.0 Hz), 3.95−3.87 (m, 4H), 3.81−3.67
(m, 6H), 3.59−3.47 (m, 6H), 3.44 (s, 3H), 3.44 (m, 2H); 13C{1H}
NMR (100 MHz, D2O+CD3OD as an internal standard) δ 103.8,
103.5, 100.1, 85.2, 83.3, 76.9, 76.5, 76.5, 74.4, 74.2, 72.3, 71.8, 70.5,
69.0, 68.9, 61.6, 61.6, 61.4, 55.9; FT-IR (neat) 3380, 2925, 1635,
1373, 1156, 1075,1043 cm−1; HRMS (ESI-TOF) m/z [M + Na]+
calcd for C19H34O16Na 541.1745, found 541.1749.
Methyl 2-O-Benzyl-4,6-O-benzylidene-3-O-[3-O-(4,6-O-benzyli-
dene-β-D-glucopyranosyl)-6-O-(3,4,6-tri-O-benzyl-β-D-glucopyra-
nosyl)-β-D-glucopyranosyl]-α-D-glucopyranoside (30). To a stirred
solution of 29 (52.9 mg, 0.0324 mmol) in MeOH (1.30 mL) was
added NaOMe (7.0 mg, 0.130 mmol) at room temperature. After
being stirred in an oil bath at 85 °C for 5 h, the solution was
quenched with Dowex 50W-4X, filtered through a pad of Celite, and
concentrated in vacuo. The residue was purified by column
chromatography on silica gel (toluene/ethyl acetate = 1:2) to give
30 (26.8 mg, 0.0220 mmol, 69%) as a colorless oil: [α]2D9 +0.428 (c
2
Penta-O-acetyl-D-glucose-13C6, H7 (32). To a stirred solution of
13 (5.0 g, 25.9 mmol) in Ac2O (53.6 mL) was addde Cu(OTf)2
(0.468 g, 1.29 mmol) at room temperature. After being stirred at
room temperature for 1 h, the solution was poured into saturated
NaHCO3 (aq) and extracted with two portions of ethyl acetate. The
combined organic layer was washed with brine, dried over MgSO4,
filtered, and concentrated in vacuo. The residue was purified by
column chromatography on silica gel (hexane/ethyl acetate = 2:1) to
give 32 (10.4 g, 25.9 mmol, quant., αβ = 2:1) as a white solid: [α]D29
+20.5 (c 1.08, CHCl3); 1H NMR (400 MHz, CDCl3) δ 2.23 (s), 2.18
(s), 2.12 (s), 2.09 (s), 2.09 (s), 2.04 (s), 2.03 (s), 2.03 (s), 2.02 (s);
13C{1H} NMR (100 MHz, CDCl3): 170.5, 170.1, 169.5, 169.3, 168.7,
166.3, 91.7−90.7 (m, C-1α), 89.1−88.1 (m, C-1β), 72.8−71.3 (m),
70.4−66.7 (m), 61.4−60.3 (m), 20.7, 20.7, 20.6, 20.5, 20.4, 20.3;
HRMS (ESI-TOF) m/z [M + Na]+ calcd for C1013C6H15D7O11Na
426.1701, found 426.1696.
1
1.20, CHCl3); H NMR (400 MHz, CDCl3) δ 7.50−7.45 (m, 4H),
7.36−7.24 (m, 24H), 7.18−7.14 (m, 2H), 5.53 (s, 1H), 5.50 (s, 1H),
4.95 (d, 1H, J = 11.2 Hz), 4.81−4.76 (m, 2H), 4.73 (d, 1H, J = 11.6
Hz), 4.65 (d, 1H, J = 11.7 Hz), 4.59−4.47 (m, 6H), 4.31−4.13 (m,
5H), 3.83−3.32 (m, 20H), 3,32 (s, 3H, CH3); 13C{1H} NMR (100
MHz, CDCl3) δ 139.1, 138.4, 138.3, 137.3, 137.2, 137.0, 130.3, 129.4,
129.2, 128.9, 128.6, 128.6, 128.5, 128.5, 128.4, 128.4, 128.0, 127.9,
127.9, 127.8, 127.7, 127.7, 126.4, 126.2, 105.4, 104.3, 104.1, 102.1,
101.1, 98.4, 87.8, 84.7, 80.4, 80.2, 79.1, 78.4, 77.4, 77.4, 75.2, 75.2,
75.0, 75.0, 74.5, 73.9, 73.6, 73.5, 73.3, 69.3, 69.0, 68.9, 68.8, 68.5,
66.9, 62.5, 55.5; FT-IR (neat) 3464, 2922, 1373, 1086 cm−1; HRMS
(ESI-TOF) m/z [M + Na]+ calcd for C67H76O21Na 1239.4777, found
1239.4761.
2
Tri-O-acetyl-D-glucal-13C6, H7 (33). To a stirred solution of 32
(1.5 g, 3.72 mmol) in DCM (11.2 mL) was added 30% HBr/AcOH
(3.72 mL) at 0 °C. After being stirred at room temperature overnight,
the solution was concentrated in vacuo to give a residue, which was
used for the next reaction without further purification. To a stirred
solution of the residue in water (7.44 mL) and AcOH (10.2 mL) were
sequentially added NaOAc (3.89 g, 47.4 mmol), CuSO4·5H2O (0.315
mg, 1.30 mmol), and Zn dust (8.46 g, 129 mmol) at 0 °C. After being
stirred at room temperature overnight, the solution was poured into
saturated NaHCO3 (aq) and extracted with two portions of ethyl
acetate. The combined organic layer was washed with brine, dried
over MgSO4, filtered, and concentrated in vacuo. The residue was
purified by column chromatography on silica gel (hexane/ethyl
Methyl 3-O-[3,6-Di-O-(β-D-Glucopyranosyl)-β-D-glucopyrano-
syl]-α-D-glucopyranoside (31). To a stirred solution of 30 (16.9
mg, 13.9 μmol) in MeOH (1.40 mL) was added 10% Pd/C (21.0 mg)
at room temperature. After being stirred at room temperature under
hydrogen overnight, the solution was filtered through a pad of Celite
and concentrated in vacuo. The residue was purified by reverse-phase
column chromatography on Bond Elut-C18 (methyl alcohol) and
diluted with water. Freeze-drying of the aqueous solution gave 31 (5.7
mg, 8.55 μmol, 61%) as a white solid: [α]2D1 +4.63 (c 0.340, H2O); 1H
NMR (400 MHz, D2O) δ 4.84 (d, 1H, H-1, J1,2 = 3.2 Hz), 4.78 (d,
1H), 4.73 (d, 1H, J = 8.0 Hz), 4.53 (d, 1H, J = 8.0 Hz), 4.24 (d, 1H, J
H
https://dx.doi.org/10.1021/acs.joc.0c01939
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
pubs.acs.org/joc
Article
acetate = 1:1) to give 33 (0.908 g, 3.18 mmol, 86% in 2 steps) as a
yellow oil: [α]2D3 −6.91 (c 0.270, CHCl3); 1H NMR (400 MHz,
CDCl3) δ 2.10 (s, 3H, acetyl), 2.08 (3H, s, acetyl), 2.05 (3H, s,
acetyl); 13C{1H} NMR (100 MHz, CDCl3) δ 170.6, 170.3, 169.6,
146.0−144.7 (m, 1C, C-1), 99.2−97.5 (m, 1C, C-2), 77.3, 74.0−72.7
(m, 1C), 67.5−66.0 (m, 2C), 61.4−60.1 (m, 1C, C-6), 21.0, 20.8,
20.7; FT-IR (neat) 1741, 1637, 1371, 1244 cm−1; HRMS (ESI-TOF)
m/z [M + NH4]+ calcd for C613C6H13D7NO7 303.1881, found
303.1868.
128.6, 128.5, 128.4, 126.2, 101.6, 78.7−75.9 (m, 2C), 74.3−73.1 (m,
1C), 72.4, 66.8−63.7 (m, 2C), 53.0−51.7 (m, 1C), 31.0, 27.6, 27.5,
27.1, 22.9, 20.0; FT-IR (neat) 2962, 2859, 2252, 1731, 1373, 1272,
1096 cm−1; HRMS (ESI-TOF) m/z [M + H]+ calcd for
C3513C6H42D7O12Si 774.3634, found 774.3607. A solution of 35 in
DCM (12.7 mL) was dried over 4 Å MS (2.54 g) at room
temperature for 1 h. Then, o-1-hexynylbenzoic acid (282 mg, 1.39
mmol) was added to the solution at −20 °C. After being stirred at
room temperature overnight, the solution was filtered through a pad
of Celite. The filtrate was poured into saturated NaHCO3 (aq) and
extracted with two portions of ethyl acetate. The combined organic
layer was washed with brine, dried over MgSO4, filtered, and
concentrated in vacuo. The residue was purified by column
chromatography on silica gel (hexane/ethyl acetate = 5:1) to give
36 (491 mg, 0.503 mmol, 40% in 2 steps) as a colorless oil: [α]D28
4,6-O-tert-Butylsilylidene-D-glucal-13C6, 2H7 (12). To a stirred
solution of 33 (0.908 g, 3.18 mmol) in methyl alcohol (17.5 mL) was
added NaOMe (18.9 mg, 0.318 mmol) at room temperature. After
being stirred at room temperature for 3 h, the solution was quenched
with Dowex 50W-4X and concentrated in vacuo. The residue was
purified by flash column chromatography on silica gel (chloroform/
methyl alcohol = 9:1) to give a glucal. To a stirred solution of the
1
+6.24 (c 0.955, CHCl3); H NMR (400 MHz, CDCl3) δ 7.97−7.94
t
glucal in DMF (8.03 mL) was added Bu2Si(OTf)2 (0.817 mL, 2.52
(m, 4H), 7.52−7.27 (m, 15H), 5.79 (t, 1H, J = 9.4, 9.4 Hz), 5.57 (s,
1H), 5.51 (t, 1H, J = 7.6, 7.6 Hz), 5.29 (dd, 1H, J = 7.2, 4.3 Hz), 4.45
(dd, 1H, J = 10.4, 4.7 Hz), 4.03 (t, 1H, J = 9.5, 9.5 Hz), 3.90 (t, 1H, J
= 10.3 Hz, 10.3 Hz), 3.72 (m, 1H), 2.38 (t, 2H, J = 7.0, 7.0 Hz),
1.61−1.42 (m, 4H), 1.11 (s, 9H), 1.05 (s, 9H), 0.93 (t, 3H, J = 7.2,
7.2 Hz); 13C{1H} NMR (100 MHz, CDCl3) δ 166.3, 165.7, 136.8,
134.6, 134.3, 133.5, 133.2, 132.3, 130.9, 130.0, 129.9, 129.5, 129.3,
129.2, 128.6, 128.4, 128.3, 127.2, 126.2, 125.3, 102.1, 101.5, 96.9,
94.6−93.5 (m, 1C), 84.9−84.1 (m, 1C), 79.4, 78.7, 75.7−74.9 (m,
1C), 74.1, 72.2−70.2 (m, 2C), 69.0, 66.9, 65.3 (br, 1C), 30.7, 27.5,
27.2, 22.8, 22.1, 20.1, 19.5, 13.8; FT-IR (neat) 2933, 1732, 1272,
1099, 1069 cm−1; HRMS (ESI-TOF) m/z [M + Na]+ calcd for
C4813C6H55D7O14SiNa 998.4447, found 998.4431.
mmol) at −45 °C. After being stirred at −45 °C for 1 h, pyridine
(0.370 mL, 4.58 mmol) was added to the reaction mixture. Then, the
solution was warmed to 0 °C over 90 min and poured into saturated
NaHCO3 (aq) and extracted with two portions of ethyl acetate. The
combined organic layer was washed with brine, dried over MgSO4,
filtered, and concentrated in vacuo. The residue was purified by
column chromatography on silica gel (hexane/ethyl acetate = 2:1) to
give 12 (0.561 g, 1.87 mmol, 82%) as a colorless oil: [α]2D3 +4.47 (c
0.100, CHCl3); 1H NMR (400 MHz, CDCl3) δ 1.07 (s, 9H,
butylsilylidene), 0.99 (s, 9H, butylsilylidene); 13C{1H} NMR (100
MHz, CDCl3) δ 144.2−142.9 (m, 1C, C-1), 103.4−101.7 (m, 1C, C-
2), 77.3−76.2 (m, 1C), 72.4−71.1 (m, 1C), 70.4−69.1 (m, 1C),
65.7−64.4 (m, 1C), 27.6, 27.1, 22.9, 20.0; FT-IR (neat) 3005, 1473,
1260, 1070 cm−1; HRMS (ESI-TOF) m/z [M + NH4]+ calcd for
C813C6H23D7NO4Si 317.2585, found 317.2566.
2-O-Benzoyl-3-O-(4,6-O-benzylidene-2,3-di-O-benzoyl-β-D-glu-
copyranosyl)-4,6-O-tert-butylsilylidene-β-D-glucopyranosyl o-1-
Hexynylbenzoate-13C6, 2H7 (9). To a stirred solution of 36 (471
mg, 0.483 mmol) in pyridine (5.03 mL) were sequentially added BzCl
(0.526 mL, 4.52 mmol) and DMAP (6.10 mg, 0.0483 mmol) at room
temperature. After being stirred in an oil bath at 80 °C for 1 h, the
solution was poured into 3 M HCl (aq) and extracted with two
portions of ethyl acetate. The combined organic layer was washed
with saturated NaHCO3 (aq) and brine, dried over MgSO4, filtered,
and concentrated in vacuo. The residue was purified by column
chromatography on silica gel (hexane/ethyl acetate = 7:1) to give 9
(433 mg, 0.401 mmol, 83%) as a yellow oil: [α]2D8 +11.8 (c 0.965,
3-O-(4,6-O-Benzylidene-2,3-di-O-benzoyl-β-D-glucopyranosyl)-
2
4,6-O-tert-butylsilylidene-D-glucal-13C6, H7 (34). A solution of 12
(0.561 g, 1.87 mmol) and 11 (1.22 g, 1.97 mmol) in DCM (37.5 mL)
was dried over 4 Å MS (1.87 g) at room temperature for 1 h. Then, a
catalytic amount of TMSOTf (0.0416 mL, 0.187 mmol) was added to
the solution at −78 °C. After being warmed to −50 °C over 30 min,
the solution was quenched with NEt3, filtered through a pad of Celite.
The filtrate was poured into saturated NaHCO3 (aq) and extracted
with two portions of ethyl acetate. The combined organic layer was
washed with brine, dried over MgSO4, filtered, and concentrated in
vacuo. The residue was purified by column chromatography on silica
gel (toluene/ethyl acetate = 9:1) to give 34 (1.21 g, 1.60 mmol, 85%)
as a white solid: [α]2D8 +2.85 (c 0.800, CHCl3); 1H NMR (400 MHz,
CDCl3) δ 8.00−7.97 (m, 4H), 7.56−7.30 (m, 11H), 5.74 (t, 1H, J =
9.0, 9.0 Hz), 5.56 (s, 1H), 5.45 (dd, 1H, J = 8.6, 7.5 Hz), 5.13 (dd,
1H, J = 7.2, 4.9 Hz), 4.44 (dd, 1H, J = 10.5, 4.8 Hz), 4.08 (t, 1H, J =
9.5, 9.5 Hz), 3.91 (t, 1H, J = 10.2, 10.2 Hz), 3.77−3.72 (dt, 1H, J =
9.8, 9.8, 4.7 Hz), 1.10 (s, 9H), 1.03 (s, 9H); 13C{1H} NMR (100
MHz, CDCl3) δ 165.8, 165.3, 145.1−143.8 (m, 1C, C-1), 133.4,
133.2, 130.0, 129.9, 129.2, 128.6, 128.4, 128.4, 126.2, 125.4, 101.6,
101.3−99.6 (m, 1C, C-2), 79.2−77.9 (m, 1C), 75.4−74.2 (m, 1C),
72.8−71.6 (m, 1C), 65.2−64.8 (m, 1C), 27.6, 27.2; FT-IR (neat)
2961, 1731, 1271, 1093, 1071 cm−1; HRMS (ESI-TOF) m/z [M +
Na]+ calcd for C3513C6H41D7O11SiNa 780.3505, found 780.3479.
3-O-(4,6-O-Benzylidene-2,3-di-O-benzoyl-β-D-glucopyranosyl)-
4,6-O-tert-butylsilylidene-β-D-glucopyranosyl o-1-hexynylbenzoa-
1
CHCl3); H NMR (400 MHz, CDCl3) δ 7.86 (d, 2H, J = 7.2 Hz),
7.75 (d, 1H, J = 8.0 Hz), 7.71 (d, 2H, J = 7.2 Hz), 7.58 (d, 2H, J = 7.2
Hz), 7.51−7.13 (m, 17H), 5.61 (t, 1H, J = 9.4, 9.4 Hz), 5.54 (s, 1H),
5.43 (dd, 1H, J = 8.8, 7.5 Hz), 5.17 (dd, 1H, J = 7.4, 3.5 Hz), 4.44
(dd, 1H, J = 10.5, 4.8 Hz), 3.94 (t, 1H, J = 9.5, 9.5 Hz), 3.85 (t, 1H, J
= 10.2, 10.2 Hz), 3.69 (dt, 1H, J = 9.6, 9.6, 4.8 Hz), 2.41 (t, 2H, J =
7.0, 7.0 Hz), 1.60−1.53 (m, 2H), 1.49−1.42 (m, 2H), 1.13 (s, 9H),
1.07 (s, 9H), 0.92 (t, 3H, J = 7.3, 7.3 Hz); 13C{1H} NMR (100 MHz,
CDCl3) δ 165.7, 165.1, 136.9, 134.6, 133.7, 133.3, 133.1, 132.9, 132.5,
130.9, 130.3, 129.8, 129.8, 129.4, 129.2, 129.1, 128.6, 128.5, 128.3,
128.2, 127.3, 126.2, 125.8, 101.5, 100.9, 97.3, 92.8−91.9 (m, 1C),
80.9−80.0 (m, 1C), 79.0, 78.7, 77.4, 75.4−74.2 (m, 1C), 72.5−70.6
(m, 2C), 69.0, 66.6, 65.5 (br, 1C), 30.8, 27.5, 27.2, 22.8, 22.2, 20.1,
19.6, 13.8; FT-IR (neat) 1736, 1273, 1104, 1069 cm−1; HRMS (ESI-
TOF) m/z [M + Na]+ calcd for C5513C6H59D7O15SiNa 1102.4709,
found 1102.4664.
Methyl 3-O-[2-O-Benzoyl-3-O-(4,6-O-benzylidene-2,3-di-O-ben-
zoyl-β-D-glucopyranosyl)-4,6-O-tert-butylsilylidene-β-D-glucopyra-
nosyl]-2-O-benzyl-4,6-O-benzylidene-α-D-glucopyranoside-13C6,
2H7 (37). A solution of 9 (413 mg, 0.382 mmol) and 10 (171 mg,
0.459 mmol) in toluene (7.65 mL) was dried over 4 Å MS (383 mg)
at room temperature for 1 h. Then, a solution of PPh3·AuOTf in
DCM (0.765 mL, 0.0765 mmol) was added to the solution at −20
°C. After being stirred at room temperature overnight, the solution
was filtered through a pad of Celite. The filtrate was poured into
saturated NaHCO3 (aq) and extracted with two portions of ethyl
acetate. The combined organic layer was washed with brine, dried
over MgSO4, filtered, and concentrated in vacuo. The residue was
purified by column chromatography on silica gel (toluene/ethyl
2
te-13C6, H7 (36). To a stirred solution of 34 (961 mg, 1.27 mmol)
in DCM (6.34 mL) was added a solution of DMDO in acetone (12.7
mL, 1.27 mmol) at −20 °C. After being warmed to room temperature
overnight, the solution was concentrated in vacuo to give 35 as a white
solid, which was directly used for the next reaction: [α]2D3 +8.48 (c
1
0.390, CHCl3); H NMR (400 MHz, CDCl3) δ 7.98−7.95 (m, 4H),
7.55−7.48 (m, 2H), 7.42−7.29 (m, 9H), 5.74 (dd, 1H, J = 9.4, 8.6
Hz), 5.56 (s, 1H), 5.49 (dd, 1H, J = 8.5, 7.2 Hz), 5.13 (dd, 1H, J =
7.2, 4.3 Hz), 4.41 (dd, 1H, J = 10.4, 4.8 Hz), 4.08 (t, 1H, J = 9.4, 9.4
Hz), 3.86 (t, 1H, J = 10.2, 10.2 Hz), 3.75 (dt, 1H, J = 9.6, 9.6, 4.8
Hz), 1.08 (s, 9H), 0.99 (s, 9H); 13C{1H} NMR (100 MHz, CDCl3) δ
165.7, 165.3, 133.6, 133.3, 130.0, 129.9, 129.5, 129.2, 129.1, 128.7,
I
https://dx.doi.org/10.1021/acs.joc.0c01939
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
pubs.acs.org/joc
Article
acetate = 20:1) to 37 (369 mg, 0.295 mmol, 77%) as a colorless oil:
[α]2D8 −3.86 (c 1.02, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.84 (d,
2H, J = 8.1 Hz), 7.77 (d, 2H, J = 8.0 Hz), 7.59 (d, 2H, J = 8.0 Hz),
7.54−7.20 (m, 24H), 5.55 (t, 1H, J = 9.3 Hz, 9.3 Hz), 5.51 (s, 1H),
5.43 (s, 1H), 5.38 (dd, 1H, J = 8.8, 7.3 Hz), 5.08 (dd, 1H, J = 7.4, 3.4
Hz), 4.43−4.39 (m, 2H), 4.15−4.12 (m, 2H), 4.06 (dt, 1H, J = 9.2,
9.2, 4.0 Hz), 4.18 (d, 1H, J = 12.7 Hz), 3.91 (t, 1H, J = 9.4, 9.4 Hz),
3.81 (t, 1H, J = 10.2, 10.2 Hz), 3.72−3.58 (m, 3H), 3.42 (t, 1H, J =
9.2, 9.2 Hz), 3.22−3.19 (m, 4H), 1.09 (s, 9H), 0.97 (s, 9H); 13C{1H}
NMR (100 MHz, CDCl3) δ 165.7, 165.2, 164.6, 138.4, 138.0, 137.5,
136.9, 133.1, 133.1, 132.8, 129.9, 129.8, 129.8, 129.4, 129.4, 129.2,
129.1, 129.1, 128.5, 128.4, 128.3, 128.3, 128.3, 128.2, 127.9, 126.2,
125.4, 101.4−100.4 (m, 1C), 99.0, 81.1−79.9 (m, 1C), 79.7, 78.7,
77.4, 75.5−73.0 (m, 2C), 72.6, 70.4−69.5 (m, 1C), 69.0, 66.4, 65.9
(m, 1C), 62.2, 55.3, 27.5, 27.2, 22.8, 21.6, 20.0; FT -IR (neat) 2933,
2859, 1736, 1274, 1091 cm−1; HRMS (ESI-TOF) m/z [M + H]+
calcd for C6313C6H70D7O19Si 1250.5469, found 1250.5457.
Methyl 3-O-[2-O-Benzoyl-3-O-(4,6-O-benzylidene-2,3-di-O-ben-
zoyl-β-D-glucopyranosyl)-β-D-glucopyranosyl]-2-O-benzyl-4,6-O-
benzylidene-α-D-glucopyranoside-13C6, 2H7 (8). To a stirred solution
of 37 (350 mg, 0.280 mmol) in THF (5.60 mL) was added HF·
pyridine (0.280 mL) at 0 °C. After being stirred at 0 °C for 1 h, the
solution was poured into saturated NaHCO3 (aq) and extracted with
two portions of ethyl acetate. The combined organic layer was washed
with brine, dried over MgSO4, filtered, and concentrated in vacuo. The
residue was purified by column chromatography on silica gel (hexane/
ethyl acetate = 1:1) to give 8 (150 mg, 0.135 mmol, 48%) as a
colorless oil: [α]2D6 +1.04 (c 1.16, CHCl3); 1H NMR (400 MHz,
CDCl3) δ 7.81 (d, 2H, J = 7.2 Hz), 7.55 (t, 4H, J = 8.4, 8.4 Hz),
7.48−7.19 (m, 22H), 7.10 (t, 2H, J = 7.8, 7.8 Hz), 5.66 (t, 1H, J = 9.6,
9.6 Hz), 5.51 (s, 1H), 5.44 (dd, 1H, J = 9.5, 7.8 Hz), 5.43 (s, 1H),
4.87 (dd, 1H, J = 7.8, 3.5 Hz), 4.45 (dd, 1H, J = 10.3, 4.7 Hz), 4.32−
4.29 (d, 1H, J = 12.6 Hz), 4.17−4.10 (m, 2H), 4.05 (dt, 1H, J = 9.3,
9.3, 5.0 Hz), 3.92−3.82 (m, 3H), 3.78−3.66 (m, 2H), 3.58 (t, 1H, J =
10.2, 10.2 Hz), 3.43−3.38 (m, 2H), 3.21−3.15 (m, 4H); 13C{1H}
NMR (100 MHz, CDCl3) δ 165.5, 165.2, 164.4, 138.2, 137.4, 136.5,
133.2, 133.1, 132.7, 129.8, 129.7, 129.6, 129.2, 129.1, 128.9, 128.6,
128.4, 128.3, 128.3, 128.1, 127.9, 127.6, 126.2, 126.0, 101.8, 101.6,
101.4, 100.5−100.0 (m, 1C), 98.9, 84.5−83.7 (m, 1C), 79.9, 79.7,
78.5, 77.0, 74.6; FT-IR (neat) 3475, 2868, 1733, 1452, 1276, 1179,
1092 cm−1; HRMS (ESI-TOF) m/z [M + Na]+ calcd for
C5513C6H53D7O19Na 1132.4267, found 1132.4315.
66.7 (m, 2C), 62.5, 55.5; FT-IR (neat) 2868, 1733, 1274, 1093, 1070
cm−1 Na]+ calcd for
HRMS (ESI-TOF) m/z [M
C8913C6H85D7O25Na 1668.6466, found 1668.6493.
;
+
Methyl 2-O-Benzyl-4,6-O-benzylidene-3-O-[3-O-(4,6-O-benzyli-
dene-β-D-glucopyranosyl)-6-O-(3,4,6-tri-O-benzyl-β-D-glucopyra-
2
nosyl)-β-D-glucopyranosyl]-α-D-glucopyranoside-13C6, H7 (39). To
a stirred solution of 38 (101 mg, 0.0613 mmol) in MeOH (2.45 mL)
was added NaOMe (4.00 equiv, 13.3 mg, 0.245 mmol) at room
temperature. After being stirred in an oil bath at 60 °C overnight, the
solution was quenched with Dowex 50W-4X, filtered through a pad of
Celite, and concentrated in vacuo. The residue was purified by column
chromatography on silica gel (chloroform/methyl alcohol = 95:5) to
give 39 (73.0 mg, 0.0443 mmol, 97%) as a colorless oil: [α]2D6 +0.370
1
(c 0.810, CHCl3); H NMR (400 MHz, CDCl3) δ 7.50−7.22 (m,
28H), 7.16−7.13 (m, 2H), 5.51 (s, 1H, benzylidene), 5.45 (s, 1H,
benzylidene), 4.95 (d, 1H, benzyl, J = 11.2 Hz), 4.80−4.74 (m, 3H),
4.62 (d, 1H, benzyl, J = 11.8 Hz), 4.58−4.46 (m, 5H), 4.29−4.22 (m,
3H), 4.15 (dd, 1H, J = 10.2 Hz, J = 4.8 Hz), 3.81−3.38 (m, 19H),
3.29−3.25 (m, 4H, CH3); 13C{1H} NMR (100 MHz, CDCl3) δ
139.0, 138.4, 138.2, 137.4, 137.3, 137.0, 133.0, 129.7, 129.3, 129.1,
128.8, 128.6, 128.5, 128.5, 128.4, 128.4, 128.4, 127.9, 127.9, 127.7,
127.7, 127.6, 126.4, 126.2, 105.1, 103.6−103.2 (m, 1C), 102.0, 101.1,
98.5, 87.4−86.7 (m, 1C), 84.6, 80.2, 79.2, 78.0, 77.4, 75.2−72.7 (m,
2C), 68.8−68.0 (m, 2C), 66.9, 62.5, 55.4, 52.2; FT-IR (neat) 3470,
2869, 1374, 1078 cm−1; HRMS (ESI-TOF) m/z [M + Na]+ calcd for
C6113C6H69D7O21Na 1252.5418, found 1252.5385.
Methyl 3-O-[3,6-Di-O-(β-D-Glucopyranosyl)-β-D-glucopyrano-
2
syl]-α-D-glucopyranoside-13C6, H7 (5). To a stirred solution of 39
(64.8 mg, 0.0527 mmol) in MeOH (1.05 mL) and THF (1.05 mL)
was added 10% Pd/C (105 mg) at room temperature. After being
stirred at room temperature under hydrogen overnight, the solution
was filtered through a pad of Celite and concentrated in vacuo. The
residue was purified by reverse-phase column chromatography on
Bond Elut-C18 (methyl alcohol) and diluted with water. Freeze-
drying of the aqueous solution gave 5 (8.7 mg, 0.0128 mmol, 24%) as
a white solid: [α]2D5 +0.861 (c 0.720, H2O); H NMR (400 MHz,
1
D2O) δ 4.84 (d, 1H, J = 3.7 Hz), 4.77 (dd, 1H, J = 8.0, 3.8 Hz), 4.53
(dd, 1H, J = 7.9, 4.2 Hz), 3.95−3.85 (m, 4H), 3.81−3.68 (m, 5H),
3.56−3.30 (m, 12H); 13C{1H} NMR (100 MHz, D2O) δ 103.7−
103.1 (m, 1C), 100.1, 84.3 (br, 1C), 77.0, 76.8, 76.5, 74.8−73.5 (m,
2C), 72.3, 71.5, 70.6, 69.1−68.0 (m, 2C), 61.7, 55.9; FT-IR (neat)
3351, 1076, 1038 cm−1; HRMS (ESI-TOF) m/z [M + Na]+ calcd for
C1913C6H37D7O21Na 716.2914, found 716.2891.
Methyl 3-O-[2-O-Benzoyl-6-O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-
D-glucopyranosyl)-3-O-(4,6-O-benzylidene-2,3-di-O-benzoyl-β-D-
glucopyranosyl)-β-D-glucopyranosyl]-2-O-benzyl-4,6-O-benzyli-
Methyl 2-O-Benzyl-4,6-O-benzylidene-3-O-[3-O-(4,6-O-benzyli-
dene-β-D-glucopyranosyl)-β-D-glucopyranosyl]-α-D-glucopyranosi-
de-13C6, 2H7 (40). To a stirred solution of 8 (85.0 mg, 0.0766 mmol)
in MeOH (3.09 mL) was added NaOMe (16.7 mg, 0.309 mmol) at
room temperature. After being stirred in an oil bath at 70 °C for 4 h,
the solution was quenched with Dowex 50W-4X, filtered through a
pad of Celite, and concentrated in vacuo. The residue was
recrystallized from methyl alcohol to give 40 (33.8 mg, 0.0424
2
dene-α-D-glucopyranoside-13C6, H7 (38). A solution of 8 (132 mg,
0.119 mmol) and 7 (156 mg, 0.223 mmol) in DCM (2.70 mL) was
dried over 4 Å MS at room temperature for 1 h. Then, a catalytic
amount of TMSOTf (6 μL, 0.238 mmol) was added to the solution at
−78 °C. After being stirred at −60 °C for 40 min, the solution was
quenched with NEt3, filtered through a pad of Celite ,and
concentrated in vacuo. The residue was purified by column
chromatography on silica gel (toluene/ethyl acetate = 9:1) to give
38 (153 mg, 0.0929 mmol, 78%) as a colorless oil: [α]2D9 +4.11 (c
1
mmol, 55%) as a colorless oil: [α]2D9 +2.52 (c 0.980, CHCl3); H
NMR (400 MHz, CDCl3) δ 7.52−7.46 (m, 4H), 7.40−7.35 (m,
11H), 5.53 (s, 1H), 5.53 (s, 1H), 4.73−4.66 (m, 3H), 4.48 (dd, 1H, J
= 7.8, 5.0 Hz), 4.31 (dd, 1H, J = 10.5, 4.8 Hz), 4.27 (dd, 1H, J = 10.0,
4.6 Hz), 4.17 (dt, 1H, J = 9.5, 9.5, 5.1 Hz), 3.86−3.68 (m, 5H), 3.63
(dd, 1H, J = 9.5, 3.6 Hz), 3.59−3.48 (m, 4H), 3.37 (s, 3H), 3.30 (d,
1H, J = 1.5 Hz), 3.21−3.18 (m, 1H), 2.70 (d, 1H, J = 2.2 Hz), 1.84−
1.83 (m, 1H); 13C{1H} NMR (100 MHz, CDCl3) δ 137.1, 137.0,
129.4, 128.9, 128.7, 128.6, 128.5, 126.4, 126.0, 105.6, 104.6−103.7
(1C), 102.1, 101.6, 98.3, 88.5−87.3 (1C), 80.4, 80.2, 79.1, 75.7−74.9
(1C), 73.5−72.9 (1C), 69.0−68.2 (1C), 66.9, 62.7, 62.2−61.0 (1C),
55.5; FT-IR (neat) 3452, 1376, 1176, 1086 cm−1; HRMS (ESI-TOF)
m/z [M + Na]+ calcd for C3413C6H41D7O16Na 820.3481, found
820.3499.
1
0.970, CHCl3); H NMR (400 MHz, CDCl3) δ 8.15 (d, 2H, J = 7.1
Hz), 7.81 (d, 2H, J = 7.3 Hz), 7.53−7.08 (m, 44H), 6.97−6.95 (m,
2H), 5.61 (t, 1H, J = 9.5, 9.5 Hz), 5.49 (s, 2H), 5.39 (dd, 1H, J = 9.2,
7.8 Hz), 5.30 (t, 1H, J = 9.2, 9.2 Hz), 4.84 (dd, 1H, J = 8.0, 4.2 Hz),
4.79 (dd, 1H, J = 7.8, 3.4 Hz), 4.77 (d, 1H, J = 11.3 Hz), 4.70 (d, 1H,
J = 11.0 Hz), 4.64 (d, 1H, J = 11.0 Hz), 4.58 (d, 1H, J = 12.2 Hz),
4.54 (d, 1H, J = 11.2 Hz), 4.44−4.35 (m, 3H), 4.23 (d, 1H, J = 3.8
Hz), 4.15−4.09 (m, 2H), 4.04 (d, 1H, J = 12.8 Hz), 3.87−3.63 (m,
7H), 3.48−3.43 (m, 2H), 3.33−3.21 (m, 3H), 3.14 (dd, 1H, J = 9.2,
3.7 Hz), 3.12 (s, 3H, CH3); 13C{1H} NMR (100 MHz, CDCl3) δ
165.6, 165.4, 165.2, 138.7, 138.5, 138.4, 138.1, 137.7, 136.6, 133.2,
133.0, 132.7, 130.2, 130.2, 129.8, 129.8, 129.6, 129.4, 129.2, 129.1,
129.0, 128.9, 128.6, 128.4, 128.3, 128.3, 128.3, 128.1, 128.0, 127.9,
127.7, 127.7, 127.6, 127.6, 127.6, 126.3, 126.2, 125.4, 101.6, 101.1,
100.9, 100.0−99.4 (m, 1C), 83.9−83.1 (m, 1C), 80.0, 79.7, 78.5, 77.6,
77.4, 75.6−74.5 (m, 1C), 74.0, 73.8, 73.5, 72.9−71.9 (m, 1C), 68.9−
Methyl 3-O-[3-O-(β-D-Glucopyranosyl)-β-D-glucopyranosyl]-α-D-
2
glucopyranoside-13C6, H7 (6). To a stirred solution of 40 (20.0 mg,
0.0251 mmol) in MeOH (1.00 mL) and THF (2.0 mL) was added
10% Pd/C (50.0 mg) at room temperature. After being stirred at
room temperature under hydrogen overnight, the solution was filtered
J
https://dx.doi.org/10.1021/acs.joc.0c01939
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
pubs.acs.org/joc
Article
2
through a pad of Celite and concentrated in vacuo. The residue was
purified by reverse-phase column chromatography on Bond Elut-C18
(methyl alcohol) and diluted with water. Freeze-drying of the aqueous
solution gave 6 (5.0 mg, 0.00964 mmol, 38%) as a white solid: [α]D22
+7.03 (c 0.110, H2O); 1H NMR (400 MHz, D2O) δ 4.83 (d, 1H, J1,2
= 3.7 Hz), 4.76 (dd, 1H, J = 8.0, 3.9 Hz), 3.95−3.86 (m, 3H), 3.81−
3.67 (m, 4H), 3.56−3.34 (m, 8H); 13C{1H} NMR (100 MHz, D2O)
δ 103.7−102.5 (m, 1C), 100.1, 84.6 (m, 1C), 76.9, 76.5−75.4 (m,
1C), 74.4−72.9 (m, 1C), 72.3, 71.7, 70.5, 68.9−67.9 (m, 1C), 61.6,
60.9 (m, 1C), 55.9; FT-IR (neat) 3538, 3198, 2952, 2841, 1668,
1451, 1410, 1016 cm−1; HRMS (ESI-TOF) m/z [M + Na]+ calcd for
C1313C6H27D7O16Na 554.2386, found 554.2338.
Methyl 3-O-(β-D-Glucopyranosyl)-α-D-glucopyranoside-13C6, H7
(44). To a stirred solution of 43 (125 mg, 0.228 mmol) in MeOH
(2.50 mL) and THF (2.50 mL) was added 5% Pd/C (228 mg) at
room temperature. After being stirred at room temperature under
hydrogen overnight, the solution was filtered through a pad of Celite
and concentrated in vacuo. The residue was purified by reverse-phase
column chromatography on Bond Elut-C18 (methyl alcohol) and
diluted with water. Freeze-drying of the aqueous solution gave 44
(84.3 mg, 0.228 mmol, quant.) as a white solid: [α]2D5 +3.14 (c 0.600,
H2O); 1H NMR (400 MHz, D2O) δ 4.85 (d, 1H, J = 3.5 Hz), 3.91−
3.87 (m, 2H), 3.82−3.76 (m, 2H), 3.72−3.68 (m, 1H), 3.54 (t, 1H, J
= 9.2, 9.2 Hz), 3.46 (s, 3H); 13C{1H} NMR (100 MHz, D2O +
acetone-d6) δ 104.2−103.3 (m, 1C, C-1′), 100.5 (C-1), 84.0, 77.3−
75.8 (m, 2C), 74.9−73.7 (m, 1C), 72.8, 72.1, 71.0−69.8 (m, 1C),
69.5, 61.9−60.8, 56.3; FT-IR (neat) 3368, 1366, 1046 cm−1; HRMS
(ESI-TOF) m/z [M + Na]+ calcd for C713C6H17D7O11Na 392.1857,
found 392.1864.
2
Tetra-O-acetyl-α-D-glucopyranosyl Bromide-13C6, H7 (41). To a
stirred solution of 32 (0.553 g, 1.37 mmol) in DCM (4.11 mL) was
added 30% HBr/AcOH (1.37 mL) at 0 °C. After being stirred at
room temperature overnight, the solution was poured into saturated
NaHCO3 (aq) and extracted with two portions of ethyl acetate. The
combined organic layer was washed with brine, dried over MgSO4,
filtered, and concentrated in vacuo. The residue was purified by
column chromatography on silica gel (hexane/ethyl acetate = 2:1) to
41 (0.352 g, 0.830 mmol, 61%) as a yellow oil: [α]2D6 +57.5 (c 1.17,
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.joc.0c01939.
1
CHCl3); H NMR (400 MHz, CDCl3) δ 2.10 (s, 3H), 2.10 (s, 3H),
2.05 (s, 3H), 2.04 (s, 3H); 13C{1H} NMR (100 MHz, CDCl3) δ
170.6, 169.9, 169.9, 169.6, 86.8−85.9 (m, 1C, C-1), 72.3−71.0 (m,
1C), 70.7−69.1 (m, 2C), 67.6−66.2 (m, 1C), 61.1−59.8 (m, 1C, 6-
C), 20.8, 20.7, 20.7, 20.6; FT-IR (neat) 1748, 1372, 1241 cm−1;
HRMS (ESI-TOF) m/z [M + NH4]+ calcd for C813C6H16D7BrNO9
441.1197, found 441.1156.
1H and 13C NMR spectra for all new compounds (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Hiroshi Tanaka − Department of Chemical Science and
Engineering, Tokyo Institute of Technology, Tokyo 152-8552,
Japan; orcid.org/0000-0002-3661-9310;
Email: thiroshi@cap.mac.titech.ac.jp
Methyl 2-O-Benzyl-4,6-O-benzylidene-3-O-(tetra-O-acetyl-β-D-
2
glucopyranosyl)-α-D-glucopyranoside-13C6, H7 (42). A solution of
41 (0.229 g, 0.540 mmol) and 10 (0.261 g, 0.701 mmol) in DCM
(5.39 mL) was dried over 4 Å MS at room temperature for 1 h. Then,
a stoichiometric amount of AgOTf (1.10 equiv, 0.152 g, 0.592 mmol)
was added to the solution at −40 °C. After being warmed to −20 °C
over 2 h, the solution was quenched with NEt3 and filtered through a
pad of Celite. The filtrate was poured into saturated water and
extracted with two portions of ethyl acetate. The combined organic
layer was washed with brine, dried over MgSO4, filtered, and
concentrated in vacuo. The residue was purified by column
chromatography on silica gel (toluene/ethyl acetate = 9:1) to give
42 (0.228 g, 0.319 mmol, 59%) as a colorless oil: [α]2D9 −11.1 (c
Authors
Hiroki Hamagami − Department of Chemical Science and
Engineering, Tokyo Institute of Technology, Tokyo 152-8552,
Japan
Yoshiki Yamaguchi − RIKEN-Max-Planck Joint Research
Center for Systems Chemical Biology RIKEN Global Research
Cluster, Saitama 351-0198, Japan
1
0.900, CHCl3); H NMR (400 MHz, CDCl3) δ 7.48−7.45 (m, 2H),
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.joc.0c01939
7.37−7.35 (m, 8H), 5.52 (s, 1H), 4.77 (d, 1H, J = 12.1 Hz), 4.49 (d,
1H, J = 12.1 Hz), 4.47 (d, 1H, J = 3.8 Hz), 4.23−4.17 (m, 2H), 3.79
(dt, 1H, J = 9.7, 9.7, 4.4 Hz), 3.70 (t, 1H, J = 9.9, 9.9 Hz), 3.60 (t, 1H,
J = 9.2, 9.2 Hz), 3.51 (dd, 1H, J = 9.3, 3.8 Hz), 3.35 (s, 3H), 1.99 (s,
3H), 1.98 (s, 3H), 1.98 (s, 3H), 1.97 (s, 3H); 13C{1H} NMR (100
MHz, CDCl3) δ 170.7, 170.4, 169.6, 169.4, 138.0, 137.4, 129.3, 128.6,
128.4, 128.3, 128.2, 126.1, 101.5, 100.9−100.0 (m), 98.9, 80.6, 78.9,
78.9, 78.3, 74.1, 73.0−70.6 (m), 69.1, 68.4−67.1 (m), 62.1, 61.4−61.0
(m), 55.4, 20.8, 20.8, 20.7, 20.7; FT-IR (neat) 2913, 1751, 1371,
1244, 1050 cm−1; HRMS (ESI-TOF) m/z [M + NH4]+ calcd for
C2913C6H39D7NO15 733.3508, found 733.3457.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the JSPS KAKENHI
(18H04390).
REFERENCES
■
Methyl 2-O-Benzyl-4,6-O-benzylidene-3-O-(β-D-glucopyranosyl)-
α-D-glucopyranoside-13C6, 2H7 (43). To a stirred solution of 42
(207.1 mg, 0.289 mmol) in MeOH (0.868 mL) and THF (0.868 mL)
was added NaOMe (1.00 equiv, 15.6 mg, 0.289 mmol) at room
temperature. After being stirred at room temperature overnight, the
solution was quenched with Dowex 50W-4X, filtered through a pad of
Celite and concentrated in vacuo to give 43 (152.1 mg, 0.278 mmol,
(1) (a) Ritchie, G. E.; Moffatt, B. E.; Sim, R. B.; Morgan, B. P.;
Dwek, R. A.; Rudd, P. M. Glycosylation and the Complement System.
Chem. Rev. 2002, 102, 305−320. (b) Davis, B. G. Synthesis of
Glycoproteins. Chem. Rev. 2002, 102, 579−601. (c) Ohtsubo, K.;
Marth, J. D. Glycosylation in cellular mechanisms of health and
disease. Cell 2006, 126, 855−867. (d) Bishop, J. R.; Schuksz, M.;
Esko, J. D. Heparan sulphate proteoglycans fine-tune mammalian
physiology. Nature 2007, 446, 1030−1037.
96%) as a colorless oil: [α]2D7 +5.37 (c 1.00, CHCl3); H NMR (400
1
MHz, CDCl3) δ 7.46−7.44 (m, 2H), 7.37−7.28 (m, 8H), 5.47 (s,
1H), 4.77 (d, 1H, J = 12.1 Hz), 4.57 (d, 1H, J = 12.1 Hz), 4.78 (d,
1H, J = 3.5 Hz), 4.22−4.12 (m, 2H), 3.79−3.50 (m, 8H), 3.30 (s,
3H); 13C{1H} NMR (100 MHz, CDCl3) δ 137.6, 137.2, 129.3, 128.8,
128.6, 128.4, 126.2, 103.7−103.3 (m, 1C, C-1′), 101.5, 98.6, 80.2,
79.3, 78.0, 76.1−74.8 (m, 2C), 74.1−73.3 (m, 1C), 69.8−68.6 (m,
1C), 62.6, 60.9 (br, 1C), 55.4; FT-IR (neat) 3361, 1387, 1090, 1053
(2) Battistel, M. D.; Azurmendi, H. F.; Yu, B.; Freedberg, D. I. Prog.
Nucl. Magn. Reson. Spectrosc. 2014, 79, 48−68.
(3) (a) Klepach, T.; Zhang, W. H.; Carmichael, I.; Serianni, A. S. C-
13-H-1 and C-13-C-13 NMR J-couplings in C-13-labeled N-acetyl-
neuraminic acid: correlations with molecular structure. J. Org. Chem.
2008, 73, 4376−4387. (b) Olsson, U.; Sawen, E.; Stenutz, R.;
Widmalm, G. Conformational flexibility and dynamics of two (1→6)-
linked disaccharides related to an oligosaccharide epitope expressed
cm−1
; HRMS (ESI-TOF) m/z [M +
NH4]+ calcd for
C2113C6H31D7NO11 565.3086, found 565.3086.
K
https://dx.doi.org/10.1021/acs.joc.0c01939
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
pubs.acs.org/joc
Article
on malignant tumour cells. Chem. - Eur. J. 2009, 15, 8886−8894.
(c) Barb, A.; Freedberg, D. I.; Battistel, M.; Prestegard, J. NMR
detection and characterization of sialylated glycoproteins and cell
surface polysaccharides. J. Biomol. NMR 2011, 51, 163−171.
(11) Mydock, L. K.; Demchenko, A. V. Superarming the S-
Benzoxazolyl Glycosyl Donors by Simple 2-O-Benzoyl-3,4,6-tri-O-
benzyl Protection. Org. Lett. 2008, 10, 2103−2106.
(12) Meng, S.; Zhong, W.; Yao, W.; Li, Z. Stereoselective
Phenylselenoglycosylation of Glycals Bearing a Fused Carbonate
Moiety toward the Synthesis of 2-Deoxy-β-galactosides and β-
Mannosides. Org. Lett. 2020, 22, 2981−2986.
(13) Lefeber, D. J.; Kamerling, J. P.; Vliegenthart, J. F. G. Synthesis
of Streptococcus pneumoniae Type 3 Neoglycoproteins Varying in
Oligosaccharide Chain Length, Loading and Carrier Protein. Chem. -
Eur. J. 2001, 7, 4411−4421.
(14) Schimmel, J.; Eleuterio, M. I. P.; Ritter, G.; Schmidt, R. R.
Synthesis of Saponins with Cholestanol, Cholesterol, and Friedelanol
as Aglycones. Eur. J. Org. Chem. 2006, 2006, 1701−1721.
(15) (a) Fraser-Reid, B.; Wu, Z.; Andrews, C. W.; Skowronski, E.;
Bowen, J. P. Torsional effects in glycoside reactivity: saccharide
couplings mediated by acetal protecting groups. J. Am. Chem. Soc.
1991, 113, 1434−1435. (b) Crich, D.; Sun, S. Direct chemical
synthesis of β-mannopyranosides and other glycosides via glycosyl
triflates. Tetrahedron 1998, 54, 8321−8348. (c) Chong, P. Y.; Roush,
W. R. Concerning the Origin of the High β-Selectivity of
Glycosidation Reactions of 2-Deoxy-2-iodo-glucopyranosyl Trichlor-
oacetimidates. Org. Lett. 2002, 4, 4523−4526. (d) Crich, D.; Smith,
M. Solid-Phase Synthesis of β-Mannosides. J. Am. Chem. Soc. 2002,
124, 8867−8869. (e) Nukada, T.; Berces, A.; Whitfield, D. M. Can
the stereochemical outcome of glycosylation reactions be controlled
by the conformational preferences of the glycosyl donor? Carbohydr.
Res. 2002, 337, 765−774.
(16) Shao, N.; Guo, Z. Solution-Phase Synthesis with Solid-State
Workup of an O-Glycopeptide with a Cluster of Cancer-Related T
Antigens. Org. Lett. 2005, 7, 3589−3592.
(17) Wisse, P.; Gold, H.; Mirzaian, M.; Ferraz, M. J.; Lutteke, G.;
van den Berg, R. J. B. H. N.; van den Elst, H.; Lugtenburg, J.; van der
Marel, G. A.; Aerts, J. M. F. G.; Codee, J. D. C.; Overkleeft, H. S.
Synthesis of a Panel of Carbon-13-Labelled (Glyco)Sphingolipids.
Eur. J. Org. Chem. 2015, 2015, 2661−2677.
̈
(d) Norris, S. E.; Landstrom, J.; Weintraub, A.; Bull, T. E.;
Widmalm, G.; Freedberg, D. I. Transient hydrogen bonding in
uniformly 13C,15N labeled carbohydrates in water. Biopolymers 2012,
97, 145−154. (e) Battistel, M. D.; Shangold, M.; Trinh, L.; Shiloach,
J.; Freedberg, D. I. Evidence for helical structure in a tetramer of α2−
8 sialic acid: unveiling a structural antigen. J. Am. Chem. Soc. 2012,
134, 10717−10720. (f) Zhu, T.; Yamaguchi, T.; Satoh, T.; Kato, K. A
Hybrid Strategy for the Preparation of 13C-labeled High-mannose-
type Oligosaccharides with Terminal Glucosylation for NMR Study.
Chem. Lett. 2015, 44, 1744−1746. (g) Huang, T.-Y.; Irene, D.;
Zulueta, M. M. L.; Tai, T.-J.; Lain, S.-H.; Cheng, C.-P.; Tsai, P.-X.;
Lin, S.-Y.; Chen, Z.-G.; Ku, C.-C.; Hsiao, C.-C.; Chyan, C.-L.; Hung,
S.-C. Structure of the Complex between a Heparan Sulfate
Octasaccharide and Mycobacterial Heparin-Binding Hemagglutinin.
Angew. Chem., Int. Ed. 2017, 56, 4192−4196.
(4) Olsson, U.; Serianni, A. S.; Stenutz, R. Conformational Analysis
of β-Glycosidic Linkages in 13C-Labeled Glucobiosides Using Inter-
residue Scalar Coupling Constants. J. Phys. Chem. B 2008, 112, 4447−
4453.
(5) Delbianco, M.; Kononov, A.; Poveda, A.; Yu, Y.; Diercks, T.;
Jimenez-Barbero, J.; Seeberger, P. H. Well-Defined Oligo- and
Polysaccharides as Ideal Probes for Structural Studies. J. Am. Chem.
Soc. 2018, 140, 5421−5426.
(6) Yamaguchi, Y.; Takizawa, T.; Kato, K.; Arata, Y.; Shimada, I. 1H
and 13C NMR assignments for the glycans in glycoproteins by using
2H/13C-labeled glucose as a metabolic precursor. J. Biomol. NMR
2000, 18, 357−360.
(7) Adachi, Y.; Ishii, T.; Ikeda, Y.; Hoshino, A.; Tamura, H.;
Aketagawa, J.; Tanaka, S.; Ohno, N. Characterization of beta-glucan
recognition site on C-type lectin, dectin 1. Infect. Immun. 2004, 72,
4159−4171.
(18) (a) Liu, K. K. C.; Danishefsky, S. J. A striking example of the
interfacing of glycal chemistry with enzymatically mediated sialylation:
a concise synthesis of ganglioside GM3. J. Am. Chem. Soc. 1993, 115,
4933−4934. (b) Gervay, J.; Peterson, J. M.; Oriyama, T.; Danishefsky,
S. J. An unexpected sialylation: total syntheses of ganglioside GM4
and a positional isomer. J. Org. Chem. 1993, 58, 5465−5468.
(c) Randolph, J. T.; Danishefsky, S. J. Application of the glycal
assembly strategy to the synthesis of a branched oligosaccharide: the
first synthesis of a complex saponin. J. Am. Chem. Soc. 1993, 115,
8473−8474. (d) Randolph, J. T.; McClure, K. F.; Danishefsky, S. J.
Major Simplifications in Oligosaccharide Syntheses Arising from a
Solid-Phase Based Method: An Application to the Synthesis of the
Lewis b Antigen. J. Am. Chem. Soc. 1995, 117, 5712−5719.
(e) Halcomb, R. L.; Danishefsky, S. J. Total syntheses of ML-236A
and compactin by combining the lactonic (silyl) enolate rearrange-
ment and aldehyde-diene cyclocondensation technologies. J. Am.
Chem. Soc. 1989, 111, 6661−6666. (f) Danishefsky, S. J.; McClure, K.
F.; Randolph, J. T.; Ruggeri, R. B. A strategy for the solid-phase
synthesis of oligosaccharides. Science 1993, 260, 1307−1309.
(19) Li, Y.; Tang, P.; Chen, Y.; Yu, B. Gold(I)-Catalyzed
Glycosidation of 1,2-Anhydrosugars. J. Org. Chem. 2008, 73, 4323−
4325.
(8) Okobira, T.; Miyoshi, K.; Uezu, K.; Sakurai, K.; Shinkai, S.
Molecular Dynamics Studies of Side Chain Effect on the β-1,3-d-
Glucan Triple Helix in Aqueous Solution. Biomacromolecules 2008, 9,
783−788.
(9) (a) Tanaka, H.; Kawai, T.; Adachi, Y.; Ohno, N.; Takahashi, T.
β(1,3) Branched heptadeca- and linear hexadeca-saccharides possess-
ing an aminoalkyl group as a strong ligand to dectin-1. Chem.
Commun. 2009, 46, 8249−8251. (b) Adamo, R.; Tontini, M.;
Brogioni, G.; Romano, M. R.; Costantini, G.; Danieli, E.; Proietti, D.;
Berti, F.; Costantino, P. Synthesis of Laminarin Fragments and
Evaluation of a β-(1,3) Glucan Hexasaccaride-CRM197 Conjugate as
Vaccine Candidate against Candida albicans. J. Carbohydr. Chem.
2011, 30, 249−280. (c) Tanaka, H.; Kawai, T.; Adachi, Y.;
Hanashima, S.; Yamaguchi, Y.; Ohno, N.; Takahashi, T. Synthesis
of β(1,3) oligoglucans exhibiting a Dectin-1 binding affinity and their
biological evaluation. Bioorg. Med. Chem. 2012, 20, 3898−3914.
(d) Liao, G.; Burgula, S.; Zhou, Z.; Guo, Z. A Convergent Synthesis
of 6-O-Branched β-Glucan Oligosaccharides. Eur. J. Org. Chem. 2015,
2015, 2942−2951. (e) Yashunsky, D. V.; Tsvetkov, Y. E.; Nifantiev,
N. E. Synthesis of 3-aminopropyl glycoside of branched β-(1 → 3)-d-
glucooctaoside. Carbohydr. Res. 2016, 436, 25−30. (f) Weishaupt, M.
W.; Hahm, H. S.; Geissner, A.; Seeberger, P. H. Automated glycan
assembly of branched β-(1,3)-glucans to identify antibody epitopes.
Chem. Commun. 2017, 53, 3591−3594. (g) Hamagami, H.; Adachi,
Y.; Ohno, N.; Tanaka, H. Convergent Synthesis of Linear and
Branched β(1,3)-Glucans and Evaluation of their Binding Affinities to
Dectin-1. Asian J. Org. Chem. 2019, 8, 411−416.
(20) Cloran, F.; Carmichael, I.; Serianni, A. S. Density Functional
Calculations on Disaccharide Mimics: Studies of Molecular Geo-
3
3
metries and Trans-O-glycosidic JCOCH and JCOCC Spin-Couplings. J.
Am. Chem. Soc. 1999, 121, 9843−9851.
(21) (a) Takeda, H.; Yasuoka, N.; Kasai, N. The crystal and
molecular structure of a 3:2 mixture of laminarabiose and O-α-d-
glucopyranosyl-(1→3)-β-d-glucopyranose. Carbohydr. Res. 1977, 53,
137−152. (b) Noguchi, K.; Okuyama, K.; Kitamura, S.; Takeo, K.
Crystal structure of methyl3-O-β-d-glucopyranosyl-β-d-glucopyrano-
side (methyl β-D-laminarabioside) monohydrate. Carbohydr. Res.
1992, 237, 33−43.
(10) (a) Kinnaert, C.; Daugaard, M.; Nami, F.; Clausen, M. H.
Chemical Synthesis of Oligosaccharides Related to the Cell Walls of
Plants and Algae. Chem. Rev. 2017, 117, 11337−11405. (b) Miyagawa,
A. Chemical Synthesis of β-(1,3)-Glucan Oligosaccharide and Its
Application. Trends Glycosci. Glycotechnol. 2018, 30, 91−101.
L
https://dx.doi.org/10.1021/acs.joc.0c01939
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
pubs.acs.org/joc
Article
(22) Aguilar, J. A.; Faulkner, S.; Nilsson, M.; Morris, G. A. Pure shift
1H NMR: a resolution of the resolution problem? Angew. Chem., Int.
Ed. 2010, 49, 3901−3903.
M
https://dx.doi.org/10.1021/acs.joc.0c01939
J. Org. Chem. XXXX, XXX, XXX−XXX