Synthesis of the Tetrasaccharide Repeating Unit from Acinetobacter baumannii Serogroup O18 Capitalizing on Phosphorus-Containing Leaving Groups

Article abstract of DOI:10.1021/acs.joc.5b00139

The first convergent synthesis of the tetrasaccharide repeating unit of the polymeric O antigen isolated from Acinetobacter baumannii serogroup O18 has been achieved. The ManNAcβ1→4Gal and GalNAcβ1→3Gal units were successfully obtained through β-selective

Full text of DOI:10.1021/acs.joc.5b00139

Article
pubs.acs.org/joc
Synthesis of the Tetrasaccharide Repeating Unit from Acinetobacter
baumannii Serogroup O18 Capitalizing on Phosphorus-Containing
Leaving Groups
Ryoichi Arihara, Kosuke Kakita, Kazuhiro Yamada, Seiichi Nakamura,*,† and Shunichi Hashimoto*
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan
S
* Supporting Information
ABSTRACT: The first convergent synthesis of the tetrasaccharide repeating unit of the poly-
meric O antigen isolated from Acinetobacter baumannii serogroup O18 has been achieved. The
ManNAcβ1→4Gal and GalNAcβ1→3Gal units were successfully obtained through β-selective
glycosylation with 2-azido-4,6-O-benzylidene-2-deoxymannosyl diphenyl phosphate and Tf2NH-
promoted glycosylation with 2-acetamido-2-deoxygalactosyl diethyl phosphite, respectively. The
disaccharide units could be coupled with the aid of TMSClO4 as an activator of the diphenyl
phosphate leaving group, and global deprotection completed the synthesis of the tetrasaccharide.
employed for the glycosylation of reactive alcohols, such as 3-O-
unprotected glycoside alcohol, at −78 °C (eq 1). With regard to
the synthesis of 2-acetamido-2-deoxymannosides, van der Marel
INTRODUCTION
■
Lipopolysaccharides (LPS) are expressed at the outer membrane
of Gram-negative bacteria, and they play an important role in
bacterial virulence and resistance to innate immunity.1 Structur-
ally, LPS is characterized by a highly variable polysaccharide
attached through a core oligosaccharide to endotoxic lipid A,
which functions as an anchor to the bacterial cell. Since the
polysaccharide, comprising iterations of a repeating unit, defines
the serotype and is responsible for the O-antigenic properties,
fragments of various polysaccharides have been synthesized for
potential vaccine development and pathogen detection.2
As mentioned in the preceding paper,9b we have developed
novel glycosylation reactions capitalizing on phosphorus-
containing leaving groups.3 By use of donors with these leaving
groups, various types of glycosidic linkages could be constructed
in a stereoselective manner by appropriate choice of reaction
conditions.4−9 To demonstrate the synthetic utility of our
method, we then undertook synthesis of the tetrasaccharide
repeating unit of the polymeric O-antigen obtained from LPS
extracted from isolated, defatted cell walls of the reference strain
for Acinetobacter baumannii serogroup O18 (Figure 1).10,11 The
tetrasaccharide [ManNAcβ1→4Galα1→4(GalNAcβ1→3)Gal]
(1) contains three types of glycosidic linkages, all of which
required us to devise strategies for stereoselective construction.
As described in the preceding paper,9b we have demonstrated
that 2-acetamido-2-deoxyglycosyl diethyl phosphites could be
and co-workers,12 inspired by Crich’s β-mannosylation,13
developed an efficient approach using 2-azido-4,6-O-benzylidene-
2-deoxy-1-thiomannoside. In experiments patterned after
their reports, we explored the glycosylation with 2-azido-2-
deoxymannosyl donors carrying phosphorus-containing
leaving groups and finally achieved a high-yield coupling with
diphenyl phosphate as a leaving group (eq 2).8 We surmised
Received: January 20, 2015
Published: March 25, 2015
Figure 1. Structure of O-antigen from Acinetobacter baumannii
serogroup O18.
© 2015 American Chemical Society
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Article
that the targeted tetrasaccharide 1 would be synthesized by
taking advantage of these reactions. In this paper, we describe
a convergent and stereocontrolled approach allowing access to
the tetrasaccharide 1 by capitalizing on diethyl phosphite and
diphenyl phosphate as leaving groups.
the p-methoxybenzyl (PMB) group was oxidatively removed
with 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ)18 to give
diol 17 in 74% overall yield for the three steps. Protection of the
C3 hydroxyl group was achieved by the diacetylation−selective
deacetylation sequence: the acetate at C1 could be hydrolyzed,
with the acetate at C3 remaining intact, by employing Hauser’s
conditions (H2O2, NaHCO3)19 to furnish hemiacetal 19 with an
α:β ratio of 95:5 in 92% yield. Phosphitylation of hemiacetal 19
with diethyl chlorophosphite in the presence of Et3N gave
α-linked diethyl phosphite 11 in 82% yield.
Both galactoside acceptors 12 and 13 were prepared from
the common diol 2020 (Scheme 3). Monobenzylation of diol
20 via the corresponding stannylene acetal under modified
conditions21 provided 4-O-unprotected galactoside 1215 in
almost quantitative yield. On the other hand, 3-O-unprotected
galactoside 13 was obtained as a single isomer in 96% yield by
the reduction of acetal 22, prepared upon treatment of diol 20
with anisaldehyde dimethyl acetal, with NaBH3CN with the aid
of trifluoroacetic acid (TFA) in the presence of 4 Å molecular
sieves (MS). It is interesting that formation of undesired
4-O-unprotected galactoside was observed by the reduction of
less polar isomer 21 under identical conditions. Stereochemical
assignments for isomers 21 and 22 were determined by nuclear
Overhauser enhancement (NOE) experiments.
RESULTS AND DISCUSSION
■
Our retrosynthetic analysis of tetrasaccharide 1 is depicted in
Scheme 1. We considered that selectively removable masking
groups should be employed for protection of the hydroxyl
groups at the 3-position of GalNAc and the reducing terminus,
so that the deprotected products could be used as building
blocks for synthesis of polymeric saccharides. From the
standpoint of convergency, coupling between disaccharides
914 and 10 seemed to be attractive for the construction of
tetrasaccharide 8. The acetamido group of disaccharide 9 needs
to be masked as an azide to ensure β-selective mannosylation8
of 4-O-unprotected galactoside 12.15 On the other hand,
disaccharide 10 would be available via glycosylation9 of 3-O-
unprotected galactoside 13 with 2-acetamido-2-deoxygalactosyl
diethyl phosphite 11.
Preparation of GalNAc donor 11 began with protection of
known alcohol 1416 with BnBr, affording benzyl ether 15 in
96% yield (Scheme 2). After deprotection of the allyl glycoside
by a two-step sequence involving olefin isomerization with
t-BuOK and bromination in aqueous tetrahydrofuran (THF),17
With monosaccharide units 11−13 in hand, efforts were next
focused on glycosylation reactions (Scheme 4). As expected,
Scheme 1. Retrosynthetic Analysis of Tetrasaccharide 1
Scheme 2. Preparation of 2-Acetamido-2-deoxygalactosyl Donor 11
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Article
Scheme 3. Preparation of Galactosyl Acceptors 12 and 13
Scheme 4. Synthesis of Disaccharide Units 9 and 10 through β-Selective Glycosylations
glycosylation of 4-O-unprotected galactoside 12 with 2-azido-2-
deoxymannosyl diphenyl phosphate 5 under optimized condi-
tions8 [trimethylsilyl trifluoromethanesulfonate (TMSOTf),
4 Å molecular sieves, CH2Cl2, −30 °C] proceeded to comple-
tion within 3 h to give disaccharides in 89% combined yield
with exceptionally high β-selectivity (8:92). After separation
of the anomers, azide 23 was transformed to acetamide 24 in
97% yield by treatment with Ph3P in aqueous THF, followed
by acetylation with Ac2O in pyridine. Hemiacetal 25 (α:β =
55:45), obtained in 84% yield from allyl glycoside 24 by a
two-step procedure that entailed treatment with t-BuOK in
dimethyl sulfoxide (DMSO) at 100 °C and bromination with
N-bromosuccinimide (NBS) in aqueous THF,17 was success-
fully converted to diphenyl phosphate 9 (α:β = 67:33)
in 81% yield via oxidation22 of the corresponding phosphite
with potassium peroxymonosulfate (Oxone)23 in aqueous
acetone.24
to predominant formation of the corresponding α-glycosides.
With this precedent in mind, we first selected TMSOTf as
promoter. As expected, tetrasaccharide 8 was obtained as a sole
product by the TMSOTf-promoted reaction of diphenyl
phosphate 9 with alcohol 10 in the presence of 5 Å molecular
sieves in CH2Cl2 at 0 °C; however, the yield was only 35% even
with 2 equiv of alcohol 10 (Table 1, entry 1).28 Gratifyingly, the
product yield was improved to 75% by use of TMSClO4 instead
of TMSOTf (entry 2).29 A solvent survey revealed that slower
reaction and reduced yield (32%)28 were observed in THF
(entry 3)30 and that the use of toluene as a cosolvent had no
discernible benefit (entry 4). Examination of the temperature
profile of the reaction demonstrated that a decrease in reaction
temperature resulted in a decrease in product yield (entries 2 vs
5 and 6). Stereochemical assignment of the newly formed
stereocenter in tetrasaccharide 8 was established by a significant
1H NOE interaction and a coupling constant (J = 2.9 Hz)
between H-1′ and H-2′.
With monosaccharide units 5 and 11−13 successfully
assembled, the remaining operations necessary for synthesis
of tetrasaccharide repeating unit 1 involved removal of all
protecting groups. Exposure of tetrasaccharide 8 to NaOMe in
MeOH effected methanolysis of the acetate at the 3-position of
GalNAc to provide alcohol 27, which can be employed as an
acceptor for synthesis of the dimeric repeating unit (Scheme 5).
The allyl protecting group at the reducing terminus in 27 could
be safely removed by a two-step sequence involving
(Ph3P)3RhCl-catalyzed olefin isomerization and bromination
in aqueous THF,17 to provide hemiacetal 28 in 82% overall
yield. Finally, global deprotection employing 10% Pd/C in
MeOH under a hydrogen atmosphere afforded the target
tetrasaccharide 1 in 94% yield.
With regard to glycosylation with 2-acetamido-2-deoxyga-
lactosyl diethyl phosphite 11, 3 equiv of 3-O-unprotected
galactoside 13 was employed as an acceptor since galactoside
13 could be easily prepared from inexpensive galactose on a
large scale and the unreacted alcohol could be quantitatively
recovered after column chromatography. The Tf2NH-promoted
reaction proceeded at −78 °C to give β-linked disaccharide 26
as a single isomer in 65% yield. Oxidative removal of the PMB
18
group with DDQ in aqueous CH2Cl2 furnished alcohol 10 in
90% yield.
Having established access to disaccharide units 9 and 10, the
stage was now set for the crucial coupling reaction. During the
course of our synthesis of globotriaosylceramide (Gb3),27 it was
found that alcohols with poor nucleophilicity favored axial
attack on the oxocarbenium ion generated from the galactosyl
phosphorodiamidate with the aid of TMSOTf in CH2Cl2, leading
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Table 1. Coupling of Disaccharide Units 9 and 10
entry
promoter
solvent
CH2Cl2
temp (°C)
time (h)
yield (%)
1
2
3
4
5
6
TMSOTf
TMSClO4
TMSClO4
TMSClO4
TMSClO4
TMSClO4
0
0
1
0.5
2
35
75
32
77
65
58
CH2Cl2
THF
0
1:1 PhMe/CH2Cl2
CH2Cl2
0
0.5
24
72
−23
−45
CH2Cl2
Scheme 5. Completion of Synthesis of Tetrasaccharide Repeating Unit
with MeOH (0.5 mL), and the resulting mixture was partitioned
between AcOEt (70 mL) and saturated aqueous NH4Cl (10 mL).
The organic layer was successively washed with H2O (10 mL) and
brine (2 × 10 mL) and dried over anhydrous Na2SO4. Filtration and
evaporation in vacuo furnished the pale yellow oil, which was purified
by column chromatography (silica gel 30 g, 10:1 n-hexane/AcOEt) to
give tert-butyldimethylsilyl 2-azido-3-O-benzyl-4,6-O-benzylidene-2-
deoxy-β-D-mannopyranoside (1.23 g, 90%) as a colorless syrup. Rf
0.71 (2:1 n-hexane/AcOEt); [α]2D3 −62.1 (c 2.03, CHCl3); IR (neat)
3034, 2930, 2858, 2106, 1454, 1379, 1255, 1199, 1099, 1005 cm−1;
1H NMR (500 MHz, CDCl3) δ 0.12 (s, 3H), 0.16 (s, 3H), 0.92 (s,
9H), 3.32 (ddd, J = 5.0, 9.3, 10.2 Hz, 1H), 3.70 (dd, J = 3.8, 9.5 Hz,
1H), 3.85−3.89 (m, 2H), 3.98 (dd, J = 9.3, 9.5 Hz, 1H), 4.26 (dd,
J = 5.0, 10.5 Hz, 1H), 4.75 (d, J = 12.4 Hz, 1H), 4.87 (d, J = 12.4 Hz,
1H), 4.89 (d, J = 1.0 Hz, 1H), 5.58 (s, 1H), 7.28−7.40 (m, 8H),
7.48 (m, 2H); 13C NMR (126 MHz, CDCl3) δ −5.6, −4.3, 17.7,
25.4, 65.0, 67.1, 68.1, 72.5, 75.7, 78.1, 95.7, 101.2, 125.8, 127.4,
127.5, 127.9, 128.2, 128.7, 137.2, 137.7; HRMS (FAB) m/z [M + H]+
calcd for C26H36N3O5Si 498.2424, found 498.2416. Anal. Calcd for
C26H35N3O5Si: C, 62.75; H, 7.09; N, 8.44. Found: C, 62.57; H, 7.16;
N, 8.39.
CONCLUSION
■
We have completed the first total synthesis of the tetrasaccharide
repeating unit of the polymeric O antigen isolated from
Acinetobacter baumannii serogroup O18 (1), wherein all of the
anomeric configurations could be controlled by proper choice
of both phosphorus-containing leaving group and reaction
conditions. The use of selectively removable protective groups
for protection of the hydroxyl groups at the 3-position of
GalNAc and the reducing terminus would allow access to
polymeric saccharides. The synthesis provides a good illustration
of the utility of phosphorus-containing leaving groups in
oligosaccharide synthesis.
EXPERIMENTAL SECTION
■
2-Azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-D-manno-
pyranosyl Diphenyl Phosphate (5). NaOMe in MeOH (1.0 M,
0.6 mL, 0.6 mmol) was added to a solution of tert-butyldimethylsilyl
3,4,6-tri-O-acetyl-2-azido-2-deoxy-β-D-mannopyranoside31 (1.65 g,
3.70 mmol) in MeOH (30 mL). After 1 h of stirring, the reaction
mixture was neutralized with Amberlite IR-120B acidic resin. Filtration
and evaporation in vacuo furnished the crude product (1.19 g), which
was used without further purification.
Anhydrous p-toluenesulfonic acid (33 mg, 0.19 mmol) was added to
a stirred solution of the crude triol and benzaldehyde dimethyl acetal
(0.67 mL, 4.48 mmol) in MeCN (10 mL). After 30 min of stirring, the
reaction was quenched with Et3N (0.2 mL), and the volatile elements
were removed in vacuo. Purification of the pale yellow residue by
column chromatography (silica gel 30 g, 9:1 → 4:1 n-hexane/AcOEt)
afforded tert-butyldimethylsilyl 2-azido-4,6-O-benzylidene-2-deoxy-β-
D-mannopyranoside (1.21 g, 80%) as a white amorphous solid.
A 60% dispersion of NaH in mineral oil (143 mg, 3.58 mmol) was
added to an ice-cooled (0 °C) mixture of the benzylidene acetal
(1.12 g, 2.75 mmol), benzyl bromide (0.43 mL, 3.58 mmol), and
Bu4NI (102 mg, 0.275 mmol) in 9:1 THF/N,N-dimethylformamide
(DMF) (20 mL). After 30 min of stirring, the reaction was quenched
Bu4NF in THF (1.0 M, 2.5 mL, 2.50 mmol) was added to an
ice-cooled (0 °C) solution of the tert-butyldimethylsilyl (TBS) ether
(1.05 g, 2.11 mmol) and AcOH (0.24 mL, 4.19 mmol) in THF
(10 mL). After 20 min of stirring, saturated aqueous NaHCO3
(10 mL) was added, and the resulting mixture was extracted with
AcOEt (70 mL). The organic extract was successively washed with
saturated aqueous NaHCO3 (10 mL) and brine (2 × 10 mL) and
dried over anhydrous Na2SO4. Filtration and evaporation in vacuo
furnished the crude product (1.28 g), which was purified by column
chromatography (silica gel 30 g, 2:1 n-hexane/AcOEt) to give 2-azido-
3-O-benzyl-4,6-O-benzylidene-2-deoxy-D-mannopyranose (798 mg, 99%,
α:β = 56:44) as a white amorphous solid. The anomeric α:β ratio of the
1
product was determined by 500 MHz H NMR.
Diphenylphosphoryl chloride (0.52 mL, 2.50 mmol) was added to
an ice-cooled (0 °C) solution of the hemiacetal (798 mg, 2.08 mmol)
and 4-dimethylaminopyridine (DMAP; 508 mg, 4.16 mmol) in
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CH2Cl2 (8 mL). After 20 min, the reaction was quenched by addition
of a piece of crushed ice, followed by stirring at room temperature
for 15 min. The mixture was poured into a two-layer mixture of Et2O
(5 mL) and saturated aqueous NaHCO3 (10 mL), and the resulting
mixture was extracted with AcOEt (50 mL). The organic extract was
washed with brine (2 × 10 mL) and dried over anhydrous Na2SO4.
Filtration and evaporation in vacuo furnished the crude product
(1.97 g, pale yellow oil), which was purified by column chromato-
graphy (silica gel 40 g, 4:1 → 2:1 n-hexane/AcOEt with 0.5% Et3N) to
give diphenyl phosphate 5 (917 mg, 72%) and its β-isomer (347 mg,
27%) as pale yellow syrups.
2-Acetamido-4,6-di-O-benzyl-2-deoxy-3-O-(4-methoxyben-
zyl)-D-galactopyranose (16). Potassium tert-butoxide (571 mg,
5.09 mmol) was added to a stirred solution of allyl glycoside 15
(2.12 g, 3.77 mmol) in DMSO (10 mL). After 30 min of stirring at
100 °C, the reaction mixture was cooled with a water bath, and the
reaction was quenched with crushed ice (ca. 1 g). Addition of cold
water (10 mL) resulted in the precipitation of a white solid, which was
filtered and washed with cold water.
NBS (739 mg, 4.15 mmol) was added to a stirred solution of the
crude prop-1-enyl glycoside in 50:3 THF/H2O (31.8 mL). After 5 min
of stirring, the volatile elements were removed in vacuo, and the
residue was partitioned between AcOEt (100 mL) and 10% aqueous
Na2S2O3 (30 mL). The organic layer was successively washed with
H2O (2 × 20 mL) and brine (2 × 30 mL), and dried over anhydrous
Na2SO4. Filtration and evaporation in vacuo furnished the crude
product (2.19 g, slightly yellow solid), which was recrystallized from
i-PrOH/i-Pr2O to give hemiacetal 16 (1.78 g, 90%) as colorless
needles. Rf 0.46 (1:1 toluene/acetone); mp 190.0−190.5 °C; [α]D22
+60.7 (c 1.03, CHCl3); IR (KBr) 3369, 3311, 3031, 2928, 1648, 1554,
Data for α-phosphate 5: Rf 0.46 (2:1 n-hexane/AcOEt); [α]2D3 +37.2
(c 1.51, CHCl3); IR (neat) 3065, 2870, 2112, 1591, 1489, 1454, 1377,
1298, 1103 cm−1; 1H NMR (500 MHz, CDCl3) δ 3.71 (t, J = 10.2 Hz,
1H), 3.86 (ddd, J = 4.8, 9.8, 10.2 Hz, 1H), 3.93 (dd, J = 1.5, 3.5 Hz,
1H), 3.98 (dd, J = 4.8, 10.2 Hz, 1H), 4.07 (dd, J = 3.5, 9.8 Hz, 1H),
4.13 (t, J = 9.8 Hz, 1H), 4.67 (d, J = 12.1 Hz, 1H), 4.85 (d, J =
12.1 Hz, 1H), 5.57 (s, 1H), 5.79 (dd, J = 1.5, 6.3 Hz, 1H), 7.17−7.23
(m, 6H), 7.29−7.38 (m, 12H), 7.46 (m, 2H); 13C NMR (126 MHz,
CDCl3) δ 62.3 (d, JC−P = 10.3 Hz), 65.7, 67.9, 73.4, 74.6, 78.1, 97.7
1
1515, 1251, 1097, 1057, 819 cm−1; H NMR (400 MHz, CDCl3, data
for α-anomer) δ 1.92 (s, 3H), 3.49 (dd, J = 6.4, 9.1 Hz, 1H), 3.56 (dd,
J = 6.4, 9.1 Hz, 1H), 3.66 (br d, J = 3.2 Hz, 1H), 3.74 (dd, J = 2.6, 10.8
Hz, 1H), 3.81 (s, 3H), 3.95 (br s, 1H), 4.11 (br t, J = 6.4 Hz, 1H), 4.37
(d, J = 11.9 Hz, 1H), 4.41 (d, J = 11.8 Hz, 1H), 4.46 (ddd, J = 3.2, 8.2,
10.8 Hz, 1H), 4.49 (d, J = 11.8 Hz, 1H), 4.57 (d, J = 11.7 Hz, 1H),
4.64 (d, J = 11.9 Hz, 1H), 4.93 (d, J = 11.7 Hz, 1H), 5.30 (t, J = 3.2
Hz, 1H), 5.42 (d, J = 8.2 Hz, 1H), 6.87−6.90 (m, 2H), 7.20−7.34 (m,
12H); 13C NMR (100 MHz, CDCl3, data for α-anomer) δ 23.4, 50.0,
55.3, 69.3, 71.1, 72.8, 73.4, 74.4, 75.8, 92.2, 113.8, 127.5, 127.7, 127.9,
128.12, 128.15, 128.3, 129.3, 130.0, 137.6, 138.3, 159.2, 170.6; HRMS
(FAB) m/z [M + H]+ calcd for C30H36NO7 522.2499, found 522.2484.
2-Acetamido-4,6-di-O-benzyl-2-deoxy-D-galactopyranose
(17). DDQ (847 mg, 3.82 mmol) was added to an ice-cooled
(0 °C), biphasic mixture of PMB ether 16 (1.66 g, 3.18 mmol) in
20:1 CH2Cl2/pH 7 phosphate buffer (31.5 mL). After 1 h of stirring at
room temperature, the reaction was quenched with saturated aqueous
NaHCO3 (5 mL), and the resulting mixture was partitioned between
AcOEt/n-hexane (9:1, 100 mL) and saturated aqueous NaHCO3
(10 mL). The organic layer was successively washed with saturated
aqueous NaHCO3 (5 × 15 mL) and brine (2 × 30 mL) and dried over
anhydrous Na2SO4. Filtration and evaporation in vacuo furnished
the crude product (1.60 g, slightly yellow solid), which was purified
by column chromatography (silica gel 40 g, 2:1 CH2Cl2/acetone) to
give diol 17 (1.05 g, 82%) as a white solid. Rf 0.25 (α-anomer),
0.18 (β-anomer) (3:2 CH2Cl2/acetone); mp 188.0−188.5 °C (colorless
needles from i-PrOH/CHCl3); [α]2D1 +53.9 (c 0.10, EtOH); IR (KBr)
3384, 3321, 3033, 2929, 1640, 1552, 1451, 827 cm−1; 1H NMR
(400 MHz, CD3OD, data for α-anomer) δ 1.98 (s, 3H), 3.48 (dd, J =
6.5, 9.4 Hz, 1H), 3.52 (dd, J = 6.5, 9.4 Hz, 1H), 3.84 (br d, J = 3.2 Hz,
1H), 3.95 (dd, J = 3.2, 11.1 Hz, 1H), 4.19 (t, J = 6.5 Hz, 1H), 4.29 (dd,
J = 3.5, 11.1 Hz, 1H), 4.39 (d, J = 11.7 Hz, 1H), 4.46 (d, J = 11.7 Hz,
1H), 4.54 (d, J = 11.1 Hz, 1H), 4.91 (d, J = 11.1 Hz, 1H), 5.11 (d, J =
3.5 Hz, 1H), 7.24−7.34 (m, 10H); 13C NMR (100 MHz, CD3OD, data
for α-anomer) δ 22.7, 52.5, 70.1, 70.3, 74.3, 76.4, 78.6, 92.8, 128.5,
128.6, 128.9, 129.10, 129.12, 129.3, 139.2, 140.1, 173.8; HRMS (FAB)
m/z [M + H]+ calcd for C22H28NO6 402.1917, found 402.1910. Anal.
Calcd for C22H27NO6: C, 65.82; H, 6.78; N, 3.49. Found: C, 65.56; H,
6.69; N, 3.48.
(d, JC−P = 5.0 Hz), 101.6, 119.9 (d, JC−P = 5.0 Hz), 120.0 (d, JC−P
5.0 Hz), 125.7, 125.8, 125.9, 127.5, 127.8, 128.1, 128.4, 129.0, 129.85,
=
129.89, 137.0, 137.6, 150.0 (d, JC−P = 7.6 Hz), 150.1 (d, JC−P
=
7.6 Hz); 31P NMR (202 MHz, CDCl3) δ −13.62; HRMS (FAB) m/z
[M + H]+ calcd for C32H31N3O8P 616.1849, found 616.1860.
Data for β-phosphate: Rf 0.39 (2:1 n-hexane/AcOEt); [α]2D3 −25.7
(c 1.52, CHCl3); IR (neat) 3065, 2876, 2112, 1591, 1489, 1454, 1386,
1
1286, 1188, 1064 cm−1; H NMR (500 MHz, CDCl3) δ 3.25 (ddd,
J = 4.9, 9.5, 10.0 Hz, 1H), 3.65 (dd, J = 3.7, 10.0 Hz, 1H), 3.66 (t, J =
10.0 Hz, 1H), 3.79 (dd, J = 1.2, 3.7 Hz, 1H), 3.92 (t, J = 9.5 Hz, 1H),
4.07 (dd, J = 4.9, 9.5 Hz, 1H), 4.58 (d, J = 12.2 Hz, 1H), 4.73 (d, J =
12.2 Hz, 1H), 5.36 (dd, J = 1.2, 6.9 Hz, 1H), 5.45 (s, 1H), 7.09−7.14
(m, 6H), 7.21−7.27 (m, 12H), 7.36 (m, 2H); 13C NMR (126 MHz,
CDCl3) δ 63.3 (d, JC−P = 7.9 Hz), 67.7, 67.8, 73.1, 76.1, 77.7, 96.1
(d, JC−P = 3.8 Hz), 101.5, 120.1 (d, JC−P = 3.8 Hz), 120.2 (d, JC−P
3.8 Hz), 125.6, 125.8, 125.9, 127.6, 127.9, 128.1, 128.4, 129.0, 129.4,
=
129.6, 129.9, 136.9, 137.3, 149.9 (d, JC−P = 7.6 Hz), 150.2 (d, JC−P
=
7.6 Hz); 31P NMR (202 MHz, CDCl3) δ −13.11; HRMS (FAB) m/z
[M + H]+ calcd for C32H31N3O8P 616.1849, found 616.1851.
Allyl 2-Acetamido-4,6-di-O-benzyl-2-deoxy-3-O-(4-methox-
ybenzyl)-α-D-galactopyranoside (15). A solution of glycoside
alcohol 14 (1.94 g, 4.11 mmol) in THF (15 mL) was added to an ice-
cooled (0 °C) suspension of NaH (65% in oil, 197 mg, 5.34 mmol)
in DMF (25 mL). After 10 min of stirring at 0 °C, BnBr (0.60 mL,
4.93 mmol) was added, and the mixture was stirred at this temperature
for 1 h. The reaction was quenched with MeOH (2 mL), and the
resulting mixture was partitioned between AcOEt (150 mL) and
saturated aqueous NH4Cl (30 mL). The organic layer was successively
washed with saturated aqueous NH4Cl (30 mL) and brine (2 × 40 mL)
and dried over anhydrous Na2SO4. Filtration and evaporation in vacuo
furnished the crude product (2.66 g, white solid), which was purified by
column chromatography (silica gel 50 g, 20:1 → 10:1 CH2Cl2/acetone)
to give benzyl ether 15 (2.21 g, 96%) as a white solid. Rf 0.40 (3:1
toluene/acetone); mp 126.5−128 °C (colorless needles from AcOEt/
n-hexane); [α]2D0 +90.6 (c 1.03, CHCl3); IR (KBr) 3324, 3065, 3030,
1649, 1549, 1252, 1119, 1044, 739 cm−1; 1H NMR (500 MHz, CDCl3)
δ 1.92 (s, 3H), 3.55 (dd, J = 6.5, 9.2 Hz, 1H), 3.60 (dd, J = 2.3, 11.0 Hz,
1H), 3.62 (dd, J = 6.5, 9.2 Hz, 1H), 3.81 (s, 3H), 3.89 (t, J = 6.5 Hz,
1H), 3.94 (ddd, J = 1.1, 6.2, 13.2 Hz, 1H), 3.98 (br s, 1H), 4.11 (ddd,
J = 1.5, 6.5, 13.2 Hz, 1H), 4.39 (d, J = 11.9 Hz, 1H), 4.42 (d, J = 12.0 Hz,
1H), 4.48 (d, J = 12.0 Hz, 1H), 4.57 (d, J = 11.5 Hz, 1H), 4.65 (d, J =
11.9 Hz, 1H), 4.66 (ddd, J = 3.8, 8.9, 11.0 Hz, 1H), 4.92 (d, J = 3.8 Hz,
1H), 4.96 (d, J = 11.5 Hz, 1H), 5.15 (ddd, J = 1.1, 1.4, 10.2 Hz, 1H),
5.19 (ddd, J = 1.4, 1.5, 17.4 Hz, 1H), 5.25 (br d, J = 8.9 Hz, 1H), 5.85
(m, 1H), 6.89 (m, 2H), 7.22−7.35 (m, 12H); 13C NMR (126 MHz,
CDCl3) δ 23.4, 48.9, 55.2, 68.1, 68.9, 69.6, 70.8, 72.5, 73.4, 74.3, 76.5,
96.9, 113.7, 117.1, 127.4, 127.7, 127.8, 128.0, 128.1, 128.3, 129.2, 130.1,
133.8, 137.8, 138.5, 159.2, 169.6; HRMS (FAB) m/z [M + H]+ calcd for
C33H40NO7 562.2805, found 562.2794. Anal. Calcd for C33H39NO7: C,
70.57; H, 7.00; N, 2.49. Found: C, 70.57; H, 6.92; N, 2.41.
2-Acetamido-3-O-acetyl-4,6-di-O-benzyl-2-deoxy-D-galacto-
pyranosyl Acetate (18). Acetic anhydride (0.65 mL, 6.93 mmol)
was added to a stirred solution of diol 17 (927 mg, 2.31 mmol) and
DMAP (28 mg, 0.23 mmol) in pyridine (20 mL). After 1.5 h of
stirring, the reaction was quenched with 10% aqueous HCl (60 mL),
and the resulting mixture was extracted with AcOEt (140 mL). The
organic extract was successively washed with brine (20 mL), saturated
aqueous NaHCO3 (2 × 50 mL), and brine (2 × 50 mL) and dried
over anhydrous Na2SO4. Filtration and evaporation in vacuo furnished
the crude product (1.19 g), which was purified by column chromato-
graphy (silica gel 20 g, 4:1 CH2Cl2/acetone) to give diacetate 18
(1.11 g, 99%, α:β = 84:16) as a colorless oil. The α- and β-anomers
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Article
were separated by flash column chromatography with 5:1 CH2Cl2/
acetone.
to give diethyl phosphite 11 (611 mg, 82%) as a white solid. Rf 0.35
(3:1 n-hexane/acetone, with Et3N-doped silica gel plate); [α]2D3 +89.2
(c 1.04, CHCl3); mp 70.5−71.5 °C; IR (neat) 3290, 3064, 3031, 2978,
Data for α-anomer: Rf 0.42 (4:1 CH2Cl2/acetone); [α]2D2 +85.4
1
1
(c 1.05, CHCl3); IR (neat) 3282, 1741, 1659, 1548, 1226 cm−1; H
2928, 1741, 1651, 1237, 1027 cm−1; H NMR (500 MHz, CDCl3) δ
NMR (400 MHz, CDCl3) δ 1.93 (s, 3H), 2.04 (s, 3H), 2.14 (s, 3H),
3.53 (dd, J = 5.3, 9.1 Hz, 1H), 3.64 (dd, J = 8.3, 9.1 Hz, 1H), 4.00 (br
d, J = 2.9 Hz, 1H), 4.04 (dd, J = 5.3, 8.3 Hz, 1H), 4.40 (d, J = 11.5 Hz,
1H), 4.46 (d, J = 11.5 Hz, 1H), 4.56 (d, J = 11.4 Hz, 1H), 4.79 (d, J =
11.4 Hz, 1H), 4.87 (ddd, J = 3.5, 9.4, 11.4 Hz, 1H), 5.20 (dd, J = 2.9,
11.4 Hz, 1H), 5.50 (d, J = 9.4 Hz, 1H), 6.17 (d, J = 3.5 Hz, 1H), 7.26−
7.36 (m, 10H); 13C NMR (100 MHz, CDCl3) δ 20.9, 21.0, 23.1,
47.5, 67.6, 70.7, 71.2, 73.4, 73.8, 75.0, 91.5, 127.6, 127.7, 127.8, 128.2,
128.3, 137.4, 137.7, 169.0, 169.9, 171.3; HRMS (FAB) m/z [M + H]+
calcd for C26H32NO8 486.2128, found 486.2113. Anal. Calcd for
C26H31NO8: C, 64.32; H, 6.44; N, 2.88. Found: C, 64.04; H, 6.37;
N, 2.94.
Data for β-anomer: Rf 0.37 (4:1 CH2Cl2/acetone); mp 152.0−
152.5 °C (colorless needles from acetone/n-hexane); [α]2D1 +9.49
(c 1.00, CHCl3); IR (KBr) 3336, 1750, 1727, 1658, 1543, 1235 cm−1;
1H NMR (400 MHz, CDCl3) δ 1.92 (s, 3H), 2.01 (s, 3H), 2.08 (s,
3H), 3.57−3.65 (m, 2H), 3.80 (br t, J = 6.8 Hz, 1H), 3.94 (br d, J =
2.9 Hz, 1H), 4.41 (d, J = 11.7 Hz, 1H), 4.47 (d, J = 11.7 Hz, 1H), 4.57
(d, J = 11.7 Hz, 1H), 4.58 (ddd, J = 8.8, 9.6, 11.4 Hz, 1H), 4.76 (d, J =
11.7 Hz, 1H), 5.00 (dd, J = 2.9, 11.4 Hz, 1H), 5.39 (d, J = 9.6 Hz, 1H),
5.62 (d, J = 8.8 Hz, 1H), 7.26−7.33 (m, 10H); 13C NMR (100 MHz,
CDCl3) δ 20.8, 20.9, 50.2, 67.5, 73.1, 73.2, 74.0, 74.9, 93.1, 127.6,
127.7, 128.0, 128.1, 128.3, 137.4, 137.7, 169.6, 170.0, 170.8; HRMS
(FAB) m/z [M + H]+ calcd for C26H32NO8 486.2128, found 486.2139.
Anal. Calcd for C26H31NO8: C, 64.32; H, 6.44; N, 2.88. Found: C,
64.06; H, 6.30; N, 2.93.
1.23 (t, J = 6.9 Hz, 3H), 1.26 (t, J = 6.9 Hz, 3H), 1.94 (s, 3H), 2.03 (s,
3H), 3.53 (dd, J = 5.7, 9.1 Hz, 1H), 3.61 (dd, J = 7.5, 9.1 Hz, 1H),
3.86−3.94 (m, 4H), 3.93 (br d, J = 2.9 Hz, 1H), 4.21 (dd, J = 5.7, 7.5
Hz, 1H), 4.42 (d, J = 12.0 Hz, 1H), 4.48 (d, J = 12.0 Hz, 1H), 4.55 (d,
J = 12.0 Hz, 1H), 4.78 (ddd, J = 3.5, 9.7, 10.9 Hz, 1H), 4.79 (d, J =
12.0 Hz, 1H), 5.21 (dd, J = 2.9, 10.9 Hz, 1H), 5.53 (dd, J = 3.5, 8.0 Hz,
1H), 5.65 (d, J = 9.7 Hz, 1H), 7.26−7.35 (m, 10H); 13C NMR
(100 MHz, CDCl3) δ 16.9 (d, JC−P = 4.7 Hz), 17.1 (d, JC−P = 4.8 Hz),
21.1, 23.4, 48.8, 58.5 (d, JC−P = 10.7 Hz), 58.7 (d, JC−P = 11.9 Hz),
68.1, 70.2, 71.2, 73.3, 74.2, 74.9, 92.7 (d, JC−P = 14.3 Hz), 127.41,
127.45, 127.8, 128.0, 128.1, 137.5, 137.7, 169.4, 170.9; 31P NMR
(202.5 MHz, CDCl3) δ 139.9; HRMS (ESI) m/z [M + Na]+ calcd
for C28H38NO9PNa 586.2182, found 586.2183. Anal. Calcd for
C28H38NO9P: C, 59.67; H, 6.80; N, 2.49. Found: C, 59.43; H, 6.69;
N, 2.50.
Allyl 2,6-Di-O-benzyl-3,4-O-[(R)-4-methoxybenzylidene]-α-D-
galactopyranoside (21) and Allyl 2,6-Di-O-benzyl-3,4-O-[(S)-4-
methoxybenzylidene]-α-D-galactopyranoside (22). p-Toluene-
sulfonic acid (76.9 mg, 0.41 mmol) was added to an ice-cooled (0 °C)
solution of diol 20 (1.62 g, 4.05 mmol) and p-methoxybenzaldehyde
dimethyl acetal (0.86 mL, 4.85 mmol) in MeCN (10 mL). After
30 min of stirring at room temperature, the reaction was quenched
with Et3N (0.1 mL), and the volatile elements were removed in vacuo.
Purification of the residue (2.59 g) by flash column chromatography
(silica gel 60 g, 10:1 n-hexane/AcOEt) afforded (R)-isomer 21 (898 mg,
43%) and (S)-isomer 22 (1.05 g, 50%) as colorless oils.
Data for (R)-isomer 21: Rf 0.62 (2:1 n-hexane/AcOEt); [α]2D1 +47.1
(c 1.01, CHCl3); IR (neat) 3063, 3030, 2911, 2870, 1613, 1514, 1454,
1249, 1095, 1031 cm−1; 1H NMR (500 MHz, CDCl3) δ 3.66 (dd, J =
3.5, 7.9 Hz, 1H), 3.71−3.74 (m, 2H), 3.81 (s, 3H), 4.03 (dd, J = 6.2,
12.9 Hz, 1H), 4.18−4.24 (m, 3H), 4.50 (d, J = 12.3 Hz, 1H), 4.61 (d,
J = 12.3 Hz, 1H), 4.68 (dd, J = 5.0, 7.9 Hz, 1H), 4.77 (d, J = 12.6 Hz,
1H), 4.84 (d, J = 12.6 Hz, 1H), 4.93 (d, J = 3.5 Hz, 1H), 5.23 (d, J =
10.3 Hz, 1H), 5.35 (d, J = 17.0 Hz, 1H), 5.90 (s, 1H), 5.94 (m, 1H),
6.87−6.90 (m, 2H), 7.24−7.41 (m, 12H); 13C NMR (100 MHz,
CDCl3) δ 55.3, 66.8, 68.3, 69.6, 72.2, 73.1, 73.3, 73.6, 77.1, 95.7, 102.4,
113.5, 117.7, 127.2, 127.26, 127.34, 127.5, 127.6, 127.7, 128.0, 128.1,
128.2, 131.0, 133.3, 137.7, 137.8, 159.8; HRMS (FAB) m/z [M + H]+
calcd for C31H35O7 519.2383, found 519.2375. Anal. Calcd for
C31H34O7: C, 71.80; H, 6.61. Found: C, 71.73; H, 6.65.
2-Acetamido-3-O-acetyl-4,6-di-O-benzyl-2-deoxy-D-galacto-
pyranose (19). NaHCO3 (281 mg, 3.34 mmol) and 35% aqueous
H2O2 (1.0 mL) were added to a stirred solution of diacetate 18
(813 mg, 1.67 mmol) in 5:1 THF/MeOH (24 mL). After 24 h of
stirring, the reaction was quenched with 10% aqueous Na2S2O3
(20 mL), and the resulting mixture was extracted with AcOEt
(100 mL). The organic extract was successively washed with half-
saturated brine (2 × 20 mL) and brine (30 mL) and dried over
anhydrous Na2SO4. Filtration and evaporation in vacuo furnished the
crude product (753 mg, white amorphous solid), which was purified
by column chromatography (silica gel 15 g, 4:1 → 2:1 CH2Cl2/
acetone) to give hemiacetal 19 (679 mg, 92%, α:β = 95:5) as a white
1
solid. The anomeric α:β ratio of the product was determined by H
NMR. Rf 0.53 (α-anomer), 0.44 (β-anomer) (2:3 CH2Cl2/acetone);
mp 131.5−132.5 °C (colorless needles from 8:1 acetone/n-hexane);
[α]2D3 +49.0 (c 1.02, CHCl3); IR (KBr) 3391, 3311, 1731, 1656, 1547,
1250 cm−1; 1H NMR (500 MHz, CDCl3, data for α-anomer) δ 1.94 (s,
3H), 2.04 (s, 3H), 3.46 (dd, J = 6.8, 9.7 Hz, 1H), 3.56 (dd, J = 6.8, 9.7
Hz, 1H), 3.58 (br d, J = 3.5 Hz, 1H), 3.87 (br d, J = 2.9 Hz, 1H), 4.22
(br t, J = 6.8 Hz, 1H), 4.41 (d, J = 12.0 Hz, 1H), 4.49 (d, J = 12.0 Hz,
1H), 4.50 (d, J = 12.0 Hz, 1H), 4.71 (ddd, J = 3.5, 9.7, 11.4 Hz, 1H),
4.80 (d, J = 12.0 Hz, 1H), 5.21 (dd, J = 2.9, 11.4 Hz, 1H), 5.26 (t, J =
3.5 Hz, 1H), 5.73 (d, J = 9.7 Hz, 1H), 7.26−7.35 (m, 10H); 13C NMR
(100 MHz, CDCl3, data for α-anomer) δ 21.0, 23.2, 48.6, 68.87, 68.94,
71.3, 73.2, 74.5, 74.8, 91.9, 127.66, 127.69, 127.74, 128.1, 128.2, 128.3,
137.4, 137.8, 170.3, 171.1; HRMS (FAB) m/z [M + H]+ calcd for
C24H30NO7 444.2022, found 444.2020. Anal. Calcd for C24H29NO7: C,
65.00; H, 6.59; N, 3.16. Found: C, 64.86; H, 6.54; N, 3.18.
2-Acetamido-3-O-acetyl-4,6-di-O-benzyl-2-deoxy-α-D-galac-
topyranosyl Diethyl Phosphite (11). Diethyl chlorophosphite
(0.23 mL, 1.60 mmol) was added to an ice-cooled (0 °C) solution of
hemiacetal 19 (591 mg, 1.33 mmol) and Et3N (0.56 mL, 4.00 mmol)
in CH2Cl2 (13 mL). After 30 min of stirring at 0 °C, the reaction was
quenched with crushed ice, followed by stirring at room temperature
for 10 min. The resulting mixture was partitioned between AcOEt
(45 mL) and saturated aqueous NaHCO3 (10 mL), and the organic
layer was washed with brine (2 × 10 mL) and dried over anhydrous
Na2SO4. Filtration and evaporation in vacuo furnished the crude
product (935.2 mg, white solid), which was purified by column
chromatography (Wako gel 15 g, 3:1 n-hexane/acetone with 3% Et3N)
Data for (S)-isomer 22: Rf 0.59 (2:1 n-hexane/AcOEt); [α]2D0 +23.4
(c 1.00, CHCl3); IR (neat) 3063, 3029, 2911, 2870, 1614, 1518, 1454,
1250, 1092, 1032 cm−1; 1H NMR (500 MHz, CDCl3) δ 3.58 (dd, J =
3.5, 7.7 Hz, 1H), 3.75 (dd, J = 7.1, 10.3 Hz, 1H), 3.82 (dd, J = 5.0, 10.3
Hz, 1H), 3.83 (s, 3H), 4.03 (dd, J = 6.4, 12.9 Hz, 1H), 4.21 (dd, J =
5.0, 12.9 Hz, 1H), 4.25−4.32 (m, 2H), 4.49 (dd, J = 6.2, 7.7 Hz, 1H),
4.54 (d, J = 12.0 Hz, 1H), 4.60 (d, J = 12.6 Hz, 1H), 4.63 (d, J = 12.0
Hz, 1H), 4.73 (d, J = 12.6 Hz, 1H), 4.85 (d, J = 3.5 Hz, 1H), 5.22 (dd,
J = 1.6, 10.3 Hz, 1H), 5.34 (dd, J = 1.6, 17.3 Hz, 1H), 5.84 (s, 1H),
5.94 (m, 1H), 6.84−6.87 (m, 2H), 7.20−7.35 (m, 12H); 13C NMR
(100 MHz, CDCl3) δ 55.2, 66.5, 68.3, 69.3, 72.1, 73.3, 75.5, 76.1, 76.7,
95.9, 103.9, 113.5, 117.8, 127.38, 127.44, 127.8, 128.0, 128.1, 128.2,
129.5, 133.5, 137.9, 138.0, 160.1; HRMS (FAB) m/z [M + H]+ calcd
for C31H35O7 519.2383, found 519.2396. Anal. Calcd for C31H34O7:
C, 71.80; H, 6.61. Found: C, 71.71; H, 6.65.
Allyl 2,6-Di-O-benzyl-4-O-(4-methoxybenzyl)-α-D-galacto-
pyranoside (13). TFA (0.26 mL, 3.38 mmol) was added to a cooled
(10 °C) mixture of acetal 22 (877 mg, 1.69 mmol), NaBH3CN
(164 mg, 2.54 mmol) and pulverized 4 Å molecular sieves (800 mg) in
THF (12 mL). After 10 min of stirring at this temperature, the reaction
was quenched with Et3N (0.5 mL). The resulting mixture was diluted
with AcOEt (15 mL) and passed through a Celite pad. The filtrate was
partitioned between AcOEt (15 mL) and saturated aqueous NaHCO3
(10 mL). The organic layer was successively washed with saturated
aqueous NaHCO3 (10 mL) and brine (2 × 10 mL), and dried over
anhydrous Na2SO4. Filtration and evaporation in vacuo furnished the
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crude product (1.45 g, slightly yellow oil), which was purified by
column chromatography (silica gel 30 g, 2:1 n-hexane/AcOEt) to give
alcohol 13 (847 mg, 96%) as a white solid. Rf 0.50 (15:1 CH2Cl2/
acetone); mp 68.0−69.0 °C; [α]2D0 +54.8 (c 1.00, CHCl3); IR (neat)
3437, 2927, 2872, 1613, 1516, 1249, 1101 cm−1; 1H NMR (400 MHz,
CDCl3) δ 2.27 (d, J = 4.7 Hz, 1H), 3.54 (d, J = 6.4 Hz, 2H), 3.78 (s,
3H), 3.79 (dd, J = 3.5, 9.1 Hz, 1H), 3.89−3.94 (m, 2H), 3.99 (t, J = 6.4
Hz, 1H), 4.07 (ddd, J = 4.1, 4.7, 9.1 Hz, 1H), 4.13 (dd, J = 5.0, 12.9 Hz,
1H), 4.43 (d, J = 11.7 Hz, 1H), 4.52 (d, J = 11.7 Hz, 1H), 4.57 (d, J =
11.5 Hz, 1H), 4.64 (d, J = 11.8 Hz, 1H), 4.68 (d, J = 11.8 Hz, 1H), 4.73
(d, J = 11.5 Hz, 1H), 4.88 (d, J = 3.5 Hz, 1H), 5.18 (d, J = 10.3 Hz,
1H), 5.29 (d, J = 17.0 Hz, 1H), 5.89 (m, 1H), 6.84 (m, 2H), 7.20−7.37
(m, 12H); 13C NMR (100 MHz, CDCl3) δ 55.3, 68.4, 68.9, 69.2, 70.1,
72.7, 73.3, 74.6, 76.0, 77.3, 95.6, 113.5, 117.5, 127.38, 127.42, 127.6,
127.8, 128.1, 128.2, 129.6, 130.3, 133.6, 137.7, 137.8, 158.9; HRMS
(FAB) m/z [M + H]+ calcd for C31H37O7 521.2540, found 521.2545.
Anal. Calcd for C31H36O7: C, 71.52; H, 6.97. Found: C, 71.33; H, 6.94.
Allyl 4-O-(2-Azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-
β-D-mannopyranosyl)-2,3,6-tri-O-benzyl-α-D-galactopyrano-
side (23). A 1.0 M solution of TMSOTf in CH2Cl2 (2.25 mL,
2.25 mmol) was added to a cooled (−30 °C) mixture of diphenyl
phosphate 5 (923 mg, 1.50 mmol), 4-O-unprotected galactoside 12
(810 mg, 1.65 mmol), and pulverized 4 Å molecular sieves (920 mg)
in CH2Cl2 (15 mL). After 3 h of stirring at −30 °C, the reaction was
quenched with Et3N (3 mL). The mixture was diluted with AcOEt
(25 mL) and passed through a Celite pad. The filtrate was partitioned
between AcOEt (35 mL) and saturated aqueous NaHCO3 (10 mL).
The organic layer was successively washed with saturated aqueous
NaHCO3 (10 mL) and brine (2 × 10 mL) and dried over anhydrous
Na2SO4. Filtration and evaporation in vacuo furnished the crude
product (1.65 g, slightly yellow oil), which was purified by column
chromatography (silica gel 35 g, 9:1 → 6:1 n-hexane/AcOEt) to give a
mixture of β-linked disaccharide 23 and its α-isomer (1.192 g, 93%) as
a slightly yellow oil. The anomeric ratio of the disaccharides was
determined to be 8:92 by HPLC analysis [eluent, 6:1 n-hexane/AcOEt;
flow rate, 1.0 mL/min; tR (α-anomer) = 16.6 min, tR (β-anomer 23) =
26.0 min]. Separation of disaccharides by flash column chromatography
(silica gel 40 g, 9:1 → 6:1 n-hexane/AcOEt) afforded β-linked
disaccharide 23 (1.042 g, 81%) as a colorless oil, along with its α-isomer
(102 mg, 8%) as a colorless oil. Rf 0.41 (4:1 n-hexane/AcOEt);
[α]2D1 +5.88 (c 1.00, EtOH); IR (neat) 3030, 2868, 2106, 1497, 1454,
1379, 1098, 1057 cm−1; 1H NMR (500 MHz, CDCl3) δ 3.12 (ddd, J =
4.9, 9.6, 10.4 Hz, 1H), 3.44 (dd, J = 3.6, 9.6 Hz, 1H), 3.62 (dd, J = 5.8,
9.5 Hz, 1H), 3.74 (dd, J = 6.9, 9.5 Hz, 1H), 3.77 (t, J = 10.4 Hz, 1H),
3.91 (t, J = 9.6 Hz, 1H), 3.93 (dd, J = 3.2, 10.1 Hz, 1H), 3.95 (dd, J =
2.6, 10.1 Hz, 1H), 3.96−4.04 (m, 3H), 4.11 (dd, J = 4.9, 10.4 Hz, 1H),
4.14−4.18 (m, 2H), 4.53 (s, 2H), 4.58 (d, J = 12.2 Hz, 1H), 4.59 (d, J =
11.3 Hz, 1H), 4.67 (d, J = 12.2 Hz, 2H), 4.76 (d, J = 12.2 Hz, 1H), 4.77
(d, J = 3.6 Hz, 1H), 4.89 (d, J = 3.2 Hz, 1H), 4.92 (d, J = 11.3 Hz, 1H),
5.21 (dd, J = 1.4, 10.1 Hz, 1H), 5.30 (dd, J = 1.4, 17.2 Hz, 1H), 5.54 (s,
1H), 5.92 (m, 1H), 7.25−7.36 (m, 23H), 7.45 (m, 2H); 13C NMR
(100 MHz, CDCl3) δ 63.1, 67.3, 68.2, 68.3, 68.7, 68.8, 72.5, 73.1, 73.3,
73.9, 76.2, 76.5, 77.9, 78.0, 95.7, 101.2, 101.5, 117.9, 125.7, 127.1,
127.27, 127.34, 127.49, 127.53, 127.6, 127.8, 127.9, 128.09, 128.13,
128.3, 128.7, 133.5, 136.9, 137.5, 137.9, 138.0, 138.2; HRMS (FAB)
m/z [M + H]+ calcd for C50H54N3O10 856.3809, found 856.3815.
Anal. Calcd for C50H53N3O10: C, 70.16; H, 6.24; N, 4.91. Found: C,
69.94; H, 6.25; N, 4.89.
5.29 (dd, J = 1.8, 17.4 Hz, 1H), 5.56 (s, 1H), 5.92 (m, 1H), 7.18−7.41
(m, 23H), 7.48 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 63.0, 64.1,
67.8, 68.40, 68.44, 72.9, 73.2, 73.6, 73.9, 74.4, 76.1, 76.5, 76.9, 79.2,
96.4, 100.4, 101.2, 118.0, 126.0, 127.3, 127.4, 127.6, 127.97, 128.03,
128.18, 128.24, 128.28, 128.32, 128.5, 128.6, 133.6, 136.9, 137.6,
138.1, 138.2; HRMS (FAB) m/z [M + H]+ calcd for C50H54N3O10
856.3809, found 856.3805. Anal. Calcd for C50H53N3O10: C, 70.16;
H, 6.24; N, 4.91. Found: C, 70.03; H, 6.30; N, 4.85.
Allyl 4-O-(2-Acetamido-3-O-benzyl-4,6-O-benzylidene-2-
deoxy-β-D-mannopyranosyl)-2,3,6-tri-O-benzyl-α-D-galacto-
pyranoside (24). Triphenylphosphine (309 mg, 1.18 mmol) was
added to a stirred solution of azide 23 (916 mg, 1.07 mmol) in THF
(10 mL). After 30 min of stirring, H2O (0.2 mL) was added, and the
mixture was refluxed for 10 h. The reaction mixture was partitioned
between AcOEt (30 mL) and brine (15 mL), and the organic layer was
dried over anhydrous Na2SO4. Filtration and evaporation in vacuo
furnished the colorless oil, which was used without further purification.
Acetic anhydride (0.30 mL, 3.18 mmol) was added to a stirred
solution of the crude amine in pyridine (4 mL). After 3 h of stirring,
the reaction mixture was partitioned between AcOEt (30 mL) and
10% aqueous HCl (15 mL). The organic layer was successively washed
with H2O (5 mL), saturated aqueous NaHCO3 (10 mL), and brine
(10 mL) and dried over anhydrous Na2SO4. Filtration and evapora-
tion in vacuo furnished the crude product (1.59 g, slightly yellow
amorphous solid), which was purified by column chromatography
(silica gel 30 g, 2:1 → 2:3 n-hexane/AcOEt) to give acetamide 24
(908 mg, 97%) as a white amorphous solid. Rf 0.31 (5:1 toluene/
acetone); [α]2D1 +7.62 (c 1.00, CHCl3); IR (KBr) 3437, 3335, 2922,
2870, 1679, 1454, 1372, 1099, 1027 cm−1; 1H NMR (400 MHz,
acetone-d6) δ 1.90 (br s, 3H), 3.32 (ddd, J = 4.7, 9.7, 10.0 Hz, 1H),
3.53 (dd, J = 4.4, 9.7 Hz, 1H), 3.58 (dd, J = 6.1, 10.0 Hz, 1H), 3.68 (t,
J = 10.0 Hz, 1H), 3.72 (dd, J = 5.6, 10.0 Hz, 1H), 3.85 (t, J = 9.7 Hz,
1H), 3.95−4.01 (m, 4H), 4.07 (dd, J = 4.7, 10.0 Hz, 1H), 4.15 (ddd,
J = 1.5, 5.0, 13.2 Hz, 1H), 4.33 (d, J = 12.0 Hz, 1H), 4.37 (br s, 1H),
4.51 (d, J = 11.7 Hz, 1H), 4.56 (d, J = 11.7 Hz, 1H), 4.69 (d, J = 11.5
Hz, 1H), 4.70 (d, J = 12.0 Hz, 1H), 4.78 (d, J = 11.7 Hz, 1H), 4.80 (d,
J = 11.5 Hz, 1H), 4.82 (d, J = 2.7 Hz, 1H), 4.85 (d, J = 11.7 Hz, 1H),
5.03 (d, J = 1.8 Hz, 1H), 5.08 (ddd, J = 1.8, 4.4, 9.7 Hz, 1H), 5.12
(ddd, J = 1.5, 1.8, 11.7 Hz, 1H), 5.32 (dd, J = 1.8, 17.3 Hz, 1H), 5.89
(s, 1H), 5.94 (m, 1H), 6.99 (d, J = 9.7 Hz, 1H), 7.20−7.36 (m, 19H),
7.44−7.48 (m, 6H); 13C NMR (100 MHz, CDCl3) δ 23.3, 50.2, 66.5,
68.2, 68.7, 68.8, 69.3, 71.7, 73.2, 73.6, 73.9, 75.7, 76.2, 76.7, 78.2, 79.0,
96.0, 100.8, 101.2, 117.8, 125.7, 127.1, 127.2, 127.4, 127.5, 127.6,
127.8, 127.9, 127.96, 127.98, 128.1, 128.3, 128.7, 133.6, 136.9, 137.8,
137.9, 138.0, 138.2, 170.0; HRMS (FAB) m/z [M + H]+ calcd for
C52H58NO11 872.4010, found 872.4024.
4-O-(2-Acetamido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-
β-D-mannopyranosyl)-2,3,6-tri-O-benzyl-D-galactopyranose
(25). Potassium tert-butoxide (140 mg, 1.25 mmol) was added to a
stirred solution of allyl glycoside 24 (906 mg, 1.04 mmol) in DMSO
(5 mL), and the mixture was heated at 100 °C for 20 min. After
cooling to room temperature, the reaction was quenched with H2O
(1 mL), and the mixture was partitioned between AcOEt (25 mL) and
saturated aqueous NH4Cl (5 mL). The organic layer was washed with
brine (2 × 10 mL) and dried over anhydrous Na2SO4. Filtration and
evaporation in vacuo furnished the slightly yellow oil, which was used
without further purification.
NBS (203 mg, 1.14 mmol) was added to a stirred solution of
the crude prop-1-enyl glycoside in 20:1 THF/H2O (10.5 mL). After
5 min of stirring, the volatile elements were removed in vacuo, and the
residue was partitioned between AcOEt (30 mL) and 10% aqueous
Na2S2O3 (10 mL). The organic layer was successively washed with
H2O (2 × 10 mL) and brine (10 mL) and dried over anhydrous
Na2SO4. Filtration and evaporation in vacuo furnished the crude
product (938 mg, slightly yellow oil), which was purified by column
chromatography (silica gel 35 g, 1:1 n-hexane/AcOEt with 0.5% Et3N)
to give hemiacetal 25 (726 mg, 84%, α:β = 55:45) as a slightly
yellow amorphous solid. Rf 0.23 (3:1 toluene/acetone); [α]2D2 −21.4
Data for α-isomer: Rf 0.45 (4:1 n-hexane/AcOEt); [α]2D3 +55.7
1
(c 1.02, CH2Cl2); IR (neat) 3032, 2106, 1735, 1454, 1371 cm−1; H
NMR (400 MHz, CDCl3) δ 3.38 (t, J = 9.1 Hz, 1H), 3.46 (dd, J = 5.4,
9.1 Hz, 1H), 3.58 (t, J = 10.0 Hz, 1H), 3.70 (dd, J = 5.0, 10.0 Hz, 1H),
3.82 (dd, J = 3.7, 10.0 Hz, 1H), 3.84 (dd, J = 2.3, 3.6 Hz, 1H), 3.85
(dd, J = 2.7, 10.0 Hz, 1H), 3.88 (ddd, J = 3.6, 5.4, 9.1 Hz, 1H), 3.96
(dd, J = 3.6, 10.0 Hz, 1H), 3.97 (m, 1H), 4.03 (t, J = 10.0 Hz, 1H),
4.08 (m, 1H), 4.11 (dd, J = 2.7, 3.6 Hz, 1H), 4.22 (dt, J = 5.0, 10.0 Hz,
1H), 4.47 (d, J = 11.3 Hz, 1H), 4.53 (d, J = 11.3 Hz, 1H), 4.64 (d, J =
11.8 Hz, 1H), 4.73 (d, J = 12.2 Hz, 1H), 4.74 (d, J = 12.2 Hz, 2H),
4.79 (d, J = 3.7 Hz, 1H), 4.80 (d, J = 2.3 Hz, 1H), 4.84 (d, J = 12.2 Hz,
1H), 4.90 (d, J = 11.8 Hz, 1H), 5.22 (dd, J = 1.8, 10.5 Hz, 1H),
1
(c 1.01, MeCN); IR (Nujol) 3328, 1659, 1496, 1212 cm−1; H NMR
(500 MHz, acetone-d6) δ 1.90 (s, 3H), 3.30−3.35 (m, 1H),
4284
DOI: 10.1021/acs.joc.5b00139
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The Journal of Organic Chemistry
Article
3.52−3.57 (m, 1.55H), 3.62 (dd, J = 5.2, 8.6 Hz, 0.45H), 3.66−3.76
(m, 3.35H), 3.84−3.89 (m, 1H), 3.95 (dd, J = 3.5, 10.3 Hz, 0.55H),
4.02 (dd, J = 2.9, 10.3 Hz, 0.55H), 4.05−4.20 (m, 1H), 4.19 (br dd, J =
5.7, 6.3 Hz, 0.55H), 4.31 (br s, 0.45H), 4.33−4.38 (m, 1.55H), 4.49−
4.57 (m, 2.1H), 4.63 (br dd, J = 6.3, 6.9 Hz, 0.45H), 4.70−4.86 (m,
4.45H), 4.98 (d, J = 11.5 Hz, 0.45H), 5.01−5.10 (m, 1H), 5.04 (d, J =
1.7 Hz, 0.55H), 5.07 (d, J = 1.7 Hz, 0.45H), 5.20 (dd, J = 3.5, 4.0 Hz,
0.55H), 5.34 (d, J = 4.0 Hz, 0.55H), 5.50 (s, 0.55H), 5.51 (s, 0.45H),
5.91 (d, J = 6.9 Hz, 0.45H), 6.94 (d, J = 9.8 Hz, 0.45H), 6.98 (d, J =
9.7 Hz, 0.55H), 7.20−7.48 (m, 25H); 13C NMR (100 MHz, acetone-
d6) δ 22.4, 24.2, 51.75, 51.79, 68.8, 70.0, 70.3, 71.3, 72.2, 74.2, 74.3,
74.37, 74.44, 74.5, 75.6, 76.2, 77.4, 78.4, 78.7, 79.7, 79.9, 82.5, 83.6, 93.1,
99.4, 102.4, 102.8, 102.9, 126.7, 127.7, 128.46, 128.52, 128.6, 128.7,
128.77, 128.79, 128.85, 128.96, 128.99, 129.1, 129.2, 129.3, 129.4, 129.5,
129.6, 129.8, 130.1, 130.3, 139.6, 140.4, 140.6, 141.0, 141.1, 171.0, 171.1;
HRMS (FAB) m/z [M + H]+ calcd for C49H54NO11 832.3697, found
832.3685. Anal. Calcd for C49H53NO11: C, 70.74; H, 6.42; N, 1.62.
Found: C, 71.04; H, 6.59; N, 1.65.
HRMS (ESI) m/z [M + Na]+ calcd for C61H62NO14PNa 1086.3806,
found 1086.3804. Anal. Calcd for C61H62NO14P: C, 68.85; H, 5.87;
N, 1.32. Found: C, 68.68; H, 6.13; N, 1.33.
Allyl 3-O-(2-Acetamido-3-O-acetyl-4,6-di-O-benzyl-2-deoxy-
β-D-galactopyranosyl)-2,6-di-O-benzyl-4-O-(4-methoxyben-
zyl)-α-D-galactopyranoside (26). A 1.0 M solution of Tf2NH in
EtCN (0.55 mL, 0.55 mmol) was added to a cooled (−78 °C) mixture
of diethyl phosphite 11 (280 mg, 0.50 mmol), 3-O-unprotected
galactoside 13 (776 mg, 1.49 mmol), and pulverized 4 Å molecular
sieves (500 mg) in CH2Cl2 (5 mL). After 2 h of stirring at this
temperature, the reaction was quenched with Et3N (0.3 mL). The
mixture was diluted with AcOEt (5 mL) and passed through a Celite
pad. The filtrate was partitioned between AcOEt (50 mL) and saturated
aqueous NaHCO3 (10 mL). The organic layer was successively washed
with saturated aqueous NaHCO3 (10 mL) and brine (2 × 10 mL), and
dried over anhydrous Na2SO4. Filtration and evaporation in vacuo
furnished the crude product (1.25 g, slightly yellow oil), which was
purified by flash column chromatography (silica gel 40 g, 8:1 → 6:1
toluene/acetone) to give β-linked disaccharide 26 (305 mg, 65%)
as a white solid, along with recovered alcohol 13 (594 mg). Rf
0.25 (4:1 toluene/acetone); mp 117.5−118.5 °C (colorless plates from
AcOEt/n-hexane); [α]2D1 −8.8 (c 1.02, CHCl3); IR (KBr) 3259, 2932,
4-O-(2-Acetamido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-
β-D-mannopyranosyl)-2,3,6-tri-O-benzyl-D-galactopyranosyl
Diphenyl Phosphate (9). Diphenyl chlorophosphite (50 mg,
0.20 mmol) was added to an ice-cooled (0 °C) solution of hemiacetal
25 (83 mg, 0.10 mmol) and Et3N (55 μL, 0.39 mmol) in CH2Cl2
(2 mL). After 10 min of stirring at 0 °C, the reaction was quenched
by addition of a piece of crushed ice, followed by stirring at room
temperature for 10 min. The mixture was partitioned between AcOEt
(15 mL) and saturated aqueous NaHCO3 (5 mL), and the organic
layer was washed with brine (2 × 10 mL) and dried over anhydrous
Na2SO4. Filtration and evaporation in vacuo furnished the crude
product (164 mg, slightly yellow oil), which was purified by column
chromatography (Wako gel 4 g, 2:1 → 1:1 n-hexane/AcOEt) to give
the corresponding diphenyl phosphite (92 mg, 88%) as a colorless oil.
Potassium peroxymonosulfate (Oxone, 162 mg, 0.26 mmol) was
added to an ice-cooled (0 °C) solution of diphenyl phosphite (92 mg,
0.088 mmol) and NaHCO3 (36.9 mg, 0.44 mmol) in 2:1 acetone/
H2O (3 mL). After 30 min of stirring at 0 °C, the reaction mixture was
diluted with Et2O (2 mL) and partitioned between AcOEt (20 mL)
and saturated aqueous NaHCO3 (5 mL). The organic layer was
successively washed with saturated aqueous NaHCO3 (5 mL) and
brine (2 × 5 mL) and dried over anhydrous Na2SO4. Filtration and
evaporation in vacuo furnished the crude product (95 mg), which was
purified by column chromatography (Wako gel 2.5 g, 1:1 n-hexane/
AcOEt with 2% Et3N) to give diphenyl phosphate 9 (86.2 mg, 92%,
α:β = 67:33) as a colorless foam. Rf 0.39 (α-anomer), 0.59 (β-anomer)
(3:1 toluene/acetone); [α]2D2 +7.67 (c 1.05, MeCN); IR (KBr) 3331,
3063, 3032, 2871, 1676, 1591, 1490, 1190 cm−1; 1H NMR (500 MHz,
acetone-d6) δ 1.89 (br s, 1H), 1.90 (br s, 2H), 3.32−3.36 (m, 1H),
3.44 (dd, J = 5.7, 9.2 Hz, 0.67H), 3.55−3.60 (m, 1H), 3.63 (dd, J = 6.5,
10.1 Hz, 0.33H), 3.67−3.73 (m, 1.67H), 3.77 (dd, J = 5.8, 10.1 Hz,
0.33H), 3.85−3.91 (m, 1.33H), 3.95−4.00 (m, 1.33H), 4.06−4.14 (m,
1.67H), 4.18 (dt, J = 9.7, 3.4 Hz, 0.67H), 4.34 (d, J = 12.0 Hz, 0.67H),
4.38 (d, J = 12.0 Hz, 0.33H), 4.40 (br d, J = 1.7 Hz, 0.33H), 4.45 (br d,
J = 0.9 Hz, 0.67H), 4.48 (d, J = 12.1 Hz, 0.67H), 4.52 (d, J = 12.1 Hz,
0.67H), 4.57 (s, 0.67H), 4.68−4.72 (m, 1.33H), 4.76−4.85 (m,
3.67H), 5.03 (d, J = 1.7 Hz, 0.67H), 5.04 (d, J = 1.7 Hz, 0.33H), 5.06−
5.14 (m, 1H), 5.42 (dd, J = 6.9, 8.0 Hz, 0.33H), 5.51 (s, 0.67H), 5.52
(s, 0.33H), 5.99 (dd, J = 3.4, 6.3 Hz, 0.67H), 7.03 (br d, J = 10.3 Hz,
0.33H), 7.04 (br d, J = 10.3 Hz, 0.67H), 7.15−7.48 (m, 35H); 13C
NMR (100 MHz, acetone-d6) δ 22.5, 26.1, 51.2, 68.3, 69.5, 69.9, 70.0,
70.3, 70.9, 71.2, 71.7, 72.8, 73.8, 73.9, 74.7, 75.0, 75.3, 76.1, 76.2, 76.7
(d, JC−P = 6.7 Hz), 77.8, 78.8, 79.4, 80.0 (d, JC−P = 9.3 Hz), 82.7, 99.5
(d, JC−P = 5.8 Hz), 101.2 (d, JC−P = 5.7 Hz), 102.2, 102.5, 116.4, 120.4,
121.18, 121.21, 121.31, 121.35, 121.55, 121.59, 121.67, 121.70, 121.85,
121.89, 126.0, 126.37, 126.40, 126.47, 126.51, 127.4, 128.2, 128.27,
128.33, 128.38, 128.44, 128.50, 128.53, 128.6, 128.7, 128.8, 128.9,
129.0, 129.1, 129.2, 129.25, 129.33, 129.38, 129.42, 129.5, 129.6, 129.8,
130.5, 130.7, 130.75, 130.82, 130.9, 131.0, 139.37, 139.40, 139.8, 139.9,
140.0, 140.07, 140.10, 140.16, 140.19, 140.25, 151.80, 151.85, 151.91,
151.95, 151.97, 152.01, 152.05, 152.36, 152.42, 158.6, 171.1; 31P NMR
(202.5 MHz, acetone-d6) δ −11.3 (α-anomer), −11.6 (β-anomer);
1
2862, 1743, 1646, 1510, 1241, 735, 697 cm−1; H NMR (500 MHz,
CDCl3) δ 1.71 (s, 3H), 2.00 (s, 3H), 3.34 (dd, J = 5.7, 9.8 Hz, 1H), 3.48
(dd, J = 6.3, 9.8 Hz, 1H), 3.56 (dd, J = 5.2, 8.6 Hz, 1H), 3.65−3.72 (m,
2H), 3.73 (s, 3H), 3.89 (br dd, J = 5.7, 6.3 Hz, 1H), 3.91−3.98 (m, 4H),
4.08 (dd, J = 2.9, 10.3 Hz, 1H), 4.10 (ddd, J = 1.1, 5.1, 13.1 Hz, 1H),
4.34 (d, J = 12.0 Hz, 1H), 4.38 (d, J = 11.7 Hz, 1H), 4.43 (d, J = 11.7
Hz, 1H), 4.45 (d, J = 12.0 Hz, 1H), 4.50 (ddd, J = 8.5, 9.3, 11.1 Hz, 1H),
4.54 (d, J = 11.7 Hz, 1H), 4.60 (d, J = 12.0 Hz, 1H), 4.61 (d,
J = 11.5 Hz, 1H), 4.65 (d, J = 12.0 Hz, 1H), 4.72 (d, J = 8.5 Hz, 1H),
4.78 (d, J = 3.6 Hz, 1H), 4.82 (d, J = 11.7 Hz, 1H), 4.86 (d, J = 11.5 Hz,
1H), 4.93 (dd, J = 2.9, 11.1 Hz, 1H), 5.16 (dd, J = 1.7, 10.3 Hz, 1H),
5.17 (d, J = 9.3 Hz, 1H), 5.26 (ddd, J = 1.1, 1.7, 17.2 Hz, 1H),
5.88 (m, 1H), 6.71 (d, J = 6.9 Hz, 2H), 7.18−7.38 (m, 22H); 13C NMR
(100 MHz, CDCl3) δ 21.0, 23.4, 51.6, 55.2, 68.1, 68.2, 69.5, 72.7, 73.1,
73.2, 73.4, 73.7, 73.9, 74.9, 75.5, 76.2, 78.5, 95.8, 102.8, 113.2, 117.8,
127.19, 127.22, 127.25, 127.3, 127.4, 127.5, 127.6, 128.0, 128.2, 128.3,
130.4, 130.6, 133.6, 137.5, 137.86, 137.94, 138.1, 158.6, 169.3, 170.5;
HRMS (FAB) m/z [M + H]+ calcd for C55H64NO13 946.4378, found
946.4385. Anal. Calcd for C55H63NO13: C, 69.82; H, 6.71; N, 1.48.
Found: C, 69.65; H, 6.78; N, 1.42.
Allyl 3-O-(2-Acetamido-3-O-acetyl-4,6-di-O-benzyl-2-deoxy-
β-D-galactopyranosyl)-2,6-di-O-benzyl-α-D-galactopyranoside
(10). DDQ (83 mg, 0.37 mmol) was added in three portions
to a stirred biphasic mixture of PMB ether 26 (295 mg, 0.31 mmol) in
20:1 CH2Cl2/pH 7 phosphate buffer (3.15 mL). After 1 h of stirring,
the reaction mixture was diluted with AcOEt (5 mL) and passed
through a Celite pad. The filtrate was partitioned between AcOEt
(10 mL) and saturated aqueous NaHCO3 (5 mL). The organic layer
was successively washed with saturated aqueous NaHCO3 (3 × 5 mL)
and brine (5 mL) and dried over anhydrous Na2SO4. Filtration
and evaporation in vacuo furnished the crude product (362 mg),
which was purified by column chromatography (silica gel 12 g, 5:1 →
3:1 toluene/acetone) to give alcohol 10 (232 mg, 90%) as a white
amorphous solid. Rf 0.25 (4:1 toluene/acetone); [α]2D3 +22.0 (c 1.02,
CHCl3); IR (KBr) 3472, 3290, 2929, 2865, 1725, 1660, 1496, 1314,
697 cm−1; 1H NMR (500 MHz, CDCl3) δ 1.73 (s, 3H), 1.99 (s, 3H),
2.78 (s, 1H), 3.53 (dd, J = 6.3, 9.2 Hz, 1H), 3.57 (t, J = 9.2 Hz, 1H),
3.669 (dd, J = 6.3, 9.2 Hz, 1H), 3.671 (dd, J = 5.2, 9.2 Hz, 1H), 3.68
(dd, J = 6.3, 9.2 Hz, 1H), 3.84 (dd, J = 3.4, 10.3 Hz, 1H), 3.89 (d, J =
2.9 Hz, 1H), 3.97 (br dd, J = 5.2, 6.3 Hz, 1H), 4.00 (m, 1H), 4.01 (dd,
J = 1.2, 10.3 Hz, 1H), 4.09 (dd, J = 1.2, 1.7 Hz, 1H), 4.15 (dd, J = 5.2,
13.2 Hz, 1H), 4.38 (ddd, J = 8.6, 9.2, 11.2 Hz, 1H), 4.39 (d, J = 12.0
Hz, 1H), 4.43 (d, J = 12.0 Hz, 1H), 4.50 (d, J = 12.0 Hz, 1H), 4.53 (d,
J = 11.5 Hz, 1H), 4.56 (d, J = 12.0 Hz, 1H), 4.61 (d, J = 12.1 Hz, 1H),
4.64 (d, J = 12.1 Hz, 1H), 4.70 (d, J = 8.6 Hz, 1H), 4.71 (d, J = 11.5
Hz, 1H), 4.82 (d, J = 3.4 Hz, 1H), 4.95 (dd, J = 2.9, 11.2 Hz, 1H), 5.15
(d, J = 9.2 Hz, 1H), 5.19 (dd, J = 1.7, 11.3 Hz, 1H), 5.29 (dd, J = 1.7,
17.1 Hz, 1H), 5.92 (m, 1H), 7.23−7.47 (m, 20H); 13C NMR (100 MHz,
4285
DOI: 10.1021/acs.joc.5b00139
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Article
CDCl3) δ 20.6, 23.1, 51.0, 67.9, 68.4, 68.9, 69.4, 72.7, 73.0, 73.09, 73.13,
73.2, 74.7, 75.0, 78.6, 95.7, 102.2, 117.8, 127.2, 127.4, 127.48, 127.53,
127.96, 128.00, 128.1, 128.16, 128.22, 137.3, 137.4, 138.0, 138.3, 170.0,
170.7; HRMS (FAB) m/z [M + H]+ calcd for C47H56NO12 826.3803,
found 826.3797. Anal. Calcd for C47H55NO12: C, 68.35; H, 6.71; N, 1.70.
Found: C, 68.25; H, 6.70; N, 1.67.
71.2, 71.8, 72.2, 73.6, 73.7, 73.8, 73.9, 75.0, 75.1, 75.7, 76.3, 76.4, 77.1,
77.7, 78.0, 78.2, 78.4, 78.6, 79.4, 80.0, 97.1, 99.8, 102.5, 103.0, 104.1,
117.6, 127.4, 128.2, 128.3, 128.35, 128.40, 128.5, 128.58, 128.64,
128.67, 128.71, 129.0, 129.07, 129.13, 129.2, 129.28, 129.30, 129.32,
129.4, 129.51, 129.53, 129.8, 130.0, 135.9, 139.4, 139.9, 140.03, 140.05,
140.3, 140.5, 140.6, 140.8, 170.8, 173.1; HRMS (FAB) m/z [M + H]+
calcd for C94H105N2O21 1597.7210, found 1597.7201.
(2-Acetamido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-β-D-
mannopyranosyl)-(1→4)-(2,3,6-tri-O-benzyl-α-D-galactopyra-
nosyl)-(1→4)-[2-acetamido-4,6-di-O-benzyl-2-deoxy-β-D-galac-
topyranosyl-(1→3)]-2,6-di-O-benzyl-D-galactopyranose (28).
Tris(triphenylphosphine)rhodium(I) chloride (4.8 mg, 5.2 μmol)
was added to a stirred solution of allyl glycoside 27 (56.7 mg,
0.034 mmol) and 1,4-diazabicyclo[2.2.2]octane (DABCO; 1.2 mg,
0.011 mmol) in 9:1 EtOH/H2O (1 mL), and the mixture was heated
at 100 °C for 2 h. After cooling to room temperature, the reaction
mixture was diluted with AcOEt (2 mL) and passed through a
Celite pad. The filtrate was partitioned between AcOEt (15 mL) and
saturated aqueous NH4Cl (3 mL). The organic layer was washed with
brine (2 × 3 mL) and dried over anhydrous Na2SO4. Filtration and
evaporation in vacuo furnished the colorless oil, which was used
without further purification.
Allyl (2-Acetamido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-
β-D-mannopyranosyl)-(1→4)-(2,3,6-tri-O-benzyl-α-D-galacto-
pyranosyl)-(1→4)-[2-acetamido-3-O-acetyl-4,6-di-O-benzyl-2-
deoxy-β-D-galactopyranosyl-(1→3)]-2,6-di-O-benzyl-α-D-galac-
topyranoside (8). A 1.0 M solution of TMSClO4 in dioxane
(0.12 mL, 0.12 mmol) was added to an ice-cooled (0 °C) mixture of
diphenyl phosphate 9 (42.6 mg, 0.04 mmol), alcohol 10 (66.1 mg,
0.08 mmol), and pulverized 5 Å molecular sieves (80 mg) in CH2Cl2
(0.6 mL). After 30 min of stirring, the reaction was quenched with
Et3N (0.2 mL). The mixture was diluted with AcOEt (4 mL) and
passed through a Celite pad. The filtrate was partitioned between
AcOEt (20 mL) and saturated aqueous NaHCO3 (5 mL). The organic
layer was successively washed with saturated aqueous NaHCO3
(5 mL) and brine (2 × 5 mL) and dried over anhydrous Na2SO4.
Filtration and evaporation in vacuo furnished the crude product (104.5
mg, white solid), which was purified by flash column chromatography
(silica gel 12 g, 5:1 toluene/acetone) to give tetrasaccharide 8
(49.5 mg, 75%) as a colorless oil. Rf 0.50 (2:1 toluene/acetone); [α]D22
+29.0 (c 1.03, CHCl3); IR (KBr) 3329, 3031, 2925, 2870, 1746, 1681,
NBS (7.6 mg, 0.041 mmol) was added to a stirred solution of the
crude prop-1-enyl glycoside in 20:1 THF/H2O (1.26 mL). After 10 min
of stirring, the volatile elements were removed in vacuo, and the residue
was partitioned between AcOEt (15 mL) and 10% aqueous Na2S2O3
(3 mL). The organic layer was successively washed with 10% aqueous
Na2S2O3 (3 mL) and brine (2 × 3 mL) and dried over anhydrous
Na2SO4. Filtration and evaporation in vacuo furnished the crude product
(67.2 mg, slightly yellow oil), which was purified by column
chromatography (silica gel 6 g, 4:1 CH2Cl2/acetone with 1% Et3N) to
give hemiacetal 28 (44.2 mg, 82% for three steps, α:β = 79:21) as a
colorless viscous oil. Rf 0.41 (2:1 toluene/acetone); [α]2D0 +21.2 (c 1.01,
CHCl3); IR (Nujol) 3324, 2922, 2853, 1657, 1455, 1376, 1076, 734,
1
1454, 1368, 1239 cm−1; H NMR (500 MHz, acetone-d6) δ 1.56 (s,
3H), 1.90 (br s, 3H), 1.91 (s, 3H), 3.33 (ddd, J = 4.6, 9.2, 9.7 Hz, 1H),
3.50 (dd, J = 6.9, 9.7 Hz, 1H), 3.53 (dd, J = 4.9, 10.2 Hz, 1H), 3.48−
3.70 (m, 3H), 3.79−4.05 (m, 14H), 4.09 (ddd, J = 1.7, 5.2, 13.2 Hz,
1H), 4.21 (dd, J = 2.9, 10.3 Hz, 1H), 4.25 (br d, J = 2.9 Hz, 1H),
4.29−4.35 (m, 3H), 4.45 (d, J = 12.0 Hz, 1H), 4.51 (d, J = 12.0 Hz,
1H), 4.51−4.60 (m, 5H), 4.64−4.83 (m, 8H), 5.05−5.16 (m, 4H),
5.19 (d, J = 8.6 Hz, 1H), 5.28 (dt, J = 17.2, 1.7 Hz, 1H), 5.38 (dd, J =
3.5, 10.8 Hz, 1H), 5.50 (s, 1H), 5.90 (m, 1H), 6.64 (br d, J = 8.0 Hz,
1H), 7.00 (br d, J = 9.8 Hz, 1H), 7.21−7.51 (m, 45H); 13C NMR
(126 MHz, CDCl3) δ 20.7, 23.09, 23.11, 50.4, 52.4, 66.5, 67.9, 68.2,
68.6, 68.8, 69.7, 71.7, 72.4, 72.7, 72.8, 72.9, 73.1, 73.2, 74.0, 74.1, 75.0,
75.1, 75.4, 75.9, 76.6, 76.8, 78.6, 95.4, 97.9, 101.1, 101.3, 101.6, 118.1,
125.2, 126.0, 127.3, 127.38, 127.42, 127.5, 127.55, 127.64, 127.7,
127.8, 128.0, 128.13, 128.15, 128.2, 128.26, 128.34, 128.4, 128.5,
128.9, 129.0, 133.8, 137.2, 137.4, 137.8, 138.0, 138.3, 138.4, 138.5,
138.6, 138.7, 169.5, 170.3, 170.5; HRMS (FAB) m/z [M + H]+ calcd
for C96H107N2O22 1639.7316, found 1639.7300. Anal. Calcd for
C96H106N2O22: C, 70.31; H, 6.52; N, 1.71. Found: C, 70.08; H, 6.56;
N, 1.70.
1
696 cm−1; H NMR (500 MHz, acetone-d6) δ 1.50 (s, 2.4H), 1.52 (s,
0.6H), 1.88 (br s, 3H), 3.32 (m, 1H), 3.42 (dd, J = 6.3, 9.7 Hz, 0.8H),
3.52 (m, 1H), 3.58 (m, 0.2H), 3.64−3.73 (m, 4.4H), 3.76−3.91 (m,
7.8H), 3.95−4.03 (m, 3H), 4.17 (t, J = 6.3 Hz, 1H), 4.20 (t, J = 6.3 Hz,
0.2H), 4.24−4.33 (m, 5.4H), 4.39−4.55 (m, 5.4H), 4.60−4.80 (m,
7.2H), 4.84−4.86 (m, 1.6H), 4.91 (d, J = 8.0 Hz, 0.2H), 5.01−5.11 (m,
4H), 5.18 (d, J = 3.4 Hz, 0.2H), 5.34 (t, J = 3.7 Hz, 0.8H), 5.42 (br s,
0.8H), 5.48 (d, J = 3.4 Hz, 0.8H), 5.51 (s, 1H), 6.00 (d, J = 6.3 Hz,
0.2H), 6.63 (d, J = 4.6 Hz, 0.2H), 6.74 (d, J = 4.0 Hz, 0.8H), 6.98 (d, J =
9.7 Hz, 1H), 7.21−7.49 (m, 45H); 13C NMR (100 MHz, acetone-d6) δ
23.38, 23.44, 23.6, 51.3, 55.8, 57.2, 57.4, 68.3, 68.6, 69.5, 69.8, 69.9,
70.0, 70.06, 70.10, 70.3, 70.6, 71.8, 73.58, 73.64, 73.7, 73.8, 73.9,
74.0, 74.2, 74.6, 74.9, 75.0, 75.1, 75.2, 75.7, 76.3, 76.4, 76.5, 77.1, 77.3,
77.66, 77.72, 78.0, 78.2, 78.3, 78.4, 79.4, 79.6, 80.1, 80.3, 80.9, 91.7,
99.2, 99.7, 99.8, 102.5, 102.9, 103.0, 103.1, 104.1, 127.4, 128.2, 128.25,
128.34, 128.4, 128.5, 128.6, 128.66, 128.69, 129.0, 129.09, 129.14,
129.2, 129.29, 129.33, 129.4, 129.50, 129.54, 129.8, 139.4, 139.85,
139.87, 139.96, 140.00, 140.04, 140.25, 140.29, 140.32, 140.55, 140.61,
140.76, 140.81, 170.8, 173.18, 173.22; HRMS (ESI) m/z [M + Na]+
calcd for C91H100N2O21Na 1579.6716, found 1579.6704.
(2-Acetamido-2-deoxy-β-D-mannopyranosyl)-(1→4)-(α-D-
galactopyranosyl)-(1→4)-[2-acetamido-2-deoxy-β-D-galacto-
pyranosyl-(1→3)]-D-galactopyranose (1). Palladium on carbon
(10%, 10 mg) was added to a stirred solution of hemiacetal 28
(21.5 mg, 0.014 mmol) in MeOH (1 mL), and the mixture was stirred
under 1 atm of hydrogen for 14 h. The reaction mixture was diluted
with MeOH (2 mL) and passed through a Celite pad. The filtrate was
evaporated in vacuo to furnish the crude product (9.8 mg), which was
purified by column chromatography (Sephadex 5 g, eluting with
MeOH) to give tetrasaccharide 1 (9.7 mg, 94%, α:β = 1:1) as a
colorless amorphous solid. Rf 0.13 (9:1 EtOH/H2O); [α]2D1 +35.7
(c 0.62, MeOH); IR (KBr) 3376, 1642, 1562, 1376, 1065 cm−1;
1H NMR (500 MHz, CD3OD) δ 1.98 (s, 1.5H), 1.99 (s, 1.5H), 2.02
(s, 3H), 3.20−3.25 (m, 1H), 3.43 (t, J = 9.7 Hz, 1H), 3.44−3.88 (m,
16H), 3.89−3.98 (m, 1.5H), 4.07 (t, J = 7.4 Hz, 0.5H), 4.12−4.16
(m, 1H), 4.20 (d, J = 2.9 Hz, 0.5H), 4.26 (d, J = 2.9 Hz, 0.5H),
Allyl (2-Acetamido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-
β-D-mannopyranosyl)-(1→4)-(2,3,6-tri-O-benzyl-α-D-galacto-
pyranosyl)-(1→4)-[2-acetamido-4,6-di-O-benzyl-2-deoxy-β-D-
galactopyranosyl-(1→3)]-2,6-di-O-benzyl-α-D-galactopyrano-
side (27). A 1.0 M solution of NaOMe in MeOH (35 μL, 0.035 mmol)
was added to an ice-cooled (0 °C) solution of tetrasaccharide 8
(56.5 mg, 0.034 mmol) in MeOH (0.5 mL). After 1.5 h of stirring at
this temperature, the reaction mixture was neutralized with Amberlite
IR-120 acidic resin. Filtration and evaporation in vacuo furnished
alcohol 27 (56.7 mg), which was used without further purification.
An analytical sample was obtained by silica gel column chromatography
with 4:1 → 3:1 toluene/acetone. Rf 0.44 (2:1 toluene/acetone);
[α]2D2 +28.0 (c 0.97, CHCl3); IR (neat) 3333, 3063, 3031, 2870, 1667,
1
1539, 1496, 1099 cm−1; H NMR (500 MHz, acetone-d6) δ 1.54 (s,
3H), 1.89 (br s, 3H), 3.32 (t, J = 11.8 Hz, 1H), 3.49 (dd, J = 6.3, 9.5
Hz, 1H), 3.51 (m, 1H), 3.63−3.75 (m, 4H), 3.77−4.05 (m, 14H), 4.13
(dd, J = 5.0, 13.1 Hz, 1H), 4.18 (dd, J = 2.7, 10.4 Hz, 1H), 4.20 (br d,
J = 2.7 Hz, 1H), 4.28 (s, 2H), 4.31 (d, J = 11.8 Hz, 1H), 4.32 (d, J =
11.3 Hz, 1H), 4.45−4.52 (m, 3H), 4.53 (d, J = 11.3 Hz, 1H), 4.64 (d,
J = 11.8 Hz, 1H), 4.70 (d, J = 11.8 Hz, 1H), 4.71−4.75 (m, 3H), 4.72
(d, J = 11.3 Hz, 1H), 4.77 (d, J = 11.3 Hz, 1H), 4.84 (d, J = 7.5 Hz,
1H), 4.86 (d, J = 11.3 Hz, 1H), 4.96 (d, J = 3.2 Hz, 1H), 5.04−5.07 (m,
4H), 5.09 (d, J = 10.4 Hz, 1H), 5.29 (dd, J = 1.4, 17.2 Hz, 1H), 5.39 (br
s, 1H), 5.52 (s, 1H), 5.92 (m, 1H), 6.74 (br d, J = 3.2 Hz, 1H), 7.00 (br
d, J = 9.9 Hz, 1H), 7.21−7.50 (m, 45H); 13C NMR (100 MHz,
acetone-d6) δ 23.4, 23.6, 51.3, 57.3, 68.3, 69.2, 69.48, 69.54, 69.9, 70.3,
4286
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The Journal of Organic Chemistry
Article
4.48 (d, J = 7.4 Hz, 0.5H), 4.51−4.55 (br q, J = 7.4 Hz, 1H), 4.58−
4.64 (m, 2H), 4.82−4.84 (d, J = 3.4 Hz, 1H), 4.93 (d, J = 4.0 Hz,
0.5H), 4.97 (d, J = 3.4 Hz, 0.5H), 5.14 (d, J = 4.0 Hz, 0.5H); 13C
NMR (100 MHz, CD3OD) δ 22.72, 22.73, 23.09, 23.11, 54.7, 54.8,
60.8, 61.0, 61.3, 62.6, 62.7, 68.8, 69.80, 69.83, 70.4, 70.7, 70.9, 71.1,
71.2, 71.4, 71.5, 71.9, 73.4, 73.7, 73.8, 74.2, 74.8, 75.6, 78.2, 78.3, 78.4,
79.3, 82.1, 94.3, 98.9, 100.3, 100.5, 102.0, 102.1, 105.1, 174.5, 174.7,
175.0, 175.1; HRMS (FAB) m/z [M + H]+ calcd for C28H49N2O21
749.2828, found 749.2835.
(7) For use of diethyl phosphite as a leaving group, see Hashimoto,
S.; Umeo, K.; Sano, A.; Watanabe, N.; Nakajima, M.; Ikegami, S.
Tetrahedron Lett. 1995, 36, 2251−2254.
(8) For β-selective glycosylation with 2-azido-2-deoxymannosyl
diphenyl phosphate, see Nakamura, S.; Tsuda, T.; Suzuki, N.;
Hashimoto, S. Heterocycles 2009, 77, 843−848.
(9) For glycosylation with 2-acetamido-2-deoxyglycosyl diethyl
phosphites, see (a) Arihara, R.; Nakamura, S.; Hashimoto, S. Angew.
Chem., Int. Ed. 2005, 44, 2245−2249. (b) Arihara, R.; Kakita, K.;
Suzuki, N.; Nakamura, S.; Hashimoto, S. J. Org. Chem. 2015,
DOI: 10.1021/acs.joc.5b00138.
ASSOCIATED CONTENT
■
(10) Haseley, S.; Wilkinson, S. G. Carbohydr. Res. 1997, 301, 187−
192. While the chemical structure of the polymeric antigen was
reported, its biological repeating unit has not been defined.
(11) Synthesis of the trisaccharide ManNAcβ1→4Galα1→4Gal
involved in the tetrasaccharide repeating unit of the O18 antigen has
already been reported: Koto, S.; Shinoda, Y.; Hirooka, M.; Sekino, A.;
Ishizumi, S.; Koma, M.; Matuura, C.; Sakata, N. Bull. Chem. Soc. Jpn.
2003, 76, 1603−1615.
S
* Supporting Information
Additional text with general information and 1H and 13C NMR
spectra for all new compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*E-mail nakamura@phar.nagoya-cu.ac.jp.
*E-mail hsmt@pharm.hokudai.ac.jp.
■
(12) (a) Litjens, R. E. J. N.; Leeuwenburgh, M. A.; van der Marel, G.
A.; van Boom, J. H. Tetrahedron Lett. 2001, 42, 8693−8696.
́
(b) Codee, J. D. C.; Litjens, R. E. J. N.; den Heeten, R.; Overkleeft,
Present Address
H. S.; van Boom, J. H.; van der Marel, G. A. Org. Lett. 2003, 5, 1519−
1522. (c) Codee, J. D. C.; van den Bos, L. J.; Litjens, R. E. J. N.;
†(S.N.) Graduate School of Pharmaceutical Sciences, Nagoya
City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-
8603, Japan.
́
Overkleeft, H. S.; van Boeckel, C. A. A.; van Boom, J. H.; van der
Marel, G. A. Tetrahedron 2004, 60, 1057−1064. (d) Litjens, R. E. J. N.;
den Heeten, R.; Timmer, M. S. M.; Overkleeft, H. S.; van der Marel,
G. A. Chem.Eur. J. 2005, 11, 1010−1016. (e) Litjens, R. E. J. N.; van
Notes
The authors declare no competing financial interest.
́
den Bos, L. J.; Codee, J. D. C.; van den Berg, R. J. B. H. N.; Overkleeft,
H. S.; van der Marel, G. A. Eur. J. Org. Chem. 2005, 918−924. (f) van
den Bos, L. J.; Duivenvoorden, B. A.; de Koning, M. C.; Filippov, D.
V.; Overkleeft, H. S.; van der Marel, G. A. Eur. J. Org. Chem. 2007,
116−124.
(13) For reviews, see (a) Crich, D. Acc. Chem. Res. 2010, 43, 1144−
1153. (b) Crich, D. J. Org. Chem. 2011, 76, 9193−9209.
(14) We selected diphenyl phosphate as the leaving group of
disaccharide 9 with the expectation that higher α-selectivity could be
obtained as compared with diethyl phosphite leaving group when
treated with a Lewis acid. See ref 4b.
ACKNOWLEDGMENTS
■
This research was supported in part by a Grant-in-Aid for
Scientific Research from the Ministry of Education, Culture,
Sports, Science and Technology, Japan. We also acknowledge
the Japan Society for the Promotion of Science for graduate
fellowships to R.A. and K.K. We thank S. Oka and M. Kiuchi of
the Center for Instrumental Analysis, Hokkaido University, for
mass measurements and elemental analysis.
(15) Nashed, M. A.; Anderson, L. Tetrahedron Lett. 1976, 3503−
3506.
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