Total Synthesis of the Repeating Unit of Bacteroides fragilis Zwitterionic Polysaccharide A1

Article abstract of DOI:10.1021/acs.joc.0c02935

Zwitterionic polysaccharides isolated from commensal bacteria are endowed with unique immunological properties and are emerging as immunotherapeutic agents as well as vaccine carriers. Reported herein is a total synthesis of the repeating unit of Bacteroides fragilis zwitterionic polysaccharide A1 (PS A1). The structurally complex tetrasaccharide unit contains a rare sugar 2-acetamido-4-amino-2,4,6-trideoxy-d-galactose (AAT) and two consecutive 1,2-cis glycosidic linkages. The repeating unit was efficiently assembled by rapid synthesis of d-galactosamine and AAT building blocks from cheap and abundant d-mannose via a one-pot SN2 displacement of 2,4-bistriflates and installation of all of the glycosidic bonds in a highly stereoselective manner. The total synthesis involves a longest linear sequence of 17 steps with 3.47% overall yield.

Full text of DOI:10.1021/acs.joc.0c02935

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Article
Total Synthesis of the Repeating Unit of Bacteroides fragilis
Zwitterionic Polysaccharide A1
Ennus K. Pathan, Bhaswati Ghosh, Ananda Rao Podilapu, and Suvarn S. Kulkarni*
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ABSTRACT: Zwitterionic polysaccharides isolated from commensal bacteria are endowed with unique immunological properties
and are emerging as immunotherapeutic agents as well as vaccine carriers. Reported herein is a total synthesis of the repeating unit of
Bacteroides fragilis zwitterionic polysaccharide A1 (PS A1). The structurally complex tetrasaccharide unit contains a rare sugar 2-
acetamido-4-amino-2,4,6-trideoxy-D-galactose (AAT) and two consecutive 1,2-cis glycosidic linkages. The repeating unit was
efficiently assembled by rapid synthesis of D-galactosamine and AAT building blocks from cheap and abundant D-mannose via a one-
pot SN2 displacement of 2,4-bistriflates and installation of all of the glycosidic bonds in a highly stereoselective manner. The total
synthesis involves a longest linear sequence of 17 steps with 3.47% overall yield.
INTRODUCTION
■
Zwitterionic polysaccharides (ZPSs) are emerging as an
important class of immunotherapeutic agents as well as
potential carriers of carbohydrate-based vaccines.1,2 ZPSs
present on the surfaces of commensal bacteria possess a
unique ability to activate major histocompatibility complex
class II (MHC-II)-mediated T-cell-dependent immune re-
sponse in the absence of proteins.3 Over the past few years, a
variety of semisynthetic polysaccharide-derived ZPSs have
been shown to display potent immunostimulatory activity.4
The promising applications of ZPSs both as adjuvants and
immunostimulators have made them attractive synthetic
targets, and as a result, a few highly complex ZPSs have
Figure 1. Structure of the zwitterionic tetrasaccharide PS A1
repeating unit.
been already synthesized.5
The most well studied natural ZPS is polysaccharide A1 (PS
A1, Figure 1),6 which is found on the capsule of the
commensal bacterium Bacteroides fragilis. Numerous reports
have highlighted the potent immunostimulating properties of
PS A1. It displays anti-inflammatory properties and plays a key
role in the development and the maintenance of a balanced
mammalian immune system.7,8 It also stimulates IL-10
secretion, modulates surgical fibrosis,9 inhibits intestinal
inflammatory disease caused by Helicobacter hepaticus,10 and
protects against central nervous system (CNS) demyelinating
Received: December 14, 2020
Published: April 12, 2021
https://doi.org/10.1021/acs.joc.0c02935
© 2021 American Chemical Society
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Scheme 1. Retrosynthetic Analysis
disease. In addition to immunostimulating properties, PS A1
can also bind to MHC-II and elicit a strong T-cell-dependent
immune response. An entirely carbohydrate-based vaccine has
been developed by employing PS A1 in place of protein
carriers for conjugation with other glycans such as the tumor-
associated cancer antigens Tn11 and STn12 as well as the
repeating unit of Streptococcus dysgalactiae 2023 polysacchar-
ide.13 Given its biological importance, there is a great interest
in developing novel, efficient strategies for the construction of
the structurally well-defined repeating unit of PS A1 for
structure−activity relationship (SAR) studies.
The tetrasaccharide repeating unit of PS A1 (Figure 1)
consists of four monosaccharide units, viz., D-galactosamine,
pyruvilated D-galactose, D-galactofuranoside, and 2-acetamido-
4-amino-2,4,6-trideoxy-D-galactose (AAT). The positive charge
on amine and negative charge on the carboxylate group are
crucial for the activation of T-cell-dependent immune
response, and the complete structure has nearly 120 repeating
units.14
Synthesis of the fully protected tetrasaccharide repeating
unit of PS A1 was carried out by van der Marel and co-workers
in 2007.15 They assembled the trisaccharide using the iterative
glycosylation method in a one-pot manner in 62% yield.
However, difficulties were encountered in the final [3 + 1]
glycosylation using the AAT donor with a trisaccharide
acceptor, and under dehydrative glycosylation conditions
using Ph2SO and Tf2O, the fully protected tetrasaccharide
was obtained in 17% yield. Due to the low yield of this step,
global deprotection was not performed, and the authors
concluded that an alternative route is desired for the assembly
of PS A1. In 2011, Seeberger and co-workers systematically
studied the preference of installation of glycosidic bonds and
established a successful route to achieve the first total synthesis
of the PS A1 tetrasaccharide in the longest linear sequence of
20 steps with 1.79% overall yield.16 The route involved
assembly of tetrasaccharide starting from the nonreducing end
AAT to the reducing end pyruvilated galactopyranose unit.
The rare sugar AAT was synthesized through a de novo
approach starting from L-threonine via azidonitration of a
glycal intermediate as a key step. Although the elegant
synthetic route leaves very little scope for alteration, the low
selectivity observed in the key azidonitration step for the
procurement of the key rare sugar AAT building block (3.5:1
dr) calls for alternate strategies.17 Also, the stereoselectivity
observed in the glycosylation of the AAT donor to obtain the
key disaccharide (α/β = 5:1) and the yield of final
glycosylation (58%) needed further improvement. Indeed, in
their subsequent study, Seeberger’s group improved the
selectivity and efficacy of azidonitration (4:1 dr) and
glycosylations (α/β = 19:1, 77%) to synthesize a conjuga-
tion-ready thioether-linked tetrasaccharide unit of PS A1.18
Recently, Andreana and co-workers reported a total synthesis
of an alternative structure of the PS A1 repeating unit as a β-
OPMP glycoside with alternating charges on adjacent
monosaccharides, by keeping the pyruvilated galactose at the
nonreducing end, linked to AAT and the GalNAc unit at the
reducing end.19 They followed a similar strategy for making
AAT via azidonitration of a D-fucal derivative (3.5:1 dr). The
synthesis of the target molecule was achieved in the longest
linear sequence of 19 steps with 0.75% overall yield. Our
laboratory has developed an expedient protocol to access
orthogonally protected D-galactosamine and bacterial rare
sugar building blocks (AAT, Bac, Fuc) via a one-pot double-
displacement of D-mannose-derived 2,4-bistriflates.20−22 Using
this methodology, we also assembled the key AAT-α (1 → 4)-
GalN3 disaccharide unit, which was however deemed
unsuitable for the total synthesis of the PS A1 tetrasaccharide
due to nonorthogonality of the protecting groups.23 Partic-
ularly, the presence of base-sensitive OAc, OBz, and
phthalimide groups in the disaccharide precluded its utilization
for the construction of repeating unit for which we would
require their selective removal in the presence of others. Thus,
we designed an alternate protecting group scheme for this
purpose by replacing Bz with Bn and Ac with 2-
naphthylmethyl (Nap) protecting groups. Herein, we report
a total synthesis of the tetrasaccharide repeating unit of PS A1
in 17 linear steps with 3.47% overall yield and exclusive
selectivity.
RESULTS AND DISCUSSION
■
Our retrosynthetic strategy for the target molecule 1, along the
lines of Seeberger’s synthesis, is outlined in Scheme 1.
Accordingly, 1 can be synthesized from fully protected
tetrasaccharide 2 by global deprotection. Tetrasaccharide 2
can be obtained via coupling of the trisaccharide donor 3 with
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D-galactopyruvate acceptor 4. It was envisaged that a
nonparticipating azido group at C2 and a bulky TBDPS
group at the C6 position on the galacto-configured sugar
would guide the entry of the donor from the less hindered
bottom face and offer α-selectivity, which can be further
augmented by use of participating solvent such as ether. The
trisaccharide 3 can be obtained by coupling of disaccharide
acceptor 5 with trichloroacetimidate donor 6. The disaccharide
acceptor 5 in turn can be furnished by glycosylation of AAT
donor 7 with D-galactosamine 4-OH acceptor 8a or 8b under
orthogonal glycosylation conditions by changing the leaving
group in AAT 7 to imidate or halide. Building blocks 7,20 8a,23
and 8b can be conveniently synthesized from D-mannose via
one-pot sequential inversions of corresponding 2,4-bistriflates
as a key step.21 Galactofuranose donor 6 and acceptor 4 can be
obtained from cheaply available D-galactose. With these
considerations, we began the total synthesis of 1.
is essential to control the ambident nucleophilicity of the
nitrite nucleophile.27 However, in this case, the reaction
proceeded selectively even in the absence of the neighboring
C3-ester group.
Synthesis of D-galactofuranose donor 6 started from known
per-acetylated galactofuranose derivative 1128 (Scheme 3).
Scheme 3. Synthesis of D-Galactofuranose Donor 6
As shown in Scheme 2, for the synthesis of the rare
deoxyamino sugar AAT 7 and 4-OH galactosamine acceptor
Scheme 2. Synthesis of Rare AAT and D-Galactosamine
Building Blocks
Nucleophilic displacement of anomeric acetate in 11 with
thiophenol in the presence of BF3·Et2O furnished the
corresponding thioglycoside in 75% yield. At this stage, to
circumvent the possible orthoester formation, the acetates
were replaced by benzoate groups by removal of the acetate
́
groups under Zemplen conditions followed by treatment with
benzoyl chloride in CH2Cl2 to get perbenzoylated thioglyco-
side 12. Subsequent hydrolysis of 12 using N-bromosuccini-
mide (NBS) in tetrahydrofuran (THF)/H2O (3:1) afforded
the corresponding anomeric hemiacetal, which was treated
with trichloroacetonitrile and cesium carbonate in CH2Cl2 to
1
8a and 8b, we started from cheaply available D-mannose. The
AAT building block 724 and D-galactosamine derivative 8a23
were easily obtained starting from D-thiomannoside 925 by
following our established protocol via sequential one-pot
displacement of corresponding 2,4-bistriflates, in good overall
yields. Synthesis of the alternative 4-OH D-galactosamine
acceptor 8b having a 3-ONap group was also achieved from D-
mannose derivative 9. The primary hydroxyl group of 9 was
protected with TBDPSCl in pyridine, followed by selective 2-
naphthylmethylation of the C-3-hydroxyl group26 using dibutyl
tin oxide, tetrabutylamonnium bromide (TBAB), and 2-
naphthylmethylbromide (NapBr), affording the desired 2,4-
diol 10 in 67% yield. The diol 10 was subjected to triflation
using triflic anhydride and pyridine in CH2Cl2 to afford the
corresponding 2,4-bistriflate, which was as such treated with a
stoichiometric amount of TBAN3 in CH3CN at −30 °C to
displace the C2-O-triflate. After the completion of the reaction
as monitored by thin-layer chromatography (TLC), TBANO2
was added in the same pot to displace the C4-OTf to obtain
the D-galactose derivative 8b in 57% yield over three steps. It
should be noted that the Lattrell−Dax reaction using an
ambident nucleophile (nitrite) smoothly delivered the desired
C4-OH product by attack of the oxygen center. The
corresponding C4-NO2 product, resulting by the attack of
the nitrogen center was not observed. Earlier, it has been
proposed that the presence of a neighboring ester functionality
afford trichloroacetimidate donor 6. The H and 13C NMR
spectral data of 6 matched well with its reported data.19
Synthesis of the appropriately protected D-galactose unit 4 is
shown in Scheme 4. Regioselective protection of the 3-OH of
the easily accessible 2,3-diol 13 was carried out using cat.
dimethyltin dichloride and FmocCl to give the desired
compound in 84% yield, and the remaining 2-OH was
benzoylated to afford the fully protected D-galactose derivative
14 in good yield. Benzylidene deprotection of 14 afforded the
desired 4,6-diol, which was subsequently masked with methyl
pyruvate to give the corresponding pyruvate derivative 15. The
stereochemistry of the pyruvate center was confirmed from
nuclear Overhauser effect (NOE) interaction between the
equatorial H-4 of D-galactose and the methyl group of the ester
(see Supporting Information (SI)). Glycosylation of donor 15
with isopropanol using NIS/AgOTf as a promoter system
afforded the desired O-glycoside (71%), which upon
concomitant deprotection of the Fmoc group using triethyl-
amine furnished acceptor 4 in 80% yield.
After having all of the building blocks in our hand, first we
went ahead for the synthesis of disaccharide acceptor 5
(Scheme 5). AAT donor 7 was converted to its corresponding
bromide using Br2 in CH2Cl2 to afford glycosyl bromide, which
was further coupled with acceptor 8a using AgOTf as a
promoter to obtain disaccharide 16a (75%), with complete α-
selectivity. The characteristic NMR signals [1H NMR δ = 5.09
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axial orientation, which may hinder the top face attack or
completely block it by neighboring group participation,18 and
(iii) the possible formation of a more reactive β-glycosyl triflate
using AgOTf and its concomitant SN2-type displacement by
the nucleophilic acceptor 8a. Removal of the acetate group in
disaccharide 16a turned out be a difficult task. We tried several
conditions to cleave the O-acetate group, none of which gave
satisfactory results (Scheme 5). For instance, use of acetyl
chloride and methanol resulted in simultaneous loss of acetate
and TBDPS groups. A similar result was obtained when we
used potassium carbonate and methanol conditions. Alter-
Scheme 4. Synthesis of Pyruvilated D-Galactose 3-OH
Acceptor 4
́
natively, Zemplen deacetylation conditions cleaved the acetate
and phthalimide groups. Likewise, the reaction with guanidium
nitrate29 was not clean, as indicated by multiple spots on TLC.
Use of a milder condition with triethylamine and methanol at
50 °C after 2 days gave only 50% product 5 and 20% recovered
starting material. The reaction did not go to completion even
after keeping it for a longer time. As the deacetylation of
compound 16a did not give satisfactory yield, we decided to
use the alternate building block 8b (Scheme 2) having a 2-
naphthylmethyl (Nap) ether protecting group at O3 in place of
Ac.
Accordingly, as shown in Scheme 6, AAT donor 7 was
treated with Br2 in CH2Cl2 to generate the corresponding
glycosyl bromide, which was further coupled with acceptor 8b
using AgOTf as a promoter to afford disaccharide 16b (78%),
with complete α-selectivity. The characteristic NMR signals
[1H NMR δ = 5.25 ppm (d, 1H, J = 3.5 Hz, H-1), 13C NMR δ
= 99.2 ppm] confirmed the α-linkage of the newly formed
glycosidic bond in 16b. Removal of the Nap group from
ppm (d, 1H, J = 3.6 Hz, H-1), 13C NMR δ = 98.9 ppm]
confirmed the α-linkage of the newly formed glycosidic bond
in 16a. The observed α-stereoselectivity in this case can be
perhaps attributed to (i) the presence of the nonparticipating
azido group at the C2 position of the glycosyl donor, (ii) the
presence of the bulky phthalimide group at the C4 position in
Scheme 5. Synthesis of Disaccharide Acceptor 5
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Scheme 6. Assembly of PS A1 Tetrasaccharide and Global Deprotection
disaccharide 16 using DDQ, CH2Cl2/H2O (9:1) afforded the
disaccharide acceptor 5 in 70% yield. At this stage, the D-Galf
imidate 6 (perbenzoylated trichloroacetimidate) was glycosy-
lated with acceptor 5 in the presence of the TMSOTf
promoter at −30 °C to furnish trisaccharide 3 in 82% yield.
The characteristic NMR signals (1H NMR δ = 5.72 ppm, 13C
NMR δ = 105.7 ppm) confirmed the β-linkage of the newly
formed glycosidic bond in 3. Glycosylation of trisaccharide
donor 3 with acceptor 4 in the presence of the NIS/TMSOTf
promoter and ether as a participating cosolvent afforded the
desired tetrasaccharide 2 in good yield (72%), exclusively. The
characteristic NMR signals (1H NMR δ = 5.64 ppm, 13C NMR
δ = 92.1 ppm) confirmed the α-linkage of the newly formed
glycosidic bond in 2. All of the synthetic intermediates were
thoroughly characterized by two-dimensional (2D) NMR
spectroscopy (see SI).
Global deprotection of tetrasaccharide was done in five
steps. First, conversion of azides to NHAc using AcSH in
pyridine (74% yield), followed by TBDPS deprotection using
TBAF/AcOH, removal of the phthalimide group using
ethylenediamine (EDA)/n-BuOH, removal of benzyl groups
using H2/Pd/C in methanol, and finally cleavage of esters
using NaOMe, in MeOH/THF/H2O (1:1:1) furnished
tetrasaccharide 1 in good overall yield. The final purification
was done using high-performance liquid chromatography
(HPLC) on a prep-C18 column using a 10% methanol/H2O
eluent system to obtain the zwitterionic tetrasaccharide 1. The
1H NMR data of compound 1 matched well with the data
reported by Seeberger and co-workers.16 The compound also
gave satisfactory 13C NMR and high-resolution mass
spectrometry (HRMS) data (see SI).
9 in 17 linear steps with 3.47% overall yield. Efforts are
underway to construct larger fragments of PS A1.
EXPERIMENTAL PROCEDURES
■
General Methods. All reactions were conducted under a dry
nitrogen atmosphere. Solvents (CH2Cl2 > 99%, THF 99.5%,
acetonitrile 99.8%, dimethylformamide (DMF) 99.5%) were
purchased in capped bottles and dried under sodium or CaH2. All
other solvents and reagents were used without further purification. All
glassware used was oven-dried before use. All reactions that required
elevated temperatures were carried out under traditional oil bath
heating. TLC was performed on precoated aluminum plates of silica
gel 60 F254 (0.25 mm, E. Merck). Developed TLC plates were
visualized under a short-wave UV lamp and by heating plates that
were dipped in ammonium molybdate/cerium(IV) sulfate solution.
Silica gel column chromatography was performed using silica gel
(100−200 mesh) and employed a solvent polarity correlated with
TLC mobility. NMR experiments were conducted on 600, 500, and
400 MHz instruments using CDCl3 (D, 99.8%) and D2O (D, 99.9%)
as solvents. Chemical shifts are relative to the deuterated solvent
peaks and are in parts per million (ppm). Structural assignments were
made with additional information from gCOSY, gHSQC, and
gHMBC experiments. Mass spectra were acquired in the SI mode
using a Q-TOF analyzer. Specific rotation experiments were carried
out at 589 nm (Na) and 25 °C.
Phenyl 6-O-tert-Butyldiphenylsilyl-3-O-(2-naphthylmethyl)-1-
thio-β-D-mannopyanoside (10). tert-Butyldiphenylsilyl chloride
(2.86 mL, 11.02 mmol) was added to a stirred solution of tetraol 9
(1.5 g, 5.51 mmol) in pyridine (18.6 mL). After 12 h, the reaction
mixture was concentrated, and the residue was dissolved in CHCl3
and washed with 1 N HCl and water sequentially. The organic layer
was dried over Na2SO4 and concentrated in vacuo, and the crude
product was purified by silica gel column chromatography (40:60 to
80:20 ethyl acetate/petroleum ether) to yield C6 OTBDPS triol (2.2
g, 94% yield) as a white foam. Triol (2.0 g, 3.92 mmol) was dissolved
in toluene (28 mL), and to this clear solution, Bu2SnO (1.17 g, 4.70
mmol) was added, and the reaction mixture was kept for stirring at
110 °C for 8 h. After complete consumption of the starting material,
the solvent was removed under reduced pressure and the crude
product was dried under high vacuum. The crude product was
dissolved in toluene (12 mL), then TBAB (1.89 g, 5.88 mmol) and 2-
naphthylmethylbromide (1.30 g, 5.88 mmol) were added, and the
solution was stirred at 60 °C for 12 h. After conversion of the starting
material into product, the reaction mixture was diluted with EtOAc
and washed with brine. The separated organic layer was dried over
CONCLUSIONS
■
In conclusion, an expedient access to rare sugar AAT and D-
galactosamine building blocks from D-mannose via a one-pot
SN2 displacement of 2,4-bistriflates enabled an efficient total
synthesis of PS A1 tetrasaccharide repeating unit 1. In this
synthesis, all of the glycosylations proceeded in high
stereoselectivity and yields. The repeating unit was efficiently
assembled from known and easily available β-D-thiomannoside
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Na2SO4, concentrated, and purified by column chromatography
(30:70 ethyl acetate/petroleum ether) to give 10 (1.69 g, 67% over
two steps) as a pale yellow viscous liquid. [α]D = −13.15 (c = 2.3,
89.5, 82.1, 80.4, 78.9, 76.2, 75.1, 73.2, 70.1, 69.1, 68.6, 67.3, 67.2,
66.3, 62.3, 61.9, 61.1, 20.8 (COCH3), 20.6 (COCH3), 20.5
(COCH3), 20.5 (COCH3), 20.4 (COCH3), 20.3 (COCH3); HR-
ESI-MS (m/z): [M + Na]+ calcd. for C16H22NaO11 413.1061; found,
413.1054.
25
CHCl3); IR (cm−1, CHCl3) 3455, 3050, 2959, 2929, 2857, 1112,
1067, 951, 858, 822, 758, 741, 705, 690, 635, 615, 504; 1H NMR (500
MHz, CDCl3): δ 7.87−7.81 (m, 4H, ArH), 7.72−7.70 (m, 3H, ArH),
7.52−7.49 (m, 5H, ArH), 7.43−7.35 (m, 7H, ArH), 7.26−7.21 (m,
3H, ArH), 4.95 (d, J = 13.4 Hz, 1H, CHHNap 1H), 4.85 (d, J = 14.5
Hz, 2H, H-1, CHHNap), 4.30 (s, 1H, H-2), 4.01 (t, J = 10 Hz, 1H, H-
4), 3.96−3.94 (m, 2H, H-6a & H-6b), 3.51 (dd, J = 7.2, 3.0 Hz, 1H,
H-3), 3.45−3.43 (m, 1H, H-5), 2.82 (bs, 1H, OH), 2.59 (bs, 1H,
OH), 1.07 (s, 9H), ((CH3)3); 13C{1H} NMR (125 MHz, CDCl3): δ
135.7 (ArC), 135.6 (ArC), 135.1 (ArC), 134.9 (ArC), 133.2 (ArC),
133.2 (ArC), 133.0 (ArC), 132.8 (ArC), 130.6 (ArC), 129.8 (ArC),
129.8 (ArC), 129.0 (ArC), 128.7 (ArC), 128.0 (ArC), 127.8 (ArC),
127.2 (ArC), 127.0 (ArC), 126.4 (ArC), 126.3 (ArC), 125.7 (ArC),
86.9 (C-1), 81.7, 79.4, 72.0, 69.7, 68.3, 64.9, 26.8 (TBDPS CH3), 19.2
(TBDPS quaternary C); HR-ESI-MS (m/z): [M + Na]+ calcd. for
C39H42O5NaSSi 673.2413; found, 673.2414.
Phenyl 2,3,5,6-tetra-O-Benzoyl-1-thio-α,β-D-galactofuranoside
(12). BF3·Et2O (2.6 mL, 20.5 mmol) was added dropwise to the
stirred solution of compound 11 (4.0 g, 10.25 mmol) in CH2Cl2 and
thiophenol (2.1 mL, 20.5 mmol) at 0 °C, and the reaction was
continued for 12 h at rt. After 12 h, the reaction mixture was diluted
with sat. NaHCO3 and the separated organic layer was washed with
brine, dried over Na2SO4, and concentrated. The crude product was
purified by column chromatography on silica gel (20:80 ethyl acetate/
petroleum) to obtain per-O-acetylated thiogalactofuranoside (3.4 g,
75%) as a colorless liquid. NaOMe (0.2 M) was added to a stirred
solution of per-O-acetylated thiogalactofuranoside (2.1 g, 4.77 mmol)
in MeOH (21 mL). After 1 h, the reaction mixture was neutralized
with Amberlite-IR 120 (H+) resin. The reaction mixture was filtered
and concentrated under reduced pressure to afford the desired tetraol
as a white foam. Benzoyl chloride (7.7 mL, 66.08 mL) was added to
the stirred solution of tetraol (1.5 g, 5.50 mmol) in CH2Cl2 (15 mL)
and pyridine (5.32 mL, 66.08 mmol) at 0 °C. Next, the reaction
mixture was diluted with EtOAc and washed with 1 N HCl and brine.
The separated organic layer was dried over Na2SO4, concentrated in
vacuo, and purified by column chromatography on silica gel (25:75
ethyl acetate/petroleum) to afford the desired compound 12 (2.7 g,
Phenyl 2-Azido-6-O-tert-butyldiphenylsilyl-2-deoxy-3-O-(2-
naphthylmethyl)-1-thio-β-D-galactopyranoside (8b). Pyridine
(2.01 mL, 24.96 mmol) and trifluoromethanesulfonic anhydride
(1.96 mL, 11.52 mmol) were added sequentially at 0 °C to a stirred
solution of compound 10 (1.25 g, 1.92 mmol) in CH2Cl2 (25 mL).
After 30 min, the reaction mixture was diluted with CH2Cl2 and
washed successively with 1 N HCl, aq NaHCO3, and brine. The
separated organic layer was dried over Na2SO4 and concentrated
under reduced pressure. The crude product was dissolved in
acetonitrile (26 mL); to this, TBAN3 (0.49 g, 1.72 mmol) was
added at −30 °C, and the reaction was stirred at the same
temperature for 16 h. After 16 h, TBANO2 (1. 27 g, 4.41 mmol)
was added and the reaction mixture was stirred at rt for 6 h. The
reaction mixture was diluted with EtOAc and washed with brine. The
separated organic layer was dried over Na2SO4 and concentrated in
vacuo. The crude product was purified by column chromatography on
silica gel (15:85 ethyl acetate/petroleum ether) to obtain 8b as a pale
yellow viscous liquid (0.74 g, 57%, over three steps). [α]D25 = −24.66
(c = 0.56, CHCl3); IR (cm−1, CHCl3) 3468, 3064, 2929, 2854, 2113,
25
72%, over two steps) as a white foam. [α]D = −1.788 (c = 0.56,
CHCl3); IR (cm−1, CHCl3) 3064, 2924, 2857, 1728, 1601, 1586,
1432, 1266, 1178, 1109, 1069, 1027, 757, 687; 1H NMR (CDCl3, 500
MHz): δ 8.09−7.88 (m, 5H, ArH), 7.59−7.43 (m, 10H, ArH), 7.38−
7.27 (m, 10H, ArH), 6.12−6.09 (q, J = 2.8 Hz, 1H, H-5), 5.84 (s, 1H,
H-3), 5.72−5.70 (d, J = 4.8 Hz, 1H, H-1), 5.67 (s, 1H, H-2), 4.96−
4.94 (t, J = 4.4 Hz, 1H, H-4), 4.77−4.69 (m, 2H, H-6); 13C{1H}
NMR (CDCl3, 125 MHz): δ 166.1 (COPh), 165.7 (COPh), 165.5
(COPh), 165.4 (COPh), 133.7 (ArC), 133.5 (ArC), 133.3 (ArC),
133.1 (ArC), 132.1 (ArC), 132.5 (ArC), 130.1 (ArC), 130.0 (ArC),
129.9 (ArC), 129.8 (ArC), 129.59 (ArC), 129.50 (ArC), 129.1 (ArC),
128.9 (ArC), 128.6 (ArC), 128.5 (ArC), 128.4 (ArC), 128.0 (ArC),
91.4 (C-1), 82.4, 81.6, 77.9, 70.3, 63.4; HR-ESI-MS (m/z): [M +
Na]+ calcd. for C40H32NaO9S, 711.1659; found, 711.1659.
1
1590, 1474, 1366, 1282, 1112, 822, 745, 703, 670, 505; H NMR
(400 MHz, CDCl3): δ 7.87−7.81 (m, 4H, ArH), 7.72−7.66 (m, 5H,
ArH), 7.60−7.58 (m, 2H, ArH), 7.54−7.48 (m, 3H, ArH), 7.43−7.26
(m, 8H, ArH), 4.92−4.85 (m, 2H, CH2Nap), 4.37 (d, J = 10.0 Hz,
1H, H-1), 4.12 (s, 1H, H-4), 3.99−3.89 (m, 2H, H-6a & H-6b), 3.75
(t, J = 9.6 Hz, 1H, H-2), 3.43−3.41 (m, 1H, H-5), 3.40−3.38 (m, 1H,
H-3), 2.77 (bs, 1H, OH), 1.05 (s, 9H, (CH3)3); 13C{1H} NMR (100
MHz, CDCl3): δ 135.7 (ArC), 135.6 (ArC), 134.6 (ArC), 133.2
(ArC), 133.0 (ArC), 132.9 (ArC), 132.8 (ArC), 131.8 (ArC), 129.9
(ArC), 129.0 (ArC), 128.6 (ArC), 128.1 (ArC), 128.0 (ArC), 127.8
(ArC), 127.8 (ArC), 127.0 (ArC), 126.3 (ArC), 126.2 (ArC), 125.8
(ArC), 86.4 (C-1), 81.0, 78.0, 72.0, 66.0, 68.4, 63.7, 61.0, 26.8
(TBDPS CH3), 19.2 (TBDPS quaternary C); HR-ESI-MS (m/z): [M
+ Na]+ calcd. for C39H41N3O4NaSSi 698.2477; found, 698.2479.
1,2,3,5,6-Penta-O-acetyl-D-galactofuranose (11). Compound 11
was prepared from per-O-tert-butyldimethylsilylgalactofuranoside (7.0
g, 9.31 mmol) using acetic anhydride (45 mL, 475.0 mmol) and p-
TsOH (35.4 g, 186.3 mmol) in CH2Cl2 (94 mL). After complete
consumption of the starting material (72 h, monitored by TLC), the
reaction mixture was diluted with CH2Cl2 and washed with aq
NaHCO3 and brine. The separated organic layer was dried over
Na2SO4 and concentrated in vacuo. The crude product was purified by
column chromatography on silica gel (15:85 ethyl acetate/pet ether)
to obtain per-O-acetylated galactofuranoside as an anomeric mixture
(α/β 1:5) as a colorless liquid (35:65 ethyl acetate/petroleum ether,
2,3,5,6-tetra-O-Benzoyl-α,β-D-galactofuranoside-N-trichloroace-
timidate (6). NBS (0.21 g, 1.16 mmol) was added to the stirred
solution of compound 12 (0.4 g, 0.58 mmol) in THF/H2O (3:1).
After 1 h, the reaction mixture was diluted with EtOAc and washed
with Na2S2O3 and brine. The organic layer was dried over Na2SO4
and evaporated under reduced pressure. The crude hemiacetal was
dissolved in CH2Cl2 (13 mL). CCl3CN (0.14 mL, 1.74 mmol) and
Cs2CO3 (0.57 g 1.74 mmol) were added to it at 0 °C. The mixture
was stirred for 12 h at rt. The reaction mixture was diluted with
CH2Cl2, filtered over celite, and concentrated in vacuo. The crude
compound was purified by silica gel column chromatography (20:80
ethyl acetate/petroleum) to obtain compound 6 (0.3 g, 70%, over two
25
steps). [α]D = +25.02 (c = 0.79, CHCl3); IR (cm−1, CHCl3) 3443,
3062, 3021, 2924, 2854, 1727, 1602, 1585, 1452, 1316, 1267, 1217,
1
1178, 1111, 1070, 1027, 758, 711, 686, 668; H NMR (CDCl3, 400
MHz): δ 8.74 (s, 1H, NH imidate), 8.11−7.88 (m, 7H, ArH), 7.61−
7.25 (m, 13H, ArH), 6.71 (s, 1H, H-1), 6.15−6.14 (m, 1H, H-5), 5.80
(d, J = 4 Hz, 1H, H-3), 5.77 (s, 1H, H-2), 4.88 (t, J = 4 Hz, 1H, H-4),
4.79−4.77 (m, 2H, H-6); 13C{1H} NMR (CDCl3, 100 MHz): δ 166.0
(COPh), 165.7 (COPh), 165.5 (COPh), 165.1 (COPh), 160.2
(COPh), 133.7 (ArC), 133.6 (ArC), 133.3 (ArC), 133.1 (ArC), 130.0
(ArC), 130.0 (ArC), 129.9 (ArC), 129.7 (ArC), 128.5 (ArC), 128.4
(ArC), 128.3 (ArC), 102.9 (C-1), 90.9, 84.6, 80.7, 77.0, 70.1, 63.4;
HR-ESI-MS (m/z): [M + K]+ calcd. for C36H28Cl3KNO10 778.0515;
found, 778.0410.
Phenyl 2-O-Benzoyl-4,6-O-benzylidene-3-O-fluorenylmethoxy-
carbonyl-1-thio-β-D-galactopyranoside (14). Diol 13 (1.3 g, 3.61
mmol) was dissolved in THF (30 mL). After complete dissolution of
compound 13, N,N-diisopropylethylamine (DIPEA; 1.25 mL, 7.22
mmol), dimethyltin dichloride (0.04 g 0.18 mmol), and FmocCl (1.02
25
2.5 g, 64%). [α]D = +6.637 (c = 1.8, CHCl3); IR (cm−1, CHCl3)
2942, 2441, 2110, 1748, 1435, 1372, 1235, 1049, 965, 883, 820, 757,
604, 520; 1H NMR (CDCl3, 400 MHz): δ 6.25−6.06 (m, 2H), 5.44−
4.95 (m, 6H), 4.26−3.98 (m, 6H), 2.05−1.95 (m, 30H, carbonyl
CH3); 13C{1H} NMR (CDCl3, 100 MHz): δ 170.3 (COCH3), 170.2
(COCH3), 169.9 (COCH3), 169.8 (COCH3), 169.7 (COCH3), 169.3
(COCH3), 169.1 (COCH3), 168.9 (COCH3), 98.9 (C-1), 92.9 (C-1),
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g, 3.97 mmol) were added sequentially at rt and stirred for 1 h. The
reaction mixture was diluted with ethyl acetate and washed with 1 N
HCl and brine, and the organic layer was dried over Na2SO4 and
concentrated under reduced pressure. The crude product was purified
by silica gel column chromatography (20:80 to 40:60 ethyl acetate/
petroleum) to give the C3-Fmoc-protected compound as a white
foam (1.76 g, 84% yield).
mmol) in CH2Cl2 (4 mL), and the flask was cooled to −40 °C. To
this solution, NIS (0.151 g, 0.673 mmol) and AgOTf (0.017 g, 0.067
mmol) were added sequentially. The solution was slowly warmed to rt
over 2 h. The solution was diluted with EtOAc (50 mL), washed with
aq NaHCO3 (3 × 10 mL), dried over Na2SO4, and concentrated in
vacuo. The resulting crude compound was purified by silica gel
chromatography (40:60, ethyl acetate/pet ether) to give the linker
coupled product (0.151 g, 71%) as a yellow foam. The linker coupled
product (0.151 g, 0.238 mmol) was dissolved in CH2Cl2 (1.6 mL)
and treated with NEt3 (1.5 mL, 10.43 mmol) at rt. After the solution
was stirred for 2 h, it was concentrated under reduced pressure to give
a crude oil. The crude oil was purified by silica gel column
chromatography (60:40 ethyl acetate/petroleum ether) to yield
The above obtained compound (1.2 g, 2.06 mmol) was dissolved in
pyridine (0.49 mL, 6.09 mmol) and CH2Cl2 (12 mL) and cooled to 0
°C. To this solution, BzCl (0.48 mL, 4.12 mmol) was added dropwise,
and the reaction was stirred for 2 h at rt. The reaction mixture was
diluted with CH2Cl2 and washed with 1 N HCl and brine. The
organic portion was concentrated under reduced pressure. The crude
product was purified by silica gel chromatography (20:80 to 40:60
ethyl acetate/petroleum) to yield compound 14 as a white foam (0.98
g, 70%). [α]D25 = −19.05 (c = 0.86, CHCl3); IR (cm−1, CHCl3) 3020,
2925, 2857, 1723, 1451, 1403, 1366, 1268, 1216, 1124, 1099, 815,
25
alcohol 4 as a white foam (0.078 g, 80%). [α]D = −26.10 (c =
0.58, CHCl3); IR (cm−1, CHCl3) 3456, 3018, 2926, 2857, 1732, 1452,
1
1373, 1270, 1216, 1122, 1086, 982, 758, 711, 668; H NMR (400
MHz, CDCl3): δ 8.06−8.04 (m, 2H, ArH), 7.58−7.54 (m, 1H, ArH),
7.46−7.42 (m, 2H), 5.30 (t, J = 8 Hz, 1H, H-2), 4.59 (d, J = 8 Hz,
1H, H-1), 4.17 (d, J = 13.2 Hz, 1H, H-4), 4.12−4.02 (m, 2H, H-6),
3.95 (m, 1H, CH isopropanol), 3.83 (s, 3H, Pyr. CO2CH3), 3.82−
3.79 (m, 1H, H-3), 3.42 (s, 1H, H-5), 2.70 (d, J = 10.4 Hz, 1H, OH),
1.63 (s, 3H, CH3), 1.21 (d, J = 6.2 Hz, 3H, CH3), 1.07 (d, J = 6.2 Hz,
3H, CH3); 13C{1H} NMR (100 MHz, CDCl3): δ 170.4 (CO2Me),
166.4 (COPh), 133.1 (ArC), 130.3 (ArC), 129.9 (ArC), 128.4 (ArC),
99.6 (C-1), 98.9 (pyruvate quaternary C), 73.0, 72.5, 71.9, 71.4, 65.9,
65.3, 52.9 (OCH3), 25.9 (pyruvate CH3), 23.4 (isopropanol C), 22.1
(isopropanol C); HR-ESI-MS (m/z): [M + Na]+ calcd. for
C20H26NaO9, 433.1465; found, 433.1469.
1
758, 710; H NMR (500 MHz, CDCl3): δ 8.15−8.14 (d, J = 8.1 Hz,
2H, ArH), 7.73−7.68 (m, 4H, ArH), 7.59−7.44 (m, 10 H, ArH),
7.44−7.05 (m, 7H, ArH), 5.80 (d, J = 10.0 Hz, 1H, H-3), 5.56 (s, 1H,
CHPh), 5.1 (dd, J = 3 Hz, 1H, H-3), 4.97 (d, J = 10 Hz, 1H, H-1),
4.54 (d, J = 2.5 Hz, 1H, H-4), 4.44 (d, J = 12.2 Hz 1H, −CH Fmoc),
4.31−4.29 (m, 2H, CH2 Fmoc), 3.64 (s, 1H, H-5), 4.14 (dd, 2H, H-
6a, J = 12.5 Hz, 8 Hz, H-6b); 13C{1H} NMR (126 MHz, CDCl3):
164.8 (COPh), 154.4 (Fmoc CO), 143.1 (ArC), 143.0 (ArC), 141.1
(ArC), 137.5 (ArC), 133.7 (ArC), 133.3 (ArC), 131.3 (ArC), 129.9
(ArC), 129.6 (ArC), 129.1 (ArC), 128.8 (ArC), 128.5 (ArC), 128.3
(ArC), 128.2 (ArC), 128.2 (ArC), 127.8 (ArC), 127.8 (ArC), 127.1
(ArC), 126.5 (ArC), 125.1 (ArC), 125.1 (ArC), 119.9 (ArC), 101.0
(CHPh), 85.2, 76.8, 73.3, 70.3, 69.3, 69.0, 67.4, 46.4 (Fmoc CH);
HR-ESI-MS (m/z): [M + Na]+ calcd. for C41H34NaO8S, 709.1866;
found 709.1867.
Phenyl 2-Azido-3-O-benzyl-2,4,6-trideoxy-4-phthalimido-α-D-
galactopyranosyl-(1 → 4)-3-O-acetyl-2-azido-2-deoxy-6-O-tert-bu-
tyldiphenylsilyl-1-thio-β-D-galactopyranoside (16a). Bromine (22
μL, 0.44 mmol) was added to a solution of 7 (0.10 g, 0.19 mmol) in
CH2Cl2 (3 mL) at 0 °C, and after 30 min stirring, the mixture was
concentrated and the residue was coevaporated twice with toluene. A
solution of acceptor 8a (55 mg, 0.0951 mmol) in CH2Cl2 (1.3 mL)
was added to a suspension of glycosyl bromide, 3 Å MS (0.25 g), and
sym. collidine (22 μL, 0.18 mmol) in CH2Cl2 (1.3 mL) and kept
stirring at −30 °C for 30 min. Then, silver triflate (0.10 g, 0.38 mmol)
was added and stirring was continued at the same temperature. After 2
h, triethylamine was added and the reaction mixture was diluted with
CH2Cl2, filtered through celite, and concentrated. The residue was
purified by column chromatography on silica gel (20:80 ethyl acetate/
petroleum ether) to obtain the desired product 16a as a foam (69 mg,
Phenyl 2-O-Benzoyl-3-O-fluorenylmethoxycarbonyl-4,6-O-[1-
(R)-(methoxycarbonyl)ethylidene]-1-thio-β-D-galactopyranoside
(15). p-TsOH (0.114 g, 0.66 mmol) was added to the stirred solution
of compound 14 (0.91 g, 1.32) in MeOH (7.9 mL) and CH2Cl2 (3
mL), and the reaction mixture was stirred for 5 h at 40 °C. The
reaction was quenched by addition of pyridine (1 mL), and the
solution was concentrated under reduced pressure. The crude product
was purified by silica gel column chromatography with (30:70 to
50:50 ethyl acetate/pet ether) to give 4,6 diol (0.65 g, 82% yield).
The 4,6 diol (0.87 g, 1.45 mmol) was dissolved in acetonitrile (20
mL). The resulting suspension at 0 °C was treated with methyl
pyruvate (0.26 mL, 2.91 mmol) and BF3·Et2O (0.36 mL, 2.91 mmol).
After stirring for 3 h, the reaction was quenched with pyridine (1 mL).
The solution was diluted with EtOAc, washed with aq NaHCO3,
dried over Na2SO4, and concentrated under reduced pressure. The
resulting crude material was purified with silica gel column
25
75%): [α]D +121.21 (c = 0.05, CHCl3); IR (cm−1, CHCl3) 2900,
2830, 2109, 1734, 1320, 1250, 1218, 1106, 1065, 1002, 950, 745, 710,
1
610, 543; H NMR (400 MHz, CDCl3): δ 7.86−7.84 (m, 2H, ArH),
7.76−7.66 (m, 9H, ArH), 7.47−7.42 (m, 7H, ArH), 7.29−7.20 (m,
6H, ArH), 5.09 (d, J = 3.6 Hz, 1H, H-1′), 4.89−4.84 (m, 2H, H-3, H-
4′), 4.85 (d, J = 2.4 Hz, 1H, H-3), 4.62−4.52 (m, 3H, H-2′ &
CH2Bn), 4.45 (d, J = 9.8, 1H, H-1), 4.31 (dd, J = 4 Hz, 1H, H-5′),
4.25 (d, J = 2 Hz, 1H, H-4), 4.15 (dq, J = 7.6 Hz, 1H, H-6b), 4.02−
3.97 (m, 2H, H-3′ & H6a), 3.68−3.63 (m, 2H, H-2 & H-5), 2.13 (s,
3H, CH3), 1.1 (s, 9H, (CH3)3CSi), 1.02 (d, J = 6.4 Hz, 3H, CH3);
13C{1H} NMR (100 MHz, CDCl3): δ 170.0 (COCH3), 137.1 (ArC),
chromatography (20:80 to 30:70 ethyl acetate/petroleum) to yield
25
pyruvilated galactose 15 as a white foam (0.66 g, 66%). [α]D
=
+6.329 (c = 0.43, CHCl3); IR (cm−1, CHCl3) 2928, 2864, 1744, 1647,
1449, 1274, 1115, 10895, 1026, 824, 758, 712; H NMR (400 MHz,
1
CDCl3): δ 8.10−8.09 (m, 2H, ArH), 7.22−7.70 (m, 2H, ArH), 7.62−
7.43 (m, 8H, ArH), 7.36−7.11 (m, 6H, ArH), 5.72 (t, J = 10 Hz, 1H,
H-2), 5.02 (dd, J = 10.3, 3.6 Hz, 1H, H-3), 4.91 (d, J = 10.0 Hz, 1H,
H-1), 4.57 (d, J = 2.8 Hz, 1H, H-4), 4.34−4.29 (m, 1H, CH Fmoc),
4.26−4.24 (m, 1H, H-6a), 4.22−4.17 (m, 2H, CH2 Fmoc), 4.01 (d, J
= 12.1 Hz, 1H, H-6b), 3.63 (s, 3H, Pyr. CO2CH3), 3.5 (s, 1H, H-5),
1.6 (s, 3H, Pyr. CH3); 13C{1H} NMR (100 MHz, CDCl3): δ 170.0
(CO2Me), 164.9 (COPh), 154.2 (Fmoc CO), 143.2 (ArC), 143.1
(ArC), 141.1 (ArC), 141.1 (ArC), 133.6 (ArC), 133.3 (ArC), 131.5
(ArC), 129.9 (ArC), 129.5 (ArC), 128.8 (ArC), 128.5 (ArC), 128.3
(ArC), 127.8 (ArC), 127.8 (ArC), 127.1 (ArC), 125.3 (ArC), 125.2
(ArC), 119.9 (ArC), 98.6 (pyruvate quaternary C), 85.5 (C-1), 70.5,
68.8, 68.8, 67.1, 65.3, 52.5 (OCH3), 46.3 (CH of Fmoc), 25.6
(pyruvate CH3); HR-ESI-MS (m/z): [M + Na]+ calcd. for
C38H34NaO10S, 705.1764; found, 705.1765.
135.8 (ArC), 135.7 (ArC), 134.3 (ArC), 134.1 (ArC), 133.4 (ArC),
133.2 (ArC), 133.0 (ArC), 130.9 (ArC), 130.1 (ArC), 130.0 (ArC),
129.1 (ArC), 128.9 (ArC), 128.5 (ArC), 128.4 (ArC), 128.3 (ArC),
128.2 (ArC), 128.0 (ArC), 128.0 (ArC), 127.9 (ArC), 127.9 (ArC),
123.6 (ArC), 98.9 (C-1), 85.7 (C-1′), 79.3, 75.0, 74.8, 73.3, 72.0,
64.0, 61.7, 60.8, 59.7, 51.6, 27.0 (TBDPS CH3), 21.2 (COCH3), 19.3
(quaternary C of TBDPS), 17.0 (CH3 of AAT); HR-ESI-MS (m/z):
[M + Na]+ calcd. for C51H53N7O9NaSSi, 990.3287; found, 990.3291.
Phenyl 2-Azido-3-O-benzyl-2,4,6-trideoxy-4-phthalimido-α-D-
galactopyranosyl-(1 → 4)-2-azido-2-deoxy-6-O-tert-butyldiphenyl-
silyl-3-O-(2-naphthylmethyl)-1-thio-β-D-galactopyranoside (16b).
Bromine (79 μL, 1.53 mmol) was added to a solution of 7 (0.34 g,
0.679 mmol) in CH2Cl2 (10 mL) at 0 °C, and the reaction was
continued for 30 min. After complete consumption of the starting
material (monitored by TLC), the reaction mixture was concentrated
under reduced pressure and the residue was coevaporated twice with
Isopropyl 2-O-Benzoyl-4,6-O-[1-(R)-(methoxycarbonyl)-ethyli-
dene]-β-D-galactopyranoside (4). i-PrOH (0.05 mL, 0.67 mmol)
was added to the stirred solution of compound 15 (0.23 g, 0.336
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toluene. A solution of acceptor 8b (0.21 g, 0.31 mmol) in CH2Cl2 (4
mL) was added to a suspension of glycosyl bromide, 3 Å MS (0.5 g),
and sym. collidine (79 μL, 0.62 mmol) in CH2Cl2 (4 mL) and kept
stirring at −30 °C for 30 min. Silver triflate (0.341 g, 1.33 mmol) was
added, and stirring was continued at the same temperature. After 2 h,
triethylamine was added and the reaction mixture was diluted with
CH2Cl2, filtered through celite, and concentrate under reduced
pressure. The residue was purified by silica gel column chromatog-
raphy (20:80 ethyl acetate/petroleum ether) to obtain the desired
ArH), 7.48−7.37 (m, 14H, ArH), 7.33−7.30 (m, 5H, ArH), 6.08 (q, J
= 4, 1H), 5.72 (s, 1H), 5.7 (d, J = 2 Hz, 1H), 5.62 (s, 1H), 5.19 (d, J
= 12.0, 4.0 Hz, 1H), 4.90 (dd, J = 3.5, 6 Hz, 1H), 4.84−4.78 (m, 2H),
4.75 (dd, J = 4.5, Hz, 1H), 4.61−4.59 (m, 1H), 4.56−4.50 (m, 2H),
4.43 (d, J =10 Hz, 1H), 4.36 (m, 1H), 4.24 (d, J = 2 Hz, 1H), 4.19
(dd, J = 6.5, 3.2 Hz, 1H), 4.07−4.04 (m, 1H), 4.00−3.98 (dd, J = 6,
4.5 Hz, 1H), 3.92 (dd, J = 6.1, 2.0 Hz, 1H), 3.72−3.67 (m, 2H) (s,
9H, (CH3)3CSi), 1.06 (d, J = 6.5 Hz, 3H, CH3); 13C{1H} NMR (126
MHz, CDCl3): δ 166.1 (COPh), 165.9 (COPh), 165.6 (COPh),
137.1 (ArC), 135.7 (ArC), 135.7 (ArC), 134.0 (ArC), 133.6 (ArC),
133.5 (ArC), 133.4 (ArC), 133.3 (ArC), 133.2 (ArC), 133.2 (ArC),
131.0 (ArC), 130.0 (ArC), 129.9 (ArC), 129.9 (ArC), 129.8 (ArC),
129.8 (ArC), 129.6 (ArC), 129.4 (ArC), 128.8 (ArC), 128.7 (ArC),
128.6 (ArC), 128.5 (ArC), 128.4 (ArC), 105.6 (C-1 Galf), 99.0 (C-1
of AAT), 85.8 (C-1 of GalN), 81.9, 81.8, 79.8, 78.4, 77.8, 75.1, 74.1,
71.9, 70.3, 64.2, 63.3, 62.4, 61.4, 61.0, 51.7, 51.7, 29.7, 26.9 (CH3 of
TBDPS), 19.2 (quaternary C of TBDPS), 16.8 (CH3 of AAT); HR-
ESI-MS (m/z): [M + Na]+ calcd. for C83H77N7O17NaSSi, 1526.4760;
found, 1526.4758.
25
product 16b as a foam (0.26 g, 78%). [α]D = +107.29 (c = 0.05,
CHCl3); IR (cm−1, CHCl3) 2926, 2857, 2112, 1717, 1468, 1365,
1332, 1273, 1216, 1114, 1078, 760, 702; 1H NMR (400 MHz,
CDCl3): δ 7.84−7.79 (m, 6H, ArH), 7.71−7.55 (m, 9H, ArH), 7.48−
7.30 (m, 12H, ArH), 7.22−7.19 (m, 4H, ArH), 5.25 (d, J = 3.5 Hz,
1H, H-1′), 5.01−4.84 (m, 3H, H-4′ & CH2Ph), 4.60−4.54 (m, 1H,
H-5′), 4.63−4.54 (m, 3H, H-2′ & CH2Nap), 4.27−4.18 (m, 3H, H-4,
H-3, H-6′), 4.08 (dd, J = 8 Hz, 1H, H-3), 3.92 (dd, J = 7.5 Hz, 1H, H-
6), 3.71 (t, J = 12.5 Hz, 1H, H-2), 3.31−3.27 (m, 2H, H-5), 1.09 (s,
9H, (CH3)3CSi), 0.87 (d, J = 6.5 Hz, 3H, CH3); 13C{1H} NMR (126
MHz, CDCl3): δ 137.2 (ArC), 135.7 (ArC), 135.6 (ArC), 134.7
(ArC), 134.4 (ArC), 134.0 (ArC), 133.5 (ArC), 133.4 (ArC), 133.2
(ArC), 133.3 (ArC), 133.0 (ArC), 131.5 (ArC), 130.1 (ArC), 130.0
(ArC), 129.0 (ArC), 128.9 (ArC), 128.6 (ArC), 128.4 (ArC), 128.4
(ArC), 128.2 (ArC), 128.0 (ArC), 128.0 (ArC), 127.9 (ArC), 127.8
(ArC), 126.8 (ArC), 126.4 (ArC), 126.2 (ArC), 125.6 (ArC), 123.6
(ArC), 123.3 (ArC), 99.2 (C-1), 86.0 (C-1′), 80.0, 79.1, 75.2, 72.2,
71.9, 71.8, 64.1, 61.1, 61.2, 60.8, 51.8, 27.1 (CH3 of TBDPS), 19.3
(quaternary of TBDPS), 16.8 (CH3 of AAT); HR-ESI-MS (m/z): [M
+ Na]+ calcd. for C60H59N7O8NaSSi, 1088.3807; found, 1088.3807.
Phenyl 2-Azido-3-O-benzyl-2,4,6-trideoxy-4-phthalimido-α-D-
galactopyranosyl-(1 → 4)-2-azido-2-deoxy-6-O-tert-butyldiphenyl-
silyl-1-thio-β-D-galactopyranoside (5). DDQ (0.182 g, 0.806 mmol)
was added to the stirred solution of 16b (0.43 g, 0.403 mmol) in
CH2Cl2/H2O (10 mL, 9:1) at rt. After 3 h, solvents were evaporated
and purified by silica gel chromatography (20:80 ethyl acetate/
petroleum ether) to obtain acceptor 5 (0.261g, 70%). [α]D25 = +51.52
(c = 0.435, CHCl3); IR (cm−1, CHCl3) 3417, 2924, 2857, 2111, 1645,
1363, 1215, 1093, 1046, 762; 1H NMR (400 MHz, CDCl3): δ 7.88−
7.87 (m, 2H, ArH), 7.80−7.75 (m, 7H, ArH), 7.66 (d, J = 6.4 Hz, 2H,
ArH), 7.49−7.43 (m, 8H, ArH), 7.29−7.24 (m, 5H, ArH), 5.07 (d, J
= 4 Hz, 1H, H-1′), 4.72 (dd, J = 6.4, 3.6 Hz, 1H, H-4′), 4.67−4.57
(m, 1H, H-2′ & 2H CH2Ph), 4.38 (d, J = 10 Hz, 1H, H-1), 4.19 (m,
1H, H-5), 4.03−3.99 (m, 3H, H-4 & H-3′), 3.93 (dd, J = 6.4, 3.2 Hz,
1H, H-5′), 3.65−3.57 (m, 3H, H6a, H6b, & H-3), 3.10 (t, J = 10 Hz,
1H, H-2), 1.14 (s, 9H, (CH3)3CSi), 0.92 (d, J = 6.8 Hz, 3H, CH3);
13C{1H} NMR (100 MHz, CDCl3): δ 136.8 (ArC), 135.7 (ArC),
Isopropyl 2-Azido-3-O-benzyl-2,4,6-trideoxy-4-phthalimido-α-D-
galactopyranosyl-(1 → 4)-[2,3,5,6-tetra-O-benzoyl-β-D-galactofur-
anosyl-(1 → 3)]-2-azido-6-O-tert-butyldimethylsilyl-2-deoxy-α-D-
galactopyranosyl-(1 → 3)-2-O-benzoyl-4,6-O-[1-(R)-(methoxycar-
bonyl)-ethylidene]-β-D-galactopyranoside (2). Thioglycoside 3 (70
mg, 0.046 mmol) and acceptor 4 (17 mg, 0.04 mmol) were
coevaporated with toluene (3 × 15 mL). The crude mixture was dried
under vacuum for 2 h. Then, the mixture was dissolved in CH2Cl2/
Et2O (1:1, 2 mL) and treated with preactivated 3 Å MS (0.1 g) and
NIS (0.02 g, 0.09 mmol). After 30 min, the mixture was cooled to 0
°C and TMSOTf (0.9 μL, 0.0046 mmol) was added to it dropwise.
After the addition was completed, the reaction mixture was monitored
by TLC. Upon completion (2 h), the reaction mixture was quenched
with triethylamine, diluted with CH2Cl2, and filtered on a celite bed.
The organic layer was washed with aq Na2S2O3 and brine and purified
by silica gel chromatography (30:70 ethyl acetate/petroleum ether) to
25
obtain tetrasaccharide 2 (53 mg, 72%). [α]D = +17.69 (c = 0.87,
CHCl3); IR (cm−1, CHCl3) 3023, 2927, 2855, 2111, 1721, 1453,
1
1373, 1266, 1216, 1111, 1093, 1068, 760, 711, 667; H NMR (500
MHz, CDCl3): δ 8.21−8.18 (m, 4H, ArH), 8.09−8.04 (m, 5H, ArH),
7.87−7.78 (m, 12H, ArH), 7.58−7.48 (m, 12H, ArH), 7.45−7.44 (m,
6H, ArH), 7.37−7.28 (m, 5H, ArH), 6.02 (m, 1H), 5.74−5.64 (m,
5H), 5.04 (s, 1H), 4.88−4.77 (m, 3H), 4.68−4.62 (m, 3H), 4.58−
4.52 (m, 2H), 4.46 (m, 1H), 4.33−4.30 (m, 3H), 4.27−4.21 (m, 3H),
4.10−4.09 (m, 2H), 4.02−3.99 (m, 2H), 3.96−3.93 (m, 4H), 3.88−
3.87 (m, 2H), 1.34 (s, 3H, Pyr. CH3), 1.29 (d, J = 6.1 Hz, 3H, CH3),
1.23 (s, 9H, (CH3)3CSi), 1.18 (d, J = 6.5 Hz, 3H, CH3, isopropanol),
1.13 (d, J = 6.5 Hz, 3H, CH3, isopropanol); 13C NMR {1H} (126
MHz, CDCl3): δ 170.7 (CO2Me), 165.9 (COPh), 165.7 (COPh),
165.6 (COPh), 165.3 (COPh), 164.8 (COPh), 137.0 (ArC), 135.6
(ArC), 135.5 (ArC), 133.3 (ArC), 133.3 (ArC), 133.2 (ArC), 133.1
(ArC), 133.1 (ArC), 130.1 (ArC), 130.0 (ArC), 129.9 (ArC), 129.9
(ArC), 129.8 (ArC), 129.7 (ArC), 129.5 (ArC), 129.4 (ArC), 129.3
(ArC), 128.8 (ArC), 128.6 (ArC), 128.5 (ArC), 128.4 (ArC), 128.4
(ArC), 128.3 (ArC), 128.2 (ArC), 128.1 (ArC), 127.9 (ArC), 127.7
(ArC), 127.7 (ArC), 107.0 (C-1 of Galf), 99.7 (C-1 Pyr. Gal), 99.2
(Cq Pyr.), 98.9 (C-1 AAT), 92.1 (C-1 of GalN), 81.2, 81.0, 74.4,
73.0, 72.2, 72.1, 72.0, 70.4, 70.3, 64.9, 65.5, 64.4, 63.0, 60.3, 59.0,
52.7, 51.8 (OCH3), 29.7, 27.0, 25.6, 23.2 (CH3 of isopropanol), 21.9
(CH3 of isopropanol), 19.4 (quaternary C of TBDPS), 16.8 (CH3 of
AAT); HR-ESI-MS (m/z): [M + K]+ calcd. for C97H97N7O26KSi,
1842.5880; found, 1842.5884.
135.5 (ArC), 134.5 (ArC), 133.6 (ArC), 133.5 (ArC), 130.4 (ArC),
130.1 (ArC), 130.0 (ArC), 129.9 (ArC), 128.8 (ArC), 128.6 (ArC),
128.2 (ArC), 128.1 (ArC), 127.9 (ArC), 127.9 (ArC), 123.7 (ArC),
100.1 (C-1), 84.9 (C-1′), 79.7, 79.6, 74.5, 74.0, 72.1, 65.1, 63.8, 62.8,
61.0, 51.3, 26.9 (CH3 of TBDPS), 19.4 (quaternary C of TBDPS),
16.4 (CH3 of AAT); HR-ESI-MS (m/z): [M + Na]+ calcd. for
C49H51N7O8NaSSi, 948.3180; found, 948.3181.
Phenyl 2-Azido-3-O-benzyl-2,4,6-trideoxy-4-phthalimido-α-D-
galactopyranosyl-(1 → 4)-[2,3,5,6-tetra-O-benzoyl-β-D-galactofur-
anosyl-(1 → 3)]-2-azido-6-O-tert-butyldimethylsilyl-2-deoxy-β-D-
galactopyranoside (3). Disaccharide acceptor 5 (0.094 g, 0.1015
mmol) and galactofuranose N- trichloroacetimidate 6 (0.21 g, 0.283
mmol) were coevaporated with toluene (3 × 20 mL) and subjected to
high vacuum for 1 h. The solution was then dissolved in CH2Cl2 (2.2
mL) and was cooled to −30 °C. The cooled solution was then treated
with a CH2Cl2 solution. After 10 min, TMSOTf (0.051 mL, 0.028
mmol) was added. After 1 h, the reaction was quenched with a NEt3
filter with a celite bed and concentrated under reduced pressure. The
crude product was purified by flash silica gel chromatography (30:70,
ethyl acetate/petroleum ether) to furnish trisaccharide 3 (0.125 g) in
Isopropyl 2-Acetamido-4-amino-2,4,6-trideoxy-α-D-galactopyra-
nosyl-(1 → 4)-[β-D-galactofuranosyl-(1 → 3)]-2-acetamido-2-
deoxy-α-D-galactopyranosyl-(1 → 3)-4,6-O-[1-(R)-(carboxy)-ethyl-
idene]-β-D-galactopyranoside (1). Global deprotection of tetrasac-
charide 2 (100 mg, 0.056 mmol) was carried out in five steps.
Compound 2 was dissolved in pyridine (0.3 mL) and treated with
thioacetic acid (0.3 mL, 0.05 mmol) at rt for 24 h. The solvents were
evaporated, and the crude product was coevaporated with toluene (5
mL × 3). Purification of the crude product by silica chromatography
25
82% yield. [α]D = +33.42 (c = 0.93, CHCl3); IR (cm−1, CHCl3)
2952, 2926, 2855, 2112, 1721, 1602, 1452, 1363, 1312, 1266, 1178,
1
1110, 1068, 1029, 759, 711; H NMR (500 MHz, CDCl3): δ 8.13−
8.01 (m, 8H, ArH), 7.89−7.70 (m, 11H, ArH), 7.57−7.53 (m, 6H,
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(70:30, ethyl acetate/petroleum ether) afforded the N-acetate
derivative (75 mg, 74%). The NHAc intermediate was treated with
TBAF/AcOH, (0.15 mL) in 0.6 mL of THF at rt overnight to give the
alcohol. The phthalimide group from this intermediate was removed
using EDA/n-BuOH (0.15 mL/1 mL). Benzyl groups were removed
using H2/Pd/C (0.5/mmol, 30 mg) in methanol (1.2 mL). Finally,
esters were deprotected using NaOMe (0.2 M) in MeOH/THF/H2O
(1:1:1) (4:4:4 mL) to obtain a crude product, which was purified by
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tetrasaccharide 1 (12.4 mg) in 36% yield over four steps. [α]D
=
1
+27.5. H NMR (600 MHz, D2O): δ 5.21 (d, J = 3.6 Hz, 1H), 4.95
(d, J = 3.0 Hz, 1H), 4.90 (d, J = 3.6 Hz, 1H), 4.47 (d, J = 3.6 Hz, 1H),
4.54−4.44 (m, 1H), 4.32 (d, J = 3.6 Hz, 1H), 4.26 (dd, J = 12.0, 4.2
Hz, 1H), 4.08 (t, J = 6.0 Hz, 1H), 4.05−4.05 (m, 1H), 4.02−3.97 (m,
2H), 3.95−3.91 (m, 1H), 3.90−3.79 (m, 5H), 3.69−3.64 (m, 2H),
3.63−3.59 (m, 1H), 3.58−3.48 (m, 6H), 3.44 (s, 1H), 1.95 (s, 3H),
1.93 (s, 3H), 1.33 (s, 3H), 1.18 (d, J = 6.6 Hz, 3H), 1.14 (d, J = 6.6
Hz, 3H), 1.08 (d, J = 6.6 Hz, 3H) 13C{1H} NMR (126 MHz, D2O): δ
174.6 (NHCOCH3), 174.0 (NHCOCH3), 171.1 (CO2H), 108.8 (C-1
of Galf), 100.1 (C-1 of Pyr. Gal), 98.9 (quaternary C of pyruvate),
97.7 (C-1 of AAT), 92.8 (C-1 of GalN), 81.8, 80.5, 76.9, 75.6, 75.0,
73.8, 72.9, 72.1, 70.5, 68.6, 66.9, 65.3, 65.2, 63.5, 62.7, 60.4, 58.9,
54.6, 49.9, 48.6, 25.1, 23.3 (CH3 of isopropanol), 22.3 (CH3 of
isopropanol), 21.9 (NHCOCH3), 20.9 (NHCOCH3), 16.2 (CH3 of
AAT); HR-ESI-MS (m/z): [M + Na]+ calcd. for C34H57N3O21Na,
866.3376; found, 866.3377.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.joc.0c02935.
■
sı
1H NMR; 13C NMR; DEPT; and 1H−1H COSY spectra
of all new compounds (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Suvarn S. Kulkarni − Department of Chemistry, Indian
Institute of Technology Bombay, Mumbai 400076, India;
orcid.org/0000-0003-2884-876X; Email: suvarn@
chem.iitb.ac.in
Authors
Ennus K. Pathan − Department of Chemistry, Indian Institute
of Technology Bombay, Mumbai 400076, India
Bhaswati Ghosh − Department of Chemistry, Indian Institute
of Technology Bombay, Mumbai 400076, India
Ananda Rao Podilapu − Department of Chemistry, Indian
Institute of Technology Bombay, Mumbai 400076, India
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.joc.0c02935
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the Science and Engineering Research Board,
Department of Science and Technology (Grant no. CRG/
2019/000025) and Department of Biotechnology (BT/INF/
22/SP23026/2017) for financial support. E.K.P and A.R.P
thank CSIR, New Delhi, for fellowships. B.G. thanks IIT
Bombay for institute post-doctoral fellowship.
(11) De Silva, R. A.; Wang, Q.; Chidley, T.; Appulage, D. K.;
Andreana, P. R. Immunological Response from an Entirely
Carbohydrate Antigen: Design of Synthetic Vaccines Based on Tn-
PS A1 Conjugates. J. Am. Chem. Soc. 2009, 131, 9622−9623.
(12) Shi, M.; Kleski, K. A.; Trabbic, K. R.; Bourgault, J. P.;
Andreana, P. R. Sialyl-Tn Polysaccharide A1 as an Entirely
Carbohydrate Immunogen: Synthesis and Immunological Evaluation.
J. Am. Chem. Soc. 2016, 138, 14264−14272.
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