Synthesis and structural verification of the xylomannan antifreeze substance from the freeze-tolerant Alaskan beetle Upis ceramboides

Article abstract of DOI:10.1021/jo201780e

Tetra-, hexa-, and octasaccharide subunits of the [→4)-β-d-Manp- (1→4)-β-d-Xylp-(1→]_n xylomannan motif proposed as the structure of a novel nonprotein, thermal hysteresis-producing antifreeze substance from the freeze-tolerant Alaskan beetle Up

Full text of DOI:10.1021/jo201780e

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Synthesis and Structural Verification of the Xylomannan Antifreeze
Substance from the Freeze-Tolerant Alaskan Beetle Upis ceramboides
David Crich* and Md. Yeajur Rahaman
Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, Michigan 48202, United States
S
* Supporting Information
ABSTRACT: Tetra-, hexa-, and octasaccharide subunits of the [→4)-
β-D-Manp-(1→4)-β-D-Xylp-(1→]n xylomannan motif proposed as the
structure of a novel nonprotein, thermal hysteresis-producing antifreeze
substance from the freeze-tolerant Alaskan beetle Upis ceramboides have
been accessed by total chemical synthesis. Comparison of their NMR
spectral data with data of the isolate lends strong support to the proposed structure. Synthetic tetrasaccharides representing
various linkage isomers considered (α- rather than β-manno, and linkage through mannose O3 rather than O4) show more
significant chemical shift differences with the isolate and are therefore excluded from further consideration.
INTRODUCTION
RESULTS AND DISCUSSION
■
■
We first considered the possibility that the antifreeze glycan
might consist of a mixture of xylans and mannans as entertained
originally. Comparison of the spectral data reported in the
literature for natural β-(1→3)- and β-(1→4)-xylans5 and for
natural5c,6 and synthetic7 β-(1→3)- and β-(1→4)-mannans
with the xylomannan THF data revealed considerable
discrepancies and enabled such structures to be excluded
from consideration. The range of candidates for synthesis was
therefore narrowed substantially, and attention was focused
on the [→4)-β-D-Manp-(1→4)-β-D-Xylp-(1→]n which was
considered to be the most likely structure by Walters and
co-workers.1 Uncertain as to the number of repeating units
necessary to mimic the structure of the isolated glycan, and
desiring maximum synthetic efficiency, we designed a concise,
convergent approach to tetra-, hexa-, and octasaccharyl subunits
of [→4)-β-D-Manp-(1→4)-β-D-Xylp-(1→]n, each in the form of
the methyl glycosides so as to simplify the NMR spectra.
Accordingly, the disaccharides 4 and 5 were assembled from
the β-mannosyl donor 18 and the xylopyranosyl acceptors 29
and 310 by our standard methodology11 involving preactiva-
tion of the donor at low temperature with the combination of
1-benzenesulfinyl piperidine (BSP),12 trifluoromethanesulfonic
anhydride (Tf2O), and the hindered base 2,4,6-tri-tert-
butylpyrimidine (TTBP) (Scheme 1).13 With the glycosyl
acceptor 5, itself a thioglycoside, the sulfenyl trap 1-octene was
added to the reaction mixture before it was allowed to warm
to room temperature to prevent premature activation of the
disaccharyl thioglycoside.7a,14 As anticipated,11 these reactions
were highly β-selective with only a single anomer being isolated
in each case. Acidolysis of the reactive trans-fused acetonide in
4 and 5 was then followed by benzoylation to give 6 and 7,
which, on treatment with sodium cyanoborohydride and HCl,15
underwent reductive cleavage of the benzylidene acetal units to
In 2009, Walters and co-workers described the isolation of a
thermal hysteresis-producing antifreeze substance from the
freeze-tolerant Alaskan beetle Upis ceramboides.1 This new
thermal hysteresis factor (THF) is highly unusual insofar as it
was shown to be glycan-based and to contain little or no
protein,1 whereas most THFs are protein or glycoprotein-
based.2 On the basis of extensive NMR and mass spectrometric
work coupled with both chemical and enzymatic degradative
studies, an alternating xylopyranose−mannopyranose glycan
[→4)-β-D-Manp-(1→4)-β-D-Xylp-(1→]n was proposed as the
likely structure for this glycan-based THF with both linkage
types belonging to the β-(1→4) class. However, as acknowl-
edged by the authors,1 the linkage isomer [→3)-β-D-Manp-
(1→4)-β-D-Xylp-(1→]n, in which the xylopyranose residue is
β-linked to the 3-position of the mannopyranose ring rather
than to the 4-position, could not be completely excluded on
the basis of the spectroscopic evidence available.1 Similarly, the
data did not permit the fully unambiguous assignment of the
α-configuration to the mannopyranoside moiety. The alternative
possibility that the glycan consists of alternating sections of a
mannan and a xylan could also not be excluded on the basis of
the data presented.1 In view of the highly unusual nature of this
glycan-based THF and the various ambiguities in its structure,
we have undertaken a program of organic synthesis of authentic
samples of homogeneous xylomannan oligosaccharides for
comparison purposes so as to aid in its structural elucidation.
The power of total organic synthesis in the resolution of
ambiguous structures is well-known3 and has proven to be
essential in the past for the elucidation of structural motifs
necessary for activity of certain antifreeze glycoproteins.4 We
report here on the successful outcome of this synthesis program
and on the close similarity of the spectroscopic data of the
natural isolate and a homologous series of synthetic
oligosaccharides that lends strong support to the proposed
structure of the antifreeze glycan-based THF.
Received: August 26, 2011
Published: September 28, 2011
© 2011 American Chemical Society
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Scheme 1. Synthesis of the Four Disaccharyl Units 6−9
Scheme 3. Completion of the Tetra-, Hexa-, and
Octasaccharides 15−17
give the corresponding 6′-O-benzyl ethers retaining hydroxyl
groups at the 4′-positions, 8 and 9 (Scheme 1).
disaccharide units at the core of each structure. All coupling
constants were in full agreement with the assigned structures of
these synthetic oligosaccharides. It is immediately clear from
inspection of the spectra of these three oligosaccharides
(Supporting Information) that they differ only in the relative
intensity of the signals due to the xylomannan core as compared
to those of the monosaccharides at the up- and downstream
termini. It is therefore clear that these substances have a regular
organized structure, as is expected to be the case for 1→3 and
1→4-glycans in general,16 and that these simple oligosaccharides
are adequate mimics of longer glycans for the purposes of
comparison of spectral data.
The disaccharide 7 was then coupled to the acceptors 8 and
9, giving the tetrasaccharides 10 and 11 as single anomers.
Subsequent reduction of the benzylidene acetal in 10 by the
sodium cyanoborohydride protocol then gave the tetrasaccharyl
acceptor 12 (Scheme 2).
Coupling of the tetrasaccharyl donor 11 with the disaccharyl
acceptor 8 afforded the hexasaccharide 13, while similar coupling
of the two saccharides 11 and 12 gave the octasaccharide 14
(Scheme 2). In this manner, a homologous series of protected
tetra-, hexa-, and octasaccharides for deprotection was assembled
efficiently with complete control of anomeric stereochemistry.
Cleavage of the benzoate esters in 10, 13, and 14 under
Zemplen conditions followed by hydrogenolysis then afforded
the pure, homogeneous target tetra-, hexa-, and octasaccharides
15−17 (Scheme 3).
A graphic representation17 of the 13C and 1H NMR chemical
shift differences between the core residues of the three
synthetic xylomannans and the literature data for the antifreeze
glycan under comparable conditions18 (Figures 1 and 2, left-
hand column) reveals close homology between the synthetic
and isolated material. Thus, in the 13C NMR spectra, only
minor differences in chemical shift were found for the two
anomeric carbons and for C2 of the mannose unit in 15−17
and the isolated glycan (Figure 1, left-hand column). Likewise,
1
The H (500 MHz) and 13C (125 MHz) NMR spectra of
15−17 were recorded in phosphate buffer (D2O) at pH 7.5
and 40 °C and assigned by a combination of TOCSY,
COSY, HMQC, and HMBC techniques, leading to the chemical
shift data presented in Table 1, entries 1−3, for the repeating
Scheme 2. Convergent Assembly of the Tetra-, Hexa-, and Octasaccharides 10−14
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Table 1. 13C and 1H Chemical Shifts (ppm) for the Repeating Units of Isolated and Synthetic Xylomannans Recorded in D2O at
pH 7.5 and 40 °C
assignments for internal mannopyranoside residues
glycan
C1 (H1)
C2 (H2)
C3 (H3)
C4 (H4)
C5 (H5)
C6 (H6, 6′)
ab
,
[→4)-β-D-Manp-(1→4)-β-D-Xylp-(1→]n (isolated material)
100.2 (4.92) 70.1 (4.30)
71.6 (3.98)
71.6 (4.01)
71.6 (3.99)
71.8 (3.98)
80.1 (4.14)
70.5 (4.09)
78.0 (4.28)
76.6 (3.96)
76.5 (3.84)
76.5 (3.82)
76.7 (3.82)
65.1 (3.97)
78.0 (4.01)
67.6 (4.07)
75.1 (3.74)
75.3 (3.69)
75.3 (3.69)
75.5 (3.63)
76.5 (3.72)
74.8 (3.89)
74.8 (3.98)
60.6 (4.09, 3.93)
60.4 (4.18, 3.99)
60.4 (4.16, 3.94)
60.7 (4.16, 3.97)
61.2 (4.21, 4.04)
60.7 (4.18, 4.02)
61.1 (4.20, 4.11)
c
H[→4)-β-D-Manp-(1→4)-β-D-Xylp-(1→]2OMe (15)
98.4 (5.08)
98.4 (5.07)
98.6 (5.03)
98.5 (5.09)
70.9 (4.29)
70.9 (4.27)
70.8 (4.25)
68.4 (4.48)
c
H[→4)-β-D-Manp-(1→4)-β-D-Xylp-(1→]3OMe (16)
c
H[→4)-β-D-Manp-(1→4)-β-D-Xylp-(1→]4OMe (17)
c
H[→3)-β-D-Manp-(1→4)-β-D-Xylp-(1→]2OMe (23)
c
H[→4)-α-D-Manp-(1→4)-β-D-Xylp-(1→]2OMe (31)
102.1 (5.31) 70.2 (4.34)
101.5 (5.45) 70.1 (4.58)
c
H[→3)-α-D-Manp-(1→4)-β-D-Xylp-(1→]2OMe (37)
assignments for internal xylopyranoside residues
glycan
C1 (H1)
C2 (H2)
C3 (H3)
C4 (H4)
C5 (H5, 5′)
ab
,
[→4)-β-D-Manp-(1→4)-β-D-Xylp-(1→]n (isolated material)
101.7 (4.66)
103.4 (4.69)
103.4 (4.69)
103.6 (4.65)
100.9 (4.86)
104.1 (4.67)
101.8 (4.89)
72.8 (3.48)
73.0 (3.59)
73.0 (3.61)
73.3 (3.57)
72.8 (3.69)
73.2 (3.60)
72.9 (3.74)
73.8 (3.73)
74.0 (3.84)
74.0 (3.84)
74.2 (3.85)
74.0 (3.87)
73.7 (3.85)
76.6 (4.00)
76.3 (4.12)
76.4 (4.12)
76.5 (4.11)
76.5 (4.11)
76.8 (3.95)
77.7 (4.09)
63.0 (4.28, 3.55)
63.1 (4.39, 3.65)
63.1 (4.40, 3.66)
63.2 (4.40, 3.67)
63.0 (4.41, 3.68)
64.5 (4.42, 3.65)
64.2 (4.47 3.78)
c
H[→4)-β-D-Manp-(1→4)-β-D-Xylp-(1→]2OMe (15)
c
H[→4)-β-D-Manp-(1→4)-β-D-Xylp-(1→]3OMe (16)
c
H[→4)-β-D-Manp-(1→4)-β-D-Xylp-(1→]4OMe (17)
H[→3)-β-D-Manp-(1→4)-β-D-Xylp-(1→]2OMe (23)
c
H[→4)-α-D-Manp-(1→4)-β-D-Xylp-(1→]2OMe (31)
c
H[→3)-α-D-Manp-(1→4)-β-D-Xylp-(1→]2OMe (37)
74.8 (3.97)
a
b
c
Data taken from Walters and co-workers.1 Recorded at 800 MHz for H and 200 MHz for 13C. Recorded at 500 MHz for H and 125 MHz
1
1
for 13C.
1
although not identical, the H NMR chemical shifts of the
internal residues of 15−17 differed little from those of the
isolated glycan (Figure 2, left-hand column). Most notably
though, in the mannopyranose moiety, the largest differences in
1H chemical shift between 15−17 and the isolated glycan were
found for the anomeric proton and H4, that is, for those protons
most closely associated with one of the two interglycosidic
linkages.
The differences, albeit small, in chemical shifts between the
synthetic xylomannans 15−17 and the natural isolate (Figures
1 and 2, left-hand column) prompted the synthesis of three
more systems representing the stereoisomeric and positional
isomers considered by Walters and co-workers as possible
alternative structures.1 As the close homology of the NMR data
for the xylomannans 15−17 (Figures 1 and 2, left-hand column
and Supporting Information) indicated simple tetrasaccharides
to be adequate models of the glycan for spectra purposes, we
limited the synthesis of these linkage isomers to the tetra-
saccharide level, which we considered a minimal but adequate
representations for the purposes of comparison of spectral data
with the natural glycan.
In order to prepare a linkage isomer in which the β-xylosyl
unit was linked to O3 rather than to O4 of mannnopyranose, we
began with the known β-mannosyl donor7a 18 protected on O3
with the 2-naphthylmethyl ether. After formation of the
disaccharide 19 and replacement of the isopropylidene group
with two esters in the usual manner, removal of the naphthyl-
methyl ether was achieved with DDQ under standard conditions
to give 21. Subsequent coupling of this disaccharide with the
β-xylopyranosyl donor 8 gave the tetrasaccharide 22 which on
deprotection afforded the linkage isomer 23 (Scheme 4).
A second linkage isomer 31, in which the mannopyranosyl
unit was linked (1→4)-α to the xylopyranosyl residue, was
obtained from the known α-mannosyl donor19 24 uneventfully
as outlined in Scheme 5.
Figure 1. 13C Δδ values for the chemical shifts of 15−17, 23, 31, and
37 with the isolated glycan; m = mannopyranoside, x = xylopyranoside.
Finally, a third linkage isomer 37, having the α-configuration
at the mannopyranosyl anomeric center and linked via O3 of
mannose, was prepared from the α-mannosyl donor20 32 by
standard methods as illustrated in Scheme 6.
No isomers (positional or stereochemical) of the xylopyr-
anosyl residue were prepared as the original structural assign-
ment of the xylopyranose moiety of the glycan-based THF is
considered secure.1
1
Figure 2. H Δδ values for the chemical shifts of 15−17, 23, 31, and
37 with the isolated glycan; m = mannopyranoside, x = xylopyranoside.
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Scheme 4. Synthesis of Linkage Isomer 23
Scheme 6. Synthesis of Linkage Isomer 37
Scheme 5. Synthesis of Linkage Isomer 31
Overall, we conclude that among the sequences considered
for this antifreeze xylomannan the [→4)-β-D-Manp-(1→4)-β-D-
Xylp-(1→]n structure favored by Walters and co-workers1 is the
best fit to the available data. This conclusion raises the obvious
question as to the origin of the minor chemical shift differences
at both the manno- and xylopyranoside anomeric centers and at
the mannopyranoside 2-position between the core of the models
15−17 and that of the isolate (Table 1, entries 1−4). Short of
invoking an actual assignment error in one of the two subunits
(such as mannose for talose) by Walters and collaborators,1 we
hypothesize that these might arise from agglomeration of the
actual glycan, or from its adoption of a higher order structure not
available to the short synthetic models. Alternatively, the small
chemical shift differences might simply be the consequence of
imperfect reproduction of the original NMR conditions; indeed,
the spectra of 15−17 showed considerable changes on going
from solution in deuteriomethanol to pH 7.5 D2O buffer, and
from 25 to 40 °C, thereby revealing the sensitivity of the spectral
data to the acquisition conditions.
EXPERIMENTAL SECTION
■
General Procedure 1. Synthesis of Disaccharides by the BSP
Method (Cmpds 4, 19, 26, and 33). A mixture of phenyl
thiomannopyranoside (0.3 mmol), BSP (76 mg, 0.36 mmol), TTBP
(112 mg, 0.45 mmol, used only for compounds 4 and 19), and 4 Å
molecular sieves (170 mg) was dried under vacuum for 0.5 h. Freshly
distilled dichloromethane (6 mL) was added, and the reaction mixture
was stirred for 0.5 h at room temperature under N2 before it was
cooled to −65 °C and stirred for an additional 5 min. Tf2O (61 μL,
0.36 mmol) was added dropwise and the reaction mixture stirred for
30 min while the temperature was held between −65 and −60 °C,
after which a solution of methyl xylopyranoside 2 (67 mg, 0.33 mmol)
in dichloromethane (3 mL) was added dropwise and the reaction
mixture was stirred for 2.5 h at −60 °C before it was quenched with
Et3N (0.5 mL) and then warmed to room temperature. The reaction
mixture was filtered through Celite, concentrated, and purified by
chromatography over silica gel.
The NMR data for the isomeric tetrasaccharides 23, 31, and
1
37 are presented in Tables 1−3, and the 13C and H NMR
chemical shift differences between their internal residues and
those of the natural isolate are represented graphically in Figures
1 and 2, right-hand column. The 13C NMR spectral data for
tetrasaccharide 23 differ significantly from those of the isolated
glycan at the level of carbons 3 and 4 of the mannopyranosyl
ring, strongly suggesting that the isolate is not linked through C3
1
of the mannose residue. The H and 13C spectral data for
tetrasaccharide 31 show good homology with that of the isolated
glycan except for the mannose H1, indicating that the isolate is
not an α-mannoside. Finally, tetrasaccharide 37, which differs
from the structure proposed for the isolated glycan at both the
mannose anomeric center and the linkage position to the
mannopyranose ring, shows the largest differences in spectral
data with the isolate.
General Procedure 2. Synthesis of Disaccharyl Thioglyco-
sides (Cmpds 5 and 25). A mixture of phenyl thiomannopyranoside
(0.25 mmol), BSP (63 mg, 0.3 mmol), TTBP (93 mg, 0.37 mmol,
used only for compound 5), and 4 Å molecular sieves (150 mg) was
dried under vacuum for 0.5 h. Freshly distilled dichloromethane
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1
Table 2. 13C and H Chemical Shifts (ppm) for the Terminal Residues of Synthetic Xylomannans Recorded in D2O at pH 7.5
a
and 40 °C
assignments for nonreducing end mannopyranoside residues
glycan
C1 (H1)
C2 (H2)
C3 (H3)
C4 (H4)
C5 (H5)
C6 (H6, 6′)
H[→4)-β-D-Manp-(1→4)-β-D-Xylp-(1→]2OMe (15)
H[→4)-β-D-Manp-(1→4)-β-D-Xylp-(1→]3OMe (16)
H[→4)-β-D-Manp-(1→4)-β-D-Xylp-(1→]4OMe (17)
H[→3)-β-D-Manp-(1→4)-β-D-Xylp-(1→]2OMe (23)
H[→4)-α-D-Manp-(1→4)-β-D-Xylp-(1→]2OMe (31)
H[→3)-α-D-Manp-(1→4)-β-D-Xylp-(1→]2OMe (37)
98.6 (5.06)
98.2 (5.07)
98.7 (5.02)
98.2 (5.10)
101.7 (5.32)
100.8 (5.42)
71.7 (4.25)
71.4 (4.25)
71.1 (4.23)
72.6 (4.28)
70.5 (4.30)
70.4 (4.27)
71.6 (3.82)
72.5 (3.87)
71.8 (3.89)
68.4 (3.97)
70.7 (4.03)
65.0 (4.15)
67.1 (3.82)
66.9 (3.82)
67.2 (3.78)
66.9 (3.90)
67.1 (3.95)
66.8 (4.00)
76.5 (3.63)
76.2 (3.66)
75.5 (3.62)
76.3 (3.70)
73.7 (3.86)
74.8 (3.96)
61.2 (4.17, 3.97)
61.2 (4.18, 3.97)
61.4 (4.15, 3.95)
61.2 (4.22, 4.04)
61.4 4.16, 4.01)
61.1 (4.20, 4.09)
assignments for reducing end methyl xylopyranoside residues
glycan
C1 (H1)
C2 (H2)
C3 (H3)
C4 (H4)
C5 (H5, 5′)
OCH3 (OCH3)
H[→4)-β-D-Manp-(1→4)-β-D-Xylp-(1→]2OMe (15) 104.0 (4.61)
H[→4)-β-D-Manp-(1→4)-β-D-Xylp-(1→]3OMe (16) 103.9 (4.61)
H[→4)-β-D-Manp-(1→4)-β-D-Xylp-(1→]4OMe (17) 104.1 (4.57)
H[→3)-β-D-Manp-(1→4)-β-D-Xylp-(1→]2OMe (23) 103.9 (4.65)
H[→4)-α-D-Manp-(1→4)-β-D-Xylp-(1→]2OMe (31) 103.6 (4.57)
H[→3)-α-D-Manp-(1→4)-β-D-Xylp-(1→]2OMe (37) 103.8 (4.68)
73.3 (3.56)
72.9 (3.55)
73.1 (3.50)
74.0 (3.59)
72.4 (3.54)
72.8 (3.63)
74.1 (3.84)
73.9 (3.83)
74.2 (3.83)
73.0 (3.85)
73.5 (3.84)
74.8 (3.82)
76.5 (4.06)
76.4 (4.07)
76.6 (4.08)
76.4 (4.12)
77.9 (3.91)
77.7 (4.02)
63.3 (4.37, 3.65)
63.0 (4.36, 3.63)
63.3 (4.31, 3.55)
63.0 (4.41, 3.68)
64.5 (4.39, 3.65)
64.3 (4.48, 3.77)
57.5 (3.79)
57.3 (3.80)
57.5 (3.76)
57.4 (3.78)
57.7 (3.78)
57.3 (3.87)
a
1
Recorded at 500 MHz for H and 125 MHz for 13C.
a
Table 3. Coupling Constants (Hz) for the Repeating Units of Synthetic Xylomannans Recorded in D2O at pH 7.5 and 40 °C
assignments for internal mannopyranoside residues
glycan
3JH1,H2
3JH2,H3
3JH3,H4
3JH4,H5
3JH5,H6
2JH6,H6
′
1JC1,H1
H[→4)-β-D-Manp-(1→4)-β-D-Xylp-(1→]2OMe (15)
H[→4)-β-D-Manp-(1→4)-β-D-Xylp-(1→]3OMe (16)
H[→4)-β-D-Manp-(1→4)-β-D-Xylp-(1→]4OMe (17)
H[→3)-β-D-Manp-(1→4)-β-D-Xylp-(1→]2OMe (23)
H[→4)-α-D-Manp-(1→4)-β-D-Xylp-(1→]2OMe (31)
H[→3)-α-D-Manp-(1→4)-β-D-Xylp-(1→]2OMe (37)
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
2.0
2.0
2.5
2.5
1.5
3.0
9.7
9.5
9.5
8.0
b
9.5
9.0
10.0
b
b
b
b
b
b
b
11.5
11.5
12.0
12.5
b
158.6
158.2
159.1
159.2
170.2
170.5
b
9.5
b
11.0
assignments for internal xylopyranoside residues
glycan
3JH1,H2
3JH2,H3
3JH3,H4
3JH4,H5
2JH5,H5
′
1JC1,H1
H[→4)-β-DManp-(1→4)-β-D-Xylp-(1→]2OMe (15)
H[→4)-β-D-Manp-(1→4)-β-D-Xylp-(1→]3OMe (16)
H[→4)-β-D-Manp-(1→4)-β-D-Xylp-(1→]4OMe (17)
H[→3)-β-D-Manp-(1→4)-β-D-Xylp-(1→]2OMe (23)
H[→4)-α-D-Manp-(1→4)-β-D-Xylp-(1→]2OMe (31)
8.0
7.5
7.5
8.0
7.5
8.0
9.2
7.5
7.5
8.5
8.7
8.5
9.5
9.5
9.0
9.2
8.0
9.5
b
b
b
b
b
b
12.0
11.7
11.7
11.5
12.0
11.2
164.3
162.1
160.6
155.7
161.1
160.4
H[→3)-α-D-Manp-(1→4)-β-D-Xylp-(1→]2OMe (37)
a
b
1
Recorded at 500 MHz for H and 125 MHz for 13C. Not determined.
(5 mL) was added, and the reaction mixture was stirred for 0.5 h at
ambient temperature under N2 atmosphere, then cooled to −65 °C
and stirred for an additional 5 min before the addition of Tf2O (61 μL,
0.36 mmol). The reaction mixture was stirred for 30 min at −65 to
−60 °C, then was cooled to −78 °C and stirred for 5 min before
1-octene (0.3 mL, 2 mmol) was added and stirring continued for
5 min more. A solution of thioxylopyranoside acceptor 3 (77 mg,
0.27 mmol) in dicholoromethane (3 mL) then was added dropwise,
and the reaction mixture was stirred for 2.5 h at −60 °C before it
was quenched with Et3N (0.5 mL) and warmed to room temperature.
The reaction mixture was filtered through Celite, concentrated, and
purified by chromatography over silica gel.
General Procedure 3. Acidolysis of Acetonide Groups and
Subsequent Benzoylation (Cmpds 6, 7, 20, 27, 28, and 34). A
solution of disaccharide (0.1 mmol) in glacial acetic acid (10 mL) was
stirred at 65 °C for 3 h. Upon completion of the reaction, acetic acid
was removed in vacuo at 30 °C to give a white residue that was taken
up in pyridine (5 mL) and cooled down to 0 °C before it was treated
dropwise with benzoyl chloride (47 μL, 0.4 mmol). After stirring for
2 h, the reaction mixture was concentrated in vacuo and the residue
was dissolved in EtOAc (25 mL). The organic layer was successively
washed with water, saturated aqueous NaHCO3, and brine and
dried over Na2SO4. The product was purified by chromatography over
silica gel.
General Procedure 4. Reductive Cleavage of Benzylidene
Acetals (Cmpds 8, 9, 12, and 29). Oligosaccharide (0.1 mmol) was
mixed with dry 3 Å molecular sieves (150 mg) and stirred in vacuo for
0.5 h. THF (2 mL) was added, and the reaction mixture was stirred for
5 min before it was cooled to 0 °C followed by the addition of sodium
cyanoborohydride (32 mg, 0.5 mmol) (for 12, 63 mg, 1 mmol). The
reaction mixture was stirred at 0 °C for 1 h (for 12 overnight),
followed by very slow dropwise addition of dry HCl in diethyl ether
(2 N, 1.5 mL) until effervescence ceased, after which the reaction
mixture was warmed to room temperature and diluted with
chloroform (30 mL). The organic layer was successively washed
with water and brine and dried over Na2SO4. Chromatographic
purification over silica gel afforded the pure product.
General Procedure 5. Synthesis of Oligosaccahrides by the
BSP Method (Cmpds 10, 13, 14, 22, 30, and 36). The phenyl
thioglycoside (100 μmol) was mixed with BSP (27 mg, 120 μmol) and
oven-dried 4 Å molecular sieves (150 mg) and dried under vacuum for
0.5 h before dichloromethane (3 mL) was added under N2, and the
reaction mixture was stirred for 0.5 h at room temperature then
cooled to −65 °C and stirred for an additional 5 min. Tf2O (22 μL,
120 μmol) was added dropwise and the reaction mixture stirred for
30 min between −65 and −60 °C; then it was cooled to −70 °C, and a
solution of polysaccharide acceptor (100 μmol) in dichloromethane
(2 mL) was added dropwise. The reaction mixture was stirred at
8615
dx.doi.org/10.1021/jo201780e|J. Org. Chem. 2011, 76, 8611−8620

The Journal of Organic Chemistry
Featured Article
−60 °C for 2.5 h before it was quenched by addition of Et3N (500 μL)
and warmed to room temperature. The quenched reaction mixture was
filtered through Celite, concentrated, and purified by silica gel column
chromatography.
7.44−7.37 (m, 9H), 7.35−7.27 (m, 8H), 5.74 (t, J = 8.2 Hz, 1H), 5.46
(s, 1H), 5.36 (dd, J = 6.7, 8.2 Hz, 1H), 4.91 (d, J = 12.0 Hz, 1H), 4.77
(d, J = 12.0 Hz, 1H), 4.71 (d, J = 12.5 Hz, 1H), 4.66 (d, J = 7.0 Hz,
1H), 4.62 (s, 1H), 4.61 (d, J = 11.0 Hz, 1H), 4.21 (dd, J = 4.7, 11.7 Hz,
1H), 4.17−4.13 (m, 1H), 4.03 (t, J = 9.7 Hz, 1H), 4.00 (dd, J = 5.0,
10.5 Hz, 1H), 3.90 (d, J = 3.0 Hz, 1H), 3.61−3.56 (m, 2H), 3.55 (s,
3H), 3.40 (t, J = 10.2 Hz, 1H), 3.23 (dt, J = 5.0, 9.7 Hz, 1H); 13C
NMR (125.9 MHz, CDCl3) δ 165.6, 165.5, 138.5, 138.4, 137.5, 133.2,
130.0, 129.9, 129.9, 129.8, 129.5, 128.9, 128.5, 128.4, 128.2, 128.1,
127.6, 127.5, 126.1, 101.9, 101.3, 101.0, 78.3, 77.6, 77.4, 77.2, 76.9,
76.1, 74.6, 72.3, 71.9, 71.1, 68.2, 67.6, 62.8, 56.9; ESIHRMS m/z calcd
for C47H46O12Na [M + Na]+ 825.2887, found 825.2873.
General Procedure 6. Global Deprotection of Oligosacchar-
ides by Hydrogenolysis (Cmpds 15, 16, 17, 23, 31, and 37). The
protected oligosaccharide (0.02 mmol) was dissolved in methanol
(2 mL) and treated with a 25% solution of NaOMe in methanol
(0.1 mL) at 0 °C. Upon completion of the reaction, the reaction
mixture was neutralized by the addition of Amberlite IR 120 (H+)
followed by filtration through Celite. The filtrate was concentrated
under reduced pressure at room temperature to give a white foam that
was taken forward without further purification. The saponified
oligosaccharide was dissolved in methanol (2 mL) and Pd−C (20 mg),
and 1 drop of glacial acetic acid was added to it. The reaction mixture was
shaken under 40 psi H2 at room temperature for 12 h after which it was
filtered through Celite and then concentrated under reduced pressure at
room temperature to afford the oligosaccharide.
General Procedure 7. Removal of Naphthalylmethyl Ethers
with DDQ (Cmpds 21 and 35). To a stirred solution of
disaccharide (0.07 mmol) in dichloromethane (10 mL) and water
(0.5 mL) was added DDQ (40 mg, 0.18 mmol) at room temperature.
The reaction mixture was stirred for 3 h before it was quenched with a
saturated aqueous solution of NaHCO3 and diluted with dichloro-
methane (20 mL). The organic layer was successively washed with
water and brine, dried over Na2SO4, concentrated, and purified by
chromatography over silica gel.
Phenyl 2,3-Di-O-benzyl-4,6-O-benzylidene-β-D-mannopyr-
anosyl-(1→4)-2,3-di-O-benzoyl-1-thio-β-D-xylopyranoside
(7). Obtained by protocol 3 from compound 5. Chromatographic
purification over silica gel with 20% EtOAc in hexanes as eluent
24
afforded disaccharide 7 (72 mg, 87%) as a white foam: [α]D −34.8
(c = 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 8.07−8.02 (m, 4H),
7.57−7.50 (m, 4H), 7.46−7.39 (m, 7H), 7.38−7.32 (m, 9H), 7.31−
7.26 (m, 6H), 5.74 (t, J = 7.2 Hz, 1H), 5.42 (s, 1H), 5.39 (t, J = 7.2
Hz, 1H), 5.17 (d, J = 7.0 Hz, 1H), 4.87 (d, J = 12.5 Hz, 1H), 4.74 (d,
J = 12.5 Hz, 1H), 4.69 (d, J = 12.5 Hz, 1H), 4.62 (s, 1H), 4.59 (d, J =
12.5 Hz, 1H), 4.42 (dd, J = 4.2, 12.2 Hz, 1H), 4.09−3.97 (m, 3H),
3.87 (d, J = 3.0 Hz, 1H), 3.66 (d, J = 8.0, 12.0 Hz, 1H), 3.55 (dd, J =
3.0, 9.5 Hz, 1H), 3.34 (t, J = 10.2 Hz, 1H), 3.26−3.21 (m, 1H); 13C
NMR (125.9 MHz, CDCl3) δ 165.7, 165.6, 138.7, 138.6, 137.8, 133.5,
133.3, 132.9, 130.4, 130.2, 129.9, 129.6, 129.3, 129.1, 128.7, 128.6,
1285, 128.4, 128.3, 127.8, 127.7, 126.3, 101.5, 86.9, 78.5, 77.8, 76.4,
74.9, 74.5, 72.5, 72.1, 70.5, 68.5, 67.8, 65.4; ESIHRMS m/z calcd for
C52H48O11SNa [M + Na]+ 903.2815, found 903.2803.
Methyl 2,3-Di-O-benzyl-4,6-O-benzylidene-β-D-mannopyr-
anosyl-(1→4)-2,3-O-isopropylidene-β-D-xylopyranoside (4). Ob-
tained by protocol 1 from compounds 1 and 2. Chromatographic
purification with 20% EtOAc in hexanes as eluent afforded dis-
Methyl 2,3,6-Tri-O-benzyl-β-D-mannopyranosyl-(1→4)-2,3-
di-O-benzoyl-β-D-xylopyranoside (8). Obtained by protocol 4
from compound 6. Chromatographic purification over silica gel with
30% EtOAc in hexanes as eluent afforded disaccharide 8 (68 mg, 85%)
as a white foam: [α]D24 −31.2 (c = 1.0, CHCl3); 1H NMR (500 MHz,
CDCl3) δ 8.02 (dd, J = 1.0, 8.5 Hz, 2H), 7.93 (dd, J = 1.0, 8.0 Hz, 2H),
7.51−7.44 (m, 2H), 7.37−7.27 (m, 16 H), 7.22−7.20 (m, 3H), 5.72
(t, J = 8.2 Hz, 1H), 5.34 (dd, J = 6.7, 8.2 Hz, 1H), 4.82 (d, J = 12.5 Hz,
1H), 4.63−4.58 (m, 3H), 4.52−4.43 (m, 3H), 4.38 (d, J = 12.0 Hz,
1H), 4.27−4.20 (m, 2H), 3.88 (dt, J = 1.5, 9.5 Hz, 1H), 3.84 (d, J = 3.0
Hz, 1H), 3.63−3.56 (m, 3H), 3.52 (s, 3H), 3.40−3.36 (m, 1H), 3.30
(dd, J = 3.0, 9.5 Hz, 1H), 2.65 (br s, 1H); 13C NMR (125.9 MHz,
CDCl3) δ 166.0, 165.6, 138.9, 138.2, 138.1, 133.3, 133.2, 130.2, 130.1,
129.9, 129.7, 128.7, 128.6, 128.5, 128.4, 128.3, 128.0, 127.9, 127.5,
102.2, 99.8, 81.3, 75.3, 74.2, 73.9, 73.6, 72.1, 71.4, 71.0, 68.6, 62.9,
57.0; ESIHRMS m/z calcd for C47H48O12Na [M + Na]+ 827.3043,
found 827.3055.
24
accharide 4 (151 mg, 83%) as a white foam: [α]D −47.8 (c = 1.0,
1
CH3Cl); H NMR (500 MHz, CDCl3) δ 7.53−7.50 (m, 2H), 7.48−
7.45 (m, 2H), 7.41−7.36 (m, 3H), 7.35−7.28 (m, 8H) 5.64 (s, 1H),
4.94 (d, J = 12.5 Hz, 1H), 4.89 (d, J = 12.0 Hz, 1H), 4.76 (d, J = 12.5
Hz, 1H), 4.66 (d, J = 12.0 Hz, 1H), 4.65 (s, 1H), 4.56 (d, J = 7.5 Hz,
1H), 4.28 (dd, J = 4.7, 10.2 Hz, 1H), 4.23 (t, J = 9.7 Hz, 1H), 4.05 (dd,
J = 5.2, 12.2 Hz, 1H), 4.02−3.99 (m, 1H), 3.96 (t, J = 10.2 Hz, 1H),
3.91 (d, J = 3.0 Hz, 1H), 3.76 (t, J = 9.2 Hz, 1H), 3.63 (dd, J = 2.7, 9.7
Hz, 1H), 3.54 (s, 3H), 3.42−3.34 (m, 2H), 3.31 (dd, J = 7.5, 9.5 Hz,
1H), 1.44 (s, 3H), 1.42 (s, 3H); 13C NMR (125.9 MHz, CDCl3) δ
138.5, 137.8, 129.1, 128.9, 128.6, 128.4, 128.3, 127.9, 129.8, 126.2,
111.7, 102.6, 101.7 (1JC−H = 157.6 Hz), 100.3, 79.3, 78.9, 78.3, 76.9,
76.4, 76.3, 75.0, 72.8, 68.9, 67.8, 65.4, 56.7, 27.0, 26.8; ESIHRMS m/z
calcd for C36H42O10Na [M + Na]+ 657.2676, found 657.2668.
Phenyl 2,3-Di-O-benzyl-4,6-O-benzylidene-β-D-mannopyr-
anosyl-(1→4)-2,3-O-isopropylidene-1-thio-β-D-xylopyranoside
(5). Obtained by protocol 2 from compounds 1 and 3. Chromato-
graphic purification with 20% EtOAc in hexanes as eluent afforded
disaccharide 5 (153 mg, 86%) as a white foam: [α]D24 −55.6 (c = 1.0,
Phenyl 2,3,6-Tri-O-benzyl-β-D-mannopyranosyl-(1→4)-2,3-
di-O-benzoyl-1-thio-β-D-xylopyranoside (9). Obtained by proto-
col 4 from compound 7. Chromatographic purification over silica gel
with 30% EtOAc in hexanes as eluent afforded disaccharide 9 (79 mg,
89%) as a white foam: [α]D24 −54.2 (c = 1.0, CHCl3); 1H NMR (500
MHz, CDCl3) δ 8.05 (dd, J = 1.5, 8.5 Hz, 2H), 7.99 (dd, J = 1.5, 8.5
Hz, 2H), 7.53 (dd, J = 3.0, 6.5 Hz, 2H), 7.51−7.47 (m, 2H), 7.36−
7.26 (m, 19H), 7.22−7.19 (m, 3H), 5.76 (t, J = 7.5 Hz, 1H), 5.40 (t,
J = 7.2 Hz, 1H), 5.15 (d, J = 7.0 Hz, 1H), 4.81 (d, J = 12.5 Hz, 1H),
4.61 (s, 1H), 4.60 (d, J = 14.0 Hz, 1H), 4.52−4.43 (m, 4H), 4.39 (d,
J = 12.0 Hz, 1H), 4.18−4.14 (m, 1H), 3.88 (t, J = 9.5 Hz, 1H), 3.85 (d,
J = 3.0 Hz, 1H), 3.70 (dd, J = 7.7, 12.2 Hz, 1H), 3.62−3.55 (m, 2H),
1
CHCl3); H NMR (500 MHz, CDCl3) δ 7.57 (dd, J = 3.0, 7.0 Hz,
2H), 7.53−7.50 (m, 2H), 7.46−7.43 (m, 2H), 7.41−7.36 (m, 2H),
7.35−7.29 (m, 12H), 5.63 (s, 1H), 4.92 (d, J = 12.5 Hz, 1H), 4.87 (d,
J = 12.0 Hz, 1H), 4.82 (d, J = 9.5 Hz, 1H), 4.76 (d, J = 12.5 Hz, 1H),
4.66 (d, J = 12.5 Hz, 1H), 4.62 (s, 1H), 4.27 (dd, J = 5.0, 10.5 Hz, 1H),
4.22 (t, J = 9.5 Hz, 1H), 4.12 (dd, J = 5.2, 11.7 Hz, 1H), 3.99−3.93 (m,
2H), 3.89 (d, J = 2.5 Hz, 1H), 3.73 (t, J = 9.0 Hz, 1H), 3.61 (dd, J =
3.0, 9.5 Hz, 1H), 3.37−3.31 (m, 2H), 3.20 (t, J = 9.2 Hz, 1H), 1.47 (s,
3H), 1.41 (s, 3H); 13C NMR (125.9 MHz, CDCl3) δ 138.5, 137.8,
133.2, 132, 129.1, 128.9, 128.6, 128.4, 128.3, 127.9, 127.8, 126.2, 111.2,
101.7, 100.1 (1JC−H = 157.2), 85.4, 81.2, 78.9, 78.3, 76.2, 75.6, 75.5,
75.0, 72.8, 68.8, 67.9, 67.8, 26.9, 26.8; ESIHRMS m/z calcd for
C41H44O9SNa [M + Na]+ 735.2604, found 735.2560.
3.42−3.37(m, 1H), 3.30 (dd, J = 2.7, 9.2 Hz, 1H), 2.66 (br s, 1H); 13
C
NMR (125.9 MHz, CDCl3) δ 165.8, 165.6, 138.9, 138.2, 138.1, 133.4,
133.3, 132.9, 130.3, 130.2, 129.7, 129.3, 128.7, 128.6, 128.5, 128.4,
128.3, 128.0, 127.9, 127.5, 100.2, 87.0, 81.3, 77.5, 77.3, 77.0, 75.3, 74.2,
74.0, 73.9, 73.4, 72.2, 71.4, 71.0, 70.7, 68.6, 65.4; ESIHRMS m/z calcd
for C52H50O11SNa [M + Na]+ 905.2972, found 905.2987.
Methyl 2,3-Di-O-benzyl-4,6-O-benzylidene-β-D-mannopyr-
anosyl-(1→4)-2,3-di-O-benzoyl-β-D-xylopyranoside (6). Ob-
tained by protocol 3 from compound 4. Chromatographic purification
over silica gel with 20% EtOAc in hexanes as eluent afforded
Methyl 2,3-Di-O-benzyl-4,6-O-benzylidene-β-D-mannopyra-
nosyl-(1→4)-2,3-di-O-benzoyl-β-D-xylopyranosyl-(1→4)-2,3,6-
tri-O-benzyl-β-D-mannopyranosyl-(1→4)-2,3-di-O-benzoyl-β-D-
xylopyranoside (10). Obtained by protocol 5 from compounds 7
and 8. Chromatographic purification over silica gel with 30% EtOAc in
hexanes as eluent afforded disachharide 10 (127 mg, 81%) as a white
24
disaccharide 6 (71 mg, 89%) as a white foam: [α]D −38.8 (c = 1.0,
1
CHCl3); H NMR (500 MHz, CDCl3) δ 8.05 (d, J = 7.0 Hz, 2H),
8.01 (d, J = 7.0 Hz, 2H), 7.56−7.51 (m, 2H), 7.49−7.47 (m, 2H),
8616
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The Journal of Organic Chemistry
Featured Article
foam: [α]D24 −43.6 (c = 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ
7.99 (d, J = 7.0 Hz, 2H), 7.93 (d, J = 8.0 Hz, 4H), 7.86 (d, J = 8.0 Hz,
2H), 7.54−7.44 (m, 6H), 7.39−7.32 (m, 18H), 7.29−7.23 (m, 10H),
7.21−7.14 (m, 8H), 5.67 (t, J = 8.2 Hz, 1H), 5.52 (t, J = 9.0 Hz, 1H),
5.43 (s, 1H), 5.30−5.26 (m, 2H), 4.81 (d, J = 12.5 Hz, 1H), 4.81 (d,
J = 7.0 Hz, 1H), 4.76 (d, J = 12.5 Hz, 1H), 4.67−4.63 (m, 4H), 4.58−
4.55 (m, 3H), 4.49 (s, 1H), 4.43 (s, 1H), 4.35 (d, J = 11.5 Hz, 1H),
4.18−4.13 (m, 3H), 4.07 (d, J = 12.0 Hz, 1H), 4.05−3.99 (m, 1H),
3.97−3.90 (m, 3H), 3.80 (dd, J = 3.2, 9.7 Hz, 2H), 3.54−3.46 (m,
7H), 3.43 (dd, J = 3.0, 9.5 Hz, 1H), 3.36 (t, J = 10.5 Hz, 1H), 3.24−
3.20 (m, 1H), 3.15−3.09 (m, 2H); 13C NMR (125.9 MHz, CDCl3) δ
165.7, 165.5, 165.4, 165.2, 138.7, 138.6, 138.5, 138.4, 137.5, 133.2,
133.1, 133.0 129.9, 129.8, 129.7, 129.5, 129.4, 129.3, 128.8, 128.4,
128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.5, 127.4, 127.3, 127.1,
126.0, 101.9, 101.3, 100.8, 100.6, 99.3, 80.1, 78.2, 76.8, 76.0, 75.8, 74.6,
74.3, 73.7, 73.2, 73.1, 72.7, 72.2, 72.0, 72.9, 72.3, 68.5, 68.2, 67.5, 63.0,
62.6, 56.7; ESIHRMS m/z calcd for C93H90O23Na [M + Na]+
1597.5771, found 1597.5748.
Phenyl 2,3-Di-O-benzyl-4,6-O-benzylidene-β-D-mannopyr-
anosyl-(1→4)-2,3-di-O-benzoyl-β-D-xylopyranosyl-(1→4)-2,3,6-
tri-O-benzyl-β-D-mannopyranosyl-(1→4)-2,3-di-O-benzoyl-1-
thio-β-D-xylopyranoside (11). Phenyl thioglycoside 7 (88 mg, 100
μmol), BSP (27 mg, 120 μmol), and 4 Å molecular sieves (150 mg)
were mixed and dried under vacuum for 0.5 h after which freshly
distilled dichloromethane (3 mL) was added and the reaction mixture
was stirred for 0.5 h at room temperature under N2 before it was
cooled to −65 °C and stirred for 5 min more. Tf2O (22 μL, 120 μmol)
was added dropwise, and the reaction mixture was stirred for 30 min
between −65 and −60 °C then cooled to −78 °C and stirred for 5 min
before 1-octene (0.15 mL, 1 mmol) was added. After stirring for 5 min,
a solution of the acceptor 9 (90 mg, 50 μmol) in dichloromethane
(2 mL) was added dropwise and the reaction mixture stirred for
2.5 h at −60 °C before it was quenched by addition of Et3N (0.1 mL)
and warmed to room temperature. The reaction mixture was filtered
through Celite, concentrated, and subjected to chromatographic purifi-
cation with 30% EtOAc in hexanes as eluent to afford tetrasaccharide
11 (139 mg, 79%) as a light yellow oil: [α]D24 −28.4 (c = 1.0, CHCl3);
1H NMR (500 MHz, CDCl3) δ 8.03 (d, J = 7.5 Hz, 2H), 7.95−7.91
4.17−4.10 (m, 2H), 3.96 (d, J = 9.0 Hz, 1H), 3.92−3.82 (m, 5H), 3.75
(dd, J = 5.5, 11.0 Hz, 1H), 3.63−3.53 (m, 3H), 3.51 (s, 3H), 3.50−
3.46 (m, 2H), 3.42−3.38 (m, 2H), 3.34 (dd, J = 2.7, 9.2 Hz, 1H), 2.73
(br s, 1H); 13C NMR (125.9 MHz, CDCl3) δ 165.9, 165.8, 165.4,
139.0, 138.8, 138.5, 138.3, 138.2, 133.4, 133.2, 133.1, 130.4, 130.1,
129.9, 129.8, 128.7, 128.6, 128.5, 128.4, 128.2, 128.1, 128.0, 127.9,
127.6, 127.5, 127.4, 101.5, 100.6, 100.1, 99.6, 82.2, 81.2, 75.9, 75.2,
75.1, 74.6, 74.3, 74.2, 74.1, 74.0, 73.9, 73.6, 73.4, 71.6, 71.3, 71.0, 70.9,
68.8, 68.3, 62.1, 56.8; ESIHRMS m/z calcd for C93H92O23Na [M +
Na]+ 1599.5927, found 1599.5914.
Methyl 2,3-Di-O-benzyl-4,6-O-benzylidene-β-D-mannopyra-
nosyl-(1→4)-2,3-di-O-benzoyl-β-D-xylopyranosyl-(1→4)-2,3,6-
tri-O-benzyl-β-D-mannopyranosyl-(1→4)-2,3-di-O-benzoyl-β-D-
xylopyranosyl-(1→4)-2,3,6-tri-O-benzyl-β-D-mannopyranosyl-
(1→4)-2,3-di-O-benzoyl-β-D-xylopyranoside (13). Obtained by
protocol 5 from compounds 11 and 8. Chromatographic purification
with 30% EtOAc in hexanes as eluent afforded the hexasaccharide 13
24
1
(157 mg, 67%) as a white foam: [α]D −32.7 (c = 1.0, CHCl3); H
NMR (500 MHz, CDCl3) δ 7.98 (d, J = 8.0 Hz, 2H), 7.93−7.89 (m,
6H), 7.85 (d, J = 8.0 Hz, 2H), 7.81 (d, J = 7.5 Hz, 2H), 7.54−7.41 (m,
9H), 7.40−7.30(m, 25 H), 7.29−7.23 (m, 17H), 7.21−7.11 (m, 12H),
5.65 (t, J = 8.0 Hz, 1H), 5.48 (t, J = 9.5 Hz, 1H), 5.46 (t, J = 9.5 Hz,
1H), 5.43 (s, 1H), 5.29−5.23 (m, 3H), 4.82−4.70 (m, 4H), 4.68−4.52
(m, 10H), 4.46 (s, 1H), 4.42 (s, 1H), 4.35−4.31 (m, 2H), 4.27 (s, 1H),
4.18−4.09 (m, 4H), 4.08−3.98 (m, 4H), 3.97−3.86 (m, 3H), 3.78 (s,
2H), 3.72 (s, 1H), 3.53−3.49 (m, 3H), 3.48 (s, 3H), 3.47−3.33 (m,
8H), 3.22−3.18 (m, 1H), 3.14−3.02(m, 4H); 13C NMR (125.9 MHz,
CDCl3) δ 165.7, 165.6, 165.5, 165.4, 165.2, 138.7, 138.7, 138.6, 138.4,
138.3, 137.5, 133.2, 133.1, 133.0, 132.8, 129.9, 129.8, 129.7, 129.5,
129.4, 129.2, 128.8, 128.4, 128.3, 128.2, 128.1, 127.9, 127.8, 127.5,
127.4, 127.3, 127.1, 126.0, 101.9, 101.2, 100.9, 100.8, 100.6, 99.2, 99.0,
80.0, 79.8, 78.2, 75.9, 74.5, 74.4, 74.3, 74.2, 73.7, 73.6, 73.2, 73.1, 73.0,
72.7, 72.1, 72.0, 71.9, 71.3, 68.4, 68.2, 67.5, 62.9, 62.6, 56.8; ESIHRMS
m/z calcd for C139H134O34Na [M + Na]+ 2369.8654, found 2369.8691.
Methyl 2,3-Di-O-benzyl-4,6-O-benzylidene-β-D-mannopyra-
nosyl-(1→4)-2,3-di-O-benzoyl-β-D-xylopyranosyl-(1→4)-2,3,6-
tri-O-benzyl-β-D-mannopyranosyl-(1→4)-2,3-di-O-benzoyl-β-D-
xylopyranosyl-(1→4)-2,3,6-tri-O-benzyl-β-D-mannopyranosyl-
(1→4)-2,3-di-O-benzoyl-β-D-xylopyranosyl-(1→4)-2,3,6-tri-O-
benzyl-β-D-mannopyranosyl-(1→4)-2,3-di-O-benzoyl-β-D-xylo-
pyranoside (14). Obtained by protocol 5 from compounds 11 and
12. Chromatographic purification with 40% EtOAc in hexanes as
eluent afforded the octasaccharide 14 (196 mg, 63%) as a white foam:
(m, 6H), 7.55−7.48 (m, 7H), 7.47−7.43 (m, 3H), 7.40−7.33 (m,
16H), 7,31−7.22 (m, 15H), 7.18−7.12 (m, 6H), 5.71 (t, J = 7.5 Hz,
1H), 5.52 (t, J = 9.0 Hz, 1H), 5.44 (s, 1H), 5.35 (t, J = 7.5 Hz, 1H),
5.28 (dd, J = 7.2, 8.7 Hz, 1H), 5.10 (d, J = 7.5 Hz, 1H), 4.83 (d, J =
12.0 Hz, 1H), 4.81 (d, J = 7.0 Hz, 1H), 4.76 (d, J = 12.5 Hz, 1H),
4.68−4.63 (m, 4H), 4.58 (s, 1H), 4.57 (d, J = 13.0 Hz, 1H), 4.50 (s,
1H), 4.44 (s, 1H), 4.38 (dd, J = 4.0, 12.0 Hz, 1H), 4.34 (d, J = 11.5 Hz,
1H), 4.17 (t, J = 9.5 Hz, 1H), 4.13−4.09 (m, 1H), 4.06 (d, J = 12.0 Hz,
1H), 4.05−4.00 (m, 1H), 3.98−3.89 (m, 3H), 3.80 (dd, J = 3.0, 7.5
Hz, 2H), 3.61 (dd, J = 8.0, 12.0 Hz, 1H), 3.53−3.47 (m, 3H), 3.43
(dd, J = 3.2, 9.2 Hz, 1H), 3.37 (t, J = 10.5 Hz, 1H), 3.25−3.21 (m,
1H), 3.16−3.08 (m, 2H); 13C NMR (125.9 MHz, CDCl3) δ 165.5,
165.3, 165.2, 138.7, 138.5, 138.3, 137.5, 133.2, 133.1, 133.0, 132.7,
132.4, 130.0, 129.8, 129.3, 129.3, 129.0, 128.8, 128.4, 128.3, 128.2,
128.1, 128.0, 127.9, 127.8, 127.5, 127.4, 127.2, 126.0, 101.3, 100.8,
100.6, 99.7, 86.7, 80.1, 78.2, 77.4, 75.9, 75.8, 74.6, 74.3, 73.7, 73.2,
72.9, 72.8, 72.2, 72.0, 70.5, 68.5, 68.2, 67.6, 65.2, 63.0; ESIHRMS m/z
calcd for C98H92O22SNa [M + Na]+ 1675.5699, found 1675.5793.
Methyl 2,3,6-Tri-O-benzyl-β-D-mannopyranosyl-(1→4)-2,3-
di-O-benzoyl-β-D-xylopyranosyl-(1→4)-2,3,6-tri-O-benzyl-β-D-
mannopyranosyl-(1→4)-2,3-di-O-benzoyl-β-D-xylopyranoside
(12). Obtained by protocol 4 from compound 10. Chromatographic
purification with 50% EtOAc in hexanes as eluent afforded the
24
1
[α]D −35.1 (c = 1.0, CHCl3); H NMR (500 MHz, CDCl3) δ 7.98
(d, J = 7.5 Hz, 2H), 7.94−7.89 (m, 8H), 7.86 (d, J = 7.0 Hz, 4H), 7.81
(d, J = 8.5 Hz, 2H), 7.53−7.48 (m, 5H), 7.46−7.43 (m, 3H), 7.38−
7.32 (m, 26H), 7.30−7.23 (m, 27H), 7.21−7.17 (m, 7H), 7.16−7.10
(m, 16H), 5.66 (t, J = 8.0 Hz, 1H), 5.54−5.46 (m, 2H), 5.43 (s, 1H),
5.32−5.22 (m, 4H), 4.83−4.69 (m, 8H), 4.67−4.52 (m, 12H), 4.47−
4.40 (m, 3H), 4.36−4.31 (m, 3H), 4.27 (s, 1H), 4.26 (s, 1H), 4.18−
4.08 (m, 5H), 4.07−3.95 (m, 7H), 3.92−3.86 (m, 3H), 3.82−3.76 (m,
3H), 3.71 (dd, J = 2.7, 11.2 Hz, 2H), 3.54−3.49 (m, 2H), 3.48 (s, 3H),
3.47−3.43 (m, 4H), 3.41−3.32 (m, 6H), 3.19 (dd, J = 3.7, 8.7 Hz,
1H), 3.16−3.06 (m, 5H) 3.03 (t, J = 10.7 Hz, 1H); 13C NMR (125.9
MHz, CDCl3) δ 165.7, 165.6, 165.5, 165.4, 165.2, 138.7, 138.6, 138.4,
138.3, 137.5, 133.2, 133.1, 133.0, 132.8, 129.9, 129.8, 129.7, 129.5,
129.4, 129.2, 128.8 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8,
127.6, 127.5, 127.4, 127.3, 127.2, 127.1, 126.0, 101.9, 101.2, 100.9,
100.8, 100.6, 99.2, 99.0, 80.0, 79.9, 78.2, 75.8, 74.6, 74.5, 74.4, 74.3,
74.2, 73.7, 73.6, 73.2, 73.1, 73.0, 72.7, 72.1, 72.0, 71.9, 71.3, 68.4, 68.3,
68.2, 67.5, 62.9, 62.6, 56.8; ESIHRMS m/z calcd for C185H178O45Na
[M + Na]+ 3142.1538, found 3142.1489.
24
tetrasaccharide 12 (137 mg, 87%) as a white foam: [α]D 36.2 (c =
Methyl β-D-Mannopyranosyl-(1→4)-β-D-xylopyranosyl-(1→
4)-β-D-mannopyranosyl-(1→4)-β-D-xylopyranoside (15). Ob-
tained by protocol 6 from compound 10. Removal of solvent afforded
15 (11.5 mg 93%) as white amorphous solid: [α]D24 52.4 (c = 1.0, 1:4
H2O/MeOH); ESIHRMS m/z calcd for C23H40O19Na [M + Na]+
1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 8.07 (d, J = 7.5 Hz, 2H),
8.02−7.93 (m, 4H), 7.91 (d, J = 7.5 Hz, 2H), 7.52 (t, J = 7.2 Hz, 1H),
7.47 (t, J = 7.5 Hz, 1H), 7.43−7.33 (m, 7H), 7.31−7.20 (m, 27H),
7.16−7.14 (m, 6H), 5.75 (t, J = 7.5 Hz, 1H), 5.63 (t, J = 7.7 Hz, 1H),
5.31 (t, J = 6.7 Hz, 1H), 5.16 (t, J = 6.7 Hz, 1H), 4.82 (d, J = 12.0 Hz,
1H), 4.75 (d, J = 11.0 Hz, 1H), 4.73 (d, J = 12.5 Hz, 1H), 4.71 (d, J =
8.0 Hz, 1H), 4.67−4.59 (m, 4H), 4.53 (d, J = 6.0 Hz, 1H), 4.52 (d, J =
5.5 Hz, 1H), 4.49−4.39 (6H), 4.23 (dd, J = 4.0, 12.0 Hz, 1H),
1
643.2061, found 643.2084. For H and 13C data, see Tables 1−3.
Methyl β-D-Mannopyranosyl-(1→4)-β-D-xylopyranosyl-(1→
4)-β-D-mannopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D-
mannopyranosyl-(1→4)-β-D-xylopyranoside (16). Obtained by
8617
dx.doi.org/10.1021/jo201780e|J. Org. Chem. 2011, 76, 8611−8620

The Journal of Organic Chemistry
Featured Article
protocol 6 from compound 13. Removal of solvent afforded 16
by protocol 5 from compounds 21 and 7 followed by methanolysis.
Chromatographic purification over silica gel with 3% methanol in
chloroform as eluent afforded tetrasaccharide 22 (80 mg, 72%) as a light
yellow oil: [α]D24 −53.8 (c = 1.0, CHCl3); 1H NMR (500 MHz, CDCl3)
δ 7.50 (dd, J = 2.0, 7.5 Hz, 2H), 7.42−7.37 (m, 5H), 7.35−7.28 (m,
18H), 5.63 (s, 1H), 4.89 (d, J = 11.5 Hz, 1H), 4.88 (d, J = 12.5 Hz, 1H),
4.84 (d, J = 12.5 Hz, 1H), 4.83 (d, J = 12.5 Hz, 1H), 4.76 (d, J = 12.5
Hz, 1H), 4.70 (br s, 1H), 4.64 (d, J = 12.5 Hz, 1H), 4.64 (d, J = 11.5 Hz,
1H), 4.58 (d, J = 11.5 Hz, 1H), 4.54 (d, J = 11.5 Hz, 1H), 4.53 (d, J =
11.5 Hz, 1H), 4.50 (s, 1H), 4.47 (s, 1H), 4.40 (d, J = 7.5 Hz, 1H), 4.32
(dd, J = 5.0, 10.5 Hz, 1H), 4.25−4.22 (m, 2H), 4.16 (t, J = 9.5 Hz, 1H),
3.99−3.95 (m, 2H), 3.94−3.89 (m, 3H), 3.81 (dd, J = 7.5, 10.5 Hz, 1H),
3.75 (dd, J = 4.0, 12.0 Hz, 1H), 3.61−3.56 (m, 3H), 3.55 (s, 3H), 3.54−
3.50 (m, 2H), 3.47 (dd, J = 3.0, 9.5 Hz, 1H), 3.43−3.30 (m, 3H), 3.19
(br s 1H), 3.06 (dd, J = 10.0, 11.0 Hz, 1H), 2.48 (br s, 1H); 13C NMR
(125.9 MHz, CDCl3) δ 138.3, 138.1, 138.0, 137.7, 137.5, 129.2, 128.9,
128.7, 128.6, 128.6, 128.5, 128.4, 128.2, 128.1, 128.0, 127.9, 127.9, 127.8,
126.3, 103.9, 103.2, 102.3, 101.8, 101.6, 81.0, 80.5, 78.5, 77.9, 76.1, 75.2,
75.1, 74.7, 74.4, 74.3, 74.0, 73.2, 73.0, 72.9, 72.1, 69.7, 68.3, 67.9, 63.8,
63.7, 57.1; ESIHRMS m/z calcd for C58H68O19Na [M + Na]+
1091.4252, found 1091.4265.
24
(17.2 mg, 94%) as white amorphous solid: [α]D 12.7 (c = 0.5, 1:4
H2O/MeOH); ESIHRMS m/z calcd for C34H58O28Na [M + Na]+
1
937.3012, found 937.3029. For H and 13C data, see Tables 1−3.
Methyl β-D-Mannopyranosyl-(1→4)-β-D-xylopyranosyl-(1→
4)-β-D-mannopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D-
mannopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-β-D-manno-
pyranosyl-(1→4)-β-D-xylopyranoside (17). Obtained by protocol
6 from compound 14. Removal of solvent afforded 17 (22.1 mg 94%)
24
as white amorphous solid: [α]D −16.8 (c = 0.4, 1:4 H2O:MeOH);
ESIHRMS m/z calcd for C45H76O37Na [M + Na]+ 1231.3963, found
1
1231.3958. For H and 13C data, see Tables 1−3.
Methyl 2-O-Benzyl-4,6-O-benzylidene-3-O-(2-naphthalenyl-
methyl)-β-D-mannopyranosyl-(1→4)-2,3-O-isopropylidine-β-D-
xylopyranoside (19). Obtained by protocol 1 from compounds 18
and 2. Chromatographic purification over silica gel with 20% EtOAc in
hexanes as eluent afforded disaccharide 19 (176 mg, 86%) as a light
yellow oil: [α]D −27.8 (c = 1.0, CHCl3); 1H NMR (500 MHz,
24
CDCl3) δ 7.85−7.83 (m, 1H), 7.80−7.75 (m, 2H), 7.73−7.69 (m, 1H),
7.56−7.52 (m, 2H), 7.49−7.45 (m, 4H), 7.43−7.39 (m, 4H), 7.37−
7.31 (m, 3H), 5.66 (s, 1H), 4.97 (d, J = 12.0 Hz, 1H), 4.91 (d, J = 12.0
Hz, 1H), 4.88 (d, J = 13.0 Hz, 1H), 4.81 (d, J = 13.0 Hz, 1H), 4.62 (s,
1H), 4.55 (d, J = 7.5 Hz, 1H), 4.30−4.24 (m, 2H), 4.04 (dd, J = 5.2,
11.7 Hz, 1H), 4.01−3.95 (m, 2H), 3.92 (d, J = 2.5 Hz, 1H), 3.75 (t, J =
9.2 Hz, 1H), 3.66 (dd, J = 2.7, 10.2 Hz, 1H), 3.53 (m, 3H), 3.41−3.34
Methyl β-D-Mannopyranosyl-(1→4)-β-D-xylopyranosyl-(1→
3)-β-D-mannopyranosyl-(1→4)-β-D-xylopyranoside (23). Ob-
tained by protocol 6 from compound 22. Removal of the solvent
afforded 23 (11.4 mg, 99%) as white amorphous solid: [α]D24 44.1 (c =
0.5, 1:4 H2O/MeOH); ESIHRMS m/z calcd for C23H40O19Na [M +
Na]+ 643.2061, found 643.2089. For 1H and 13C data, see Tables 1 and 2.
Phenyl 2,3-Di-O-acetyl-4,6-O-benzylidene-α-D-mannopyra-
nosyl-(1→4)-2,3-O-isopropylidene-1-thio-β-D-xylopyranoside
(25). Obtained by protocol 1 from compound 24 and 2. Chromato-
graphic purification over silica gel with 20% EtOAc in hexanes as
eluent afforded disaccharide 25 (122 mg, 79%) as a light yellow oil:
[α]D24 −26.5 (c = 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.59−
7.56 (m, 2H), 7.47−7.45 (m, 2H), 7.39−7.36 (m, 3H), 7.35−7.32 (m,
3H), 5.58 (s, 1H), 5.43 (dd, J = 1.5, 3.5 Hz, 1H), 5.35 (dd, J = 3.5,
10.5 Hz, 1H), 5.10 (d, J = 1.0 Hz, 1H), 4.81 (d, J = 9.5 Hz, 1H), 4.29
(dd, J = 3.0, 8.5 Hz, 1H), 4.18 (dd, J = 5.0, 12.0 Hz, 1H), 4.04 (t, J =
9.7 Hz, 1H), 3.99 (dt, J = 5.5, 9.0 Hz, 1H), 3.93−3.84 (m, 2H), 3.63
(t, J = 9.0 Hz, 1H), 3.39 (dd, J = 9.2, 11.7 Hz, 1H), 3.23 (t, J = 9.0 Hz,
1H), 2.18 (s, 3H), 2.02 (s, 3H), 1.49 (s, 3H), 1.42 (s, 3H); 13C NMR
(125.9 MHz, CDCl3) δ 169.9, 169.7, 136.9, 133.0, 131.6, 129.2, 128.9,
128.3, 128.2, 126.1, 111.6, 101.9, 98.1 (1JC−H = 169.7 Hz), 85.2, 81.6,
75.9, 75.2, 73.4, 69.7, 68.6, 68.1, 64.4, 26.7, 26.6, 20.9, 20.8; ESIHRMS
m/z calcd for C31H36O11SNa [M + Na]+ 639.1876, found 639.1887.
Methyl 2,3-Di-O-acetyl-4,6-O-benzylidene-α-D-mannopyr-
anosyl-(1→4)-2,3-O-isopropylidene-β-D-xylopyranoside
(26). Obtained by protocol 2 from compounds 24 and 3. Chromato-
graphic purification over silica gel with 30% EtOAc in hexanes as eluent
(m, 2H), 3.29 (dd, J = 7.7, 9.7 Hz, 1H), 1.43 (s, 3H), 1.40 (s, 3H); 13
C
NMR (125.9 MHz, CDCl3) δ 138.5, 137.8, 135.9, 133.4, 133.2, 129.1,
129.0, 128.5, 128.4, 128.1, 127.9, 126.6, 126.4, 126.3, 126.2, 125.9,
111.7, 102.6, 101.8, 100.3 (1JC−H = 157.5 Hz), 79.3, 78.9, 78.0, 76.8,
76.4, 76.1, 75.0, 72.7, 68.9, 67.8, 65.3, 56.7, 27.0, 26.8; ESIHRMS m/z
calcd for C40H44O10Na [M + Na]+ 707.2832, found 707.2813.
Methyl 2-O-Benzyl-4,6-O-benzylidene-3-O-(2-naphthalenyl-
methyl)-β-D-mannopyranosyl-(1→4)-2,3-di-O-acetyl-β-D-xylo-
pyranoside (20). Obtained by protocol 3 from compound 19.
Chromatographic purification over silica gel with 20% EtOAc in
hexanes as eluent afforded disaccharide 20 (64 mg, 88%) as a light
yellow oil: [α]D −34.9 (c = 1.0, CHCl3); 1H NMR (500 MHz,
24
CDCl3) δ 7.84−7.82 (m, 1H), 7.79−7.76 (m, 2H), 7.71−7.69 (m,
1H), 7.53 (dd, J = 2.0, 7.5 Hz, 2H), 7.50−7.46 (m, 4H), 7.42−7.38
(m, 4H), 7.36 (t, J = 7.5 Hz, 2H), 7.32 (d, J = 7.5 Hz, 1H), 5.64 (s,
1H), 5.21 (t, J = 8.7 Hz, 1H), 4.92 (d, J = 12.0 Hz, 1H), 4.87 (dd, J =
7.0, 9.0 Hz, 1H), 4.84 (d, J = 13.0 Hz, 1H), 4.79 (d, J = 12.0 Hz, 1H),
4.78 (d, J = 12.5 Hz, 1H), 4.50 (s, 1H), 4.38 (d, J = 7.0 Hz, 1H), 4.32
(dd, J = 5.0, 10.5 Hz, 1H), 4.21 (t, J = 9.5 Hz, 1H), 4.03 (dd, J = 5.0,
12.0 Hz, 1H), 3.97−3.92 (m, 1H), 3.89 (t, J = 10.0 Hz, 1H), 3.85 (d,
J = 3.0 Hz, 1H), 3.62 (dd, J = 3.2, 10.0, Hz, 1H), 3.48 (s, 3H), 3.35−
3.28 (m, 2H), 2.05 (s, 3H), 2.01 (s, 3H); 13C NMR (125.9 MHz,
CDCl3) δ 170.1, 169.7, 138.5, 137.5, 135.7, 133.2, 132.9, 128.9, 128.5,
128.3, 128.2, 128.1, 127.9, 127.7, 127.6, 126.3, 126.1, 125.9, 125.6,
101.9, 101.5, 100.1, 78.5, 75.9, 74.6, 73.5, 72.2, 72.1, 71.0, 68.5, 67.7,
62.6, 56.8, 20.9, 20.8; ESIHRMS m/z calcd for C41H44O12Na [M +
Na]+ 751.2730, found 751.2741.
24
afforded disaccharide 26 (136 mg, 84%) as a white foam: [α]D −7.6
(c = 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.48−7.46 (m, 2H),
7.39−7.36 (m, 3H), 5.59 (s, 1H), 5.44 (dd, J = 1.5, 3.0 Hz, 1H), 5.38
(dd, J = 3.5, 10.5 Hz, 1H), 5.12 (br s, 1H), 4.56 (d, J = 7.5 Hz, 1H),
4.31 (dd, J = 4.0, 10.0 Hz, 1H), 4.12 (dd, J = 5.0, 12.0 Hz, 1H), 4.08−
4.02 (m, 2H), 3.93 (dt, J = 4.0, 9.0 Hz, 1H), 3.88 (t, J = 10.0 Hz, 1H),
3.63 (t, J = 9.0 Hz, 1H), 3.55 (s, 3H), 3.41 (dd, J = 7.7, 12.2 Hz, 1H),
3.34 (t, J = 8.2 Hz, 1H), 2.19 (s, 3H), 2.03 (s, 3H), 1.46 (s, 3H), 1.44
(s, 3H); 13C NMR (125.9 MHz, CDCl3) δ 169.9, 169.8, 136.9, 129.2,
128.3, 126.1, 112.0, 102.5, 102.9, 97.9 (1JC−H = 170.2 Hz), 79.8, 76.6,
78.0, 73.9, 69.7, 68.6, 68.1, 65.4, 64.3, 56.6, 29.7, 26.6, 20.9; ESIHRMS
m/z calcd for C26H34O12Na [M + Na]+ 561.1948, found 561.1959.
Phenyl 2,3-Di-O-benzyl-4,6-O-benzylidene-α-D-mannopyra-
nosyl-(1→4)-2,3-O-di-O-acetyl-1-thio-β-D-xylopyranoside
(27). Obtained by protocol 3 from compound 25. Chromatographic
purification over silica gel with 20% EtOAc in hexanes as eluent
Methyl 2-O-Benzyl-4,6-O-benzylidene-β-D-mannopyranosyl-
(1→4)-2,3-di-O-acetyl- β-D-xylopyranoside (21). Obtained by
protocol 7 from compound 20. Chromatographic purification over
silica gel with 40% EtOAc in hexanes as eluent afforded disaccharide
21 (35 mg, 85%) as a white foam: [α]D24 −34.9 (c = 1.0, CHCl3); 1H
NMR (500 MHz, CDCl3) δ 7.48 (dd, J = 2.2, 7.2 Hz, 2H), 7.41−7.30
(m, 8H), 5.54 (s, 1H), 5.24 (t, J = 9.0 Hz, 1H), 4.99 (d, J = 11.5 Hz,
1H), 4.90 (dd, J = 7.2, 8.7 Hz, 1H), 4.64 (s, 1H), 4.57 (d, J = 12.0 Hz,
1H), 4.40 (d, J = 7.0 Hz, 1H), 4.34 (dd, J = 5.0, 10.5 Hz, 1H), 4.11
(dd, J = 5.2, 11.7 Hz, 1H), 4.01−3.96 (m, 1H), 3.87 (d, J = 2.5 Hz,
1H), 3.84−3.77 (m, 3H), 3.50 (s, 3H), 3.41 (dd, J = 9.5, 12.0 Hz, 1H),
3.35−3.31 (m, 1H), 2.43 (d, J = 8.5 Hz, 1H), 2.06 (s, 3H), 2.03
(s, 3H); 13C NMR (125.9 MHz, CDCl3) δ 170.4, 169.9, 138.3, 137.3,
129.4, 128.8, 128.5, 128.2, 126.5, 102.3, 102.2, 100.5, 79.4, 78.6, 75.7,
74.0, 72.3, 71.2, 70.9, 68.7, 67.5, 62.8, 57.1, 21.1, 21.0; ESIHRMS m/z
calcd for C30H36O12Na [M + Na]+ 611.2104, found 611.2117.
Methyl 2,3,6-Tri-O-benzyl-β-D-mannopyranosyl-(1→4)-β-
D-xylopyranosyl-(1→3)-2-O-benzyl-4,6-O-benzylidene-β-D-
mannopyranosyl-(1→4)-β-D-xylopyranoside (22). Obtained
24
afforded disaccharide 27 (55 mg, 84%) as a light yellow oil: [α]D
1
−23.2 (c = 1.0, CHCl3); H NMR (500 MHz, CDCl3) δ 7.49−7.47
(m, 2H), 7.46−7.43 (m, 2H), 7.38−7.35 (m, 3H), 7.34−7.32 (m, 3H),
5.57 (s, 1H), 5.36 (dd, J = 3.7, 10.2 Hz, 1H), 5.19−5.16 (m, 2H),
4.93−4.92 (d, J = 7.5 Hz, 1H), 4.89 (d, J = 11.0 Hz, 1H), 4.80 (d, J =
9.0 Hz, 1H), 4.24 (t, J = 11.2 Hz, 1H), 4.23 (t, J = 11.5 Hz, 1H), 4.04
(t, J = 10.0 Hz, 1H), 3.93 (dt, J = 4.5, 9.5 Hz, 1H), 3.83 (t, J = 10.5 Hz,
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1
1H), 3.79 (dt, J = 5.0, 8.5 Hz, 1H), 3.54 (dd, J = 9.0, 11.7 Hz, 1H),
2.17 (s, 3H), 2.12 (s, 3H), 2.11 (s, 3H), 2.01 (s, 3H); 13C NMR
(125.9 MHz, CDCl3) δ 169.9, 169.8, 169.7, 169.6, 136.9, 132.6, 132.4,
129.2, 129.1, 128.3, 128.2, 126.1, 101.9, 99.5, 86.4, 76.0, 74.8, 73.3,
70.2, 70.0, 68.4, 67.6, 66.9, 64.5, 20.8, 20.7; ESIHRMS m/z calcd for
C32H36O13SNa [M + Na]+ 683.1774, found 683.1785.
a colorless oil: [α]D −22.7 (c = 1.0, CHCl3); H NMR (500 MHz,
CDCl3) δ 7.85−7.80 (m, 3H), 7.73 (s, 1H), 7.52−7.46 (m, 2H), 7.38
(dd, J = 1.5, 8.5 Hz, 1H), 7.35−7.31 (m, 5H), 5.53 (br s, 1H), 5.25
(t, J = 10.2 Hz, 1H), 5.18 (s, 1H), 4.83 (d, J = 12.0 Hz, 1H), 4.59−
4.52 (m, 4H), 4.15 (dd, J = 5.5, 12.0 Hz, 1H), 4.06−4.03 (m, 1H),
3.87 (dd, J = 3.2, 9.7 Hz, 1H), 3.84−3.80 (m, 1H), 3.60 (t, J = 9.0 Hz,
1H), 3.58−3.53 (m, 2H), 3.52 (s, 3H), 3.36−3.32 (m, 2H), 2.17 (s,
3H), 1.92 (s, 3H), 1.48 (s, 3H), 1.45 (s, 3H); 13NMR (125.9 MHz,
CDCl3) δ 170.3, 169.8, 137.8, 135.3, 133.2, 132.9, 129.2, 128.8, 128.3,
128.1, 127.9, 127.7, 127.6, 126.5, 126.2, 126.0, 125.7, 112.0, 102.4, 97.5
(1JC−H = 170.6 Hz), 79.6, 74.7, 74.4, 73.6, 71.3, 70.5, 69.4, 68.1, 67.9,
65.5, 56.4, 26.7, 26.4, 21.1, 20.9; ESIHRMS m/z calcd for
C37H44O12Na [M + Na]+ 703.2730, found 703.2756.
Methyl 2,3-Di-O-acetyl-4,6-O-benzylidene-α-D-mannopyr-
anosyl-(1→4)-2,3-di-O-acetyl-β-D-xylopyranoside (28). Obtained
by protocol 3 from compound 26. Chromatographic purification over
silica gel with 20% EtOAc in hexanes as eluent afforded disachharide
1
28 (48 mg, 87%) as a colorless oil: [α]D24 −8.4 (c = 1.0, CHCl3); H
NMR (500 MHz, CDCl3) δ 7.47−7.43 (m, 2H), 7.38−7.35 (m, 3H),
5.57 (s, 1H), 5.37 (dd, J = 3.5, 10.5 Hz, 1H), 5.17 (br s, 1H), 5.14 (t,
J = 8.7 Hz, 1H), 4.92 (d, J = 1.0 Hz, 1H), 4.84 (dd, J = 7.0, 9.0 Hz,
1H), 4.37 (d, J = 7.0 Hz, 1H), 4.25 (dd, J = 4.7, 10.0 Hz, 1H), 4.08−
4.02 (m, 2H), 3.93 (dt, J = 4.5, 9.7 Hz, 1H), 3.85−3.78 (m, 2H),
3.49−3.46 (m, 1H), 3.47 (s, 3H), 2.16 (s, 3H), 2.09 (s, 3H), 2.06 (s,
3H), 2.03 (s, 3H); 13C NMR (125.9 MHz, CDCl3) δ 170.0, 169.8,
169.6, 136.9, 129.2, 128.2, 126.1, 101.8, 101.7, 99.5, 76.0, 75.2, 72.8,
71.0, 70.2, 68.4, 67.6, 64.5, 63.6, 56.8, 20.8, 20.6; ESIHRMS m/z calcd
for C27H34O14Na [M + Na]+ 605.1846, found 605.1832.
Methyl 2,4-Di-O-acetyl-6-O-benzyl-3-O-(2-naphthalenyl-
methyl)-α-D-mannopyranosyl-(1→4)-2,3-di-O-acetyl-β-D-xylo-
pyranoside (34). Obtained by protocol 3 from compound 33.
Chromatographic purification over silica gel with 25% EtOAc in
hexanes as eluent afforded disaccharide 34 (61 mg, 85%) as a colorless
24
1
oil: [α]D −15.8 (c = 1.0, CHCl3); H NMR (500 MHz, CDCl3) δ
7.87−7.81 (m, 3H), 7.74 (s, 1H), 7.52−7.47 (m, 2H), 7.38 (d, J = 8.5
Hz, 1H), 7.35−7.29 (m, 5H), 5.24 (t, J = 10 Hz, 1H), 5.20 (br d, J =
2.7 Hz, 1H), 5.13 (t, J = 9.0 Hz, 1H), 4.96 (s, 1H), 4.86 (dd, J = 7.2,
9.2 Hz, 1H), 4.77 (d, J = 12.0 Hz, 1H), 4.56 (d, J = 12.5 Hz, 1H),
4.54−4.53 (m, 2H), 4.31 (d, J = 7.5 Hz, 1H), 4.21 (dd, J = 5.0, 11.5
Hz, 1H), 3.84−3.78 (m, 3H), 3.56−3.49 (m, 2H), 3.46 (s, 3H), 3.34
(dd, J = 10.0, 11.5 Hz, 1H), 2.13 (s, 3H), 2.09 (s, 3H), 2.06 (s, 3H),
1.90 (s, 3H); 13C NMR (125.9 MHz, CDCl3) δ 170.6, 170.1, 169.9,
138.0, 135.3, 133.5, 133.2, 128.6, 128.4, 128.3, 127.9, 127.0, 126.4,
126.2, 126.0, 102.1, 99.6, 76.7, 74.3, 73.8, 73.4, 71.6, 71.4, 70.9, 69.5,
68.5, 68.1, 64.2, 57.0, 21.2, 21.1, 21.0, 20.9; ESIHRMS m/z calcd for
C38H44O14Na [M + Na]+ 747.2629, found 747.2641.
Methyl 2,4-Di-O-acetyl-6-O-benzyl-α-D-mannopyranosyl-
(1→4)-2,3-di-O-acetyl-β-D-xylopyranoside (35). Obtained by
protocol 7 from compound 34. Chromatographic purification over
silica gel with 40% EtOAc in hexanes as eluent afforded disaccharide
35 (36 mg, 88%) as a white foam: [α]D24 −11.4 (c = 1.0, CHCl3); 1H
NMR (500 MHz, CDCl3) δ 7.36−7.29 (m, 5H), 5.13 (t, J = 9.0 Hz,
1H), 5.11 (t, J = 10.0 Hz, 1H), 5.01 (d, J = 1.0 Hz, 1H), 4.90 (dd, J =
1.5, 3.5 Hz, 1H), 4.85 (dd, J = 7.5, 9.5 Hz, 1H), 4.63 (d, J = 12.0 Hz,
1H), 4.49 (d, J = 12.5 Hz, 1H), 4.33 (d, J = 7.0 Hz, 1H), 4.17 (dd, J =
5.5, 11.5 Hz, 1H), 3.96 (br d, J = 8.0 Hz, 1H), 3.85−3.78 (m, 2H),
3.57−3.51 (m, 2H), 3.47 (s, 3H), 3.36 (dd, J = 10.0, 11.5 Hz, 1H),
2.23 (br s, 1H), 2.14 (s, 3H), 2.12 (s, 3H), 2.06 (s, 3H), 1.99 (s, 3H);
13C NMR (125.9 MHz, CDCl3) δ 171.2, 170.4, 170.3, 169.7, 137.7,
Methyl 2,3-Di-O-acetyl-6-O-benzyl-α-D-mannopyranosyl-
(1→4)-2,3-di-O-acetyl-β-D-xylopyranoside (29). Obtained by
protocol 4 from compound 28. Chromatographic purification over
silica gel with 40% EtOAc in hexanes as eluent afforded disaccharide
29 (51 mg, 87%) as a colorless oil: [α]D24 −12.1 (c = 1.0, CHCl3); 1H
NMR (500 MHz, CDCl3) δ 7.36−7.32 (m, 4H), 7.31−7.28 (m, 1H),
5.13−5.09 (m, 2H), 5.03 (dd, J = 1.5, 3.0 Hz, 1H), 4.91 (br s, 1H),
4.82 (dd, J = 7.2, 9.0 Hz, 1H), 4.63 (d, J = 12.0 Hz, 1H), 4.56 (d, J =
12.0 Hz, 1H), 4.33 (d, J = 7.0 Hz, 1H), 4.12 (dd, J = 5.2, 11.7 Hz, 1H),
3.97 (dt, J = 1.5, 9.5 Hz, 1H), 3.81−3.77 (m, 2H), 3.76−3.73 (m, 2H),
3.45 (s, 3H), 3.42−3.36 (m, 1H), 2.80 (d, J = 3.5 Hz, 1H), 2.09 (s,
3H), 2.07 (s, 3H), 2.06 (s, 3H), 2.04(s, 3H); 13NMR (125.9 MHz,
CDCl3) δ 170.8, 180.2, 170.0, 169.8, 137.8, 128.4, 127.8, 127.6, 101.7,
98.8, 75.3, 73.7, 73.0, 72.0, 71.2, 71.1, 69.9, 69.7, 66.9, 63.7, 56.8, 20.9,
20.8, 20.7, 20.6; ESIHRMS m/z calcd for C27H36O14Na [M + Na]+
607.2003, found 607.1968.
Methyl 2,3-Di-O-acetyl-4,6-O-benzylidene-α-D-mannopyr-
anosyl-(1→4)-2,3-di-O-acetyl-β-D-xylopyranosyl-(1→4)-6-O-
benzyl-2,3-di-O-acetyl-α-D-mannopyranosyl-(1→4)-2,3-di-O-
acetyl-β-D-xylopyranoside (30). Obtained by protocol 5 from
compounds 27 and 29. Chromatographic purification over silica gel
with 30% EtOAc in hexanes as eluent afforded tetrasaccharide 30
24
1
(96 mg, 85%) as a colorless oil: [α]D −18.3 (c = 1.0, CHCl3); H
NMR (500 MHz, CDCl3) δ 7.47−7.44 (m, 2H), 7.43−7.41 (m, 2H),
7.40−7.36 (m, 6H), 5.58 (s, 1H), 5.38 (dd, J = 3.5, 10.5 Hz, 1H), 5.25
(dd, J = 3.0, 9.7 Hz, 1H), 5.17 (dd, J = 1.5, 3.0 Hz, 1H), 5.11 (t, J = 9.0
Hz, 1H), 5.00−4.95 (m, 3H), 4.89 (s, 1H), 4.83 (dd, J = 7.5, 9.0 Hz,
1H), 4.76 (d, J = 12.0 Hz, 1H), 4.70 (dd, J = 7.0, 8.0 Hz, 1H), 4.49 (d,
J = 12.0 Hz, 1H), 4.37 (d, J = 7.0 Hz, 1H), 4.34 (d, J = 7.5 Hz, 1H),
4.23 (dd, J = 4.0, 10.0 Hz, 1H), 4.07−4.02 (m, 3H), 3.95−3.88 (m,
2H), 3.84 (t, J = 10.0 Hz, 1H), 3.81−3.67 (m, 4H), 3.65 (d, J = 10.0
Hz, 1H), 3.45 (s, 3H), 3.44−3.39 (m, 1H), 3.26 (dd, J = 9.5, 12.0 Hz,
1H), 2.16 (s, 3H), 2.13 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 2.05 (s,
3H), 2.04 (s, 3H), 2.01 (s, 3H), 1.96 (s, 3H); 13NMR (125.9 MHz,
CDCl3) δ 170.0, 169.8, 169.6, 169.4, 137.7, 136.8, 129.2, 128.6, 128.3,
128.1, 127.9, 126.0, 101.8, 101.7, 100.4, 99.3, 98.6, 77.3, 77.2, 77.0,
76.7, 75.9, 75.1, 74.7, 73.7, 73.4, 72.9, 72.7, 71.7, 71.1, 71.0, 70.3, 70.1,
68.4, 67.7, 67.5, 64.5, 63.6, 63.4, 56.8, 20.9, 20.8, 20.7, 20.6; ESIHRMS
m/z calcd for C53H66O27Na [M + Na]+ 1157.3689, found 1157.3663.
Methyl α-D-Mannopyranosyl-(1→4)-β-D-xylopyranosyl-(1→
4)-α-D-mannopyranosyl-(1→4)-β-D-xylopyranoside (31). Ob-
tained by protocol 6 from compound 30. Removal of solvent afforded
31 (11.4 mg 92%) as white amorphous solid: [α]D24 24.5[c = 1, H2O/
MeOH(1/4)]; ESIHRMS m/z calcd for C23H40O19Na [M + Na]+
128.4, 127.8, 127.8, 101.9, 98.8, 76.0, 73.5, 73.0, 72.3, 71.2, 70.1, 69.5,
68.4, 64.0, 56.8, 21.0, 20.9, 20.8, 20.7; C27H36O14Na [M + Na]+
607.2003, found 607.2023.
Methyl 4,6-O-Benzylidene-α-D-mannopyranosyl-(1→4)-β-D-
xylopyranosyl-(1→3)-6-O-benzyl-α-D-mannopyranosyl-(1→4)-
β-D-xylopyranoside (36). Obtained by protocol 5 from compounds
28 and 35 followed by methanolysis. Chromatographic purification
over silica gel with 5% methanol in chloroform as eluent afforded
disaccharide 36 (59 mg, 74%) as a white foam: [α]D24 −21.7 (c = 1.0,
CHCl3); 1H NMR (500 MHz, CD3OD) δ 7.48−7.43 (m, 3H), 7.41−
7.35 (m, 7H), 5.58 (s, 1H), 5.58 (s, 1H), 5.40−5.33 (m, 1H), 5.30−
5.23 (m, 1H), 5.18 (s, 1H), 5.14−5.09 (m, 2H), 5.03−4.93 (m, 2H),
4.90 (s, 1H), 4.83 (t, J = 7.7 Hz, 1H), 4.78−4.67 (m, 2H), 4.51 (t, J =
6.5 Hz, 1H), 4.33 (d, J = 7.2 Hz, 1H), 4.27−4.22 (m, 2H), 4.09−3.99
(m, 4H), 3.96−3.88 (m, 2H), 3.85−3.69 (m, 6H), 3.53−3.39 (m, 4H),
3.37−3.26 (m, 1H), 2.18 (s, 3H), 2.15 (s, 3H), 2.14 (s, 3H), 2.11 (s,
3H), 2.05 (s, 3H), 2.03 (s, 3H), 2.01 (s, 3H), 1.99 (s, 3H); 13C NMR
(125.9 MHz, CD3OD) δ 138.5, 138.1, 128.7, 128.2, 127.9, 127.6,
126.3, 104.7, 103.9, 103.3, 102.2, 78.9, 78.1, 78.0, 76.8, 75.8, 75.7, 73.8,
73.6, 73.3, 71.5, 71.2, 70.1, 69.7, 69.1, 68.5, 68.2, 64.7, 64.6, 64.5, 56.1;
C53H66O27Na [M + Na]+ 821.2844, found 821.2857.
1
643.2061, found 643.2079. For H and 13C data, see Tables 1−3.
Methyl 2,4-Di-O-acetyl-6-O-benzyl-3-O-(2-naphthalenyl-
methyl)-α-D-mannopyranosyl-(1→4)-2,3-O-isopropylidene-β-
D-xylopyranoside (33). Obtained by protocol 1 from compounds
32 and 2. Chromatographic purification over silica gel with 25%
EtOAc in hexanes as eluent afforded disaccharide 33 (167 mg, 82%) as
Methyl α-D-Mannopyranosyl-(1→4)-β-D-xylopyranosyl-(1→
3)-α-D-mannopyranosyl-(1→4)-β-D-xylopyranoside (37). Ob-
tained by protocol 6 from compound 36. Removal of solvent afforded
37 (11.7 mg, 99%) as white amorphous solid: [α]D24 32.1 (c = 0.5, 1:4
8619
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H2O/MeOH); ESIHRMS m/z calcd for C23H40O19Na [M + Na]+
1
643.2061, found 643.2082. For H and 13C data, see Tables 1−3.
ASSOCIATED CONTENT
■
S
* Supporting Information
1
Copies of H, 13C, and 2D NMR spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: dcrich@chem.wayne.edu.
■
ACKNOWLEDGMENTS
■
We thank the NIH (GM 62160 and GM 57335) for support of
this work, and Dr. Bashar Ksebati (WSU) for extensive help
with NMR spectroscopy.
REFERENCES
■
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(18) NMR spectra for the synthetic oligosaccharides were recorded
under the same conditions of pH and temperature reported for the
1
spectra of the natural isolate, albeit at a different field strength: H and
13C NMR spectral data for the synthetic oligosaccharides were
obtained at 500 and 125 MHz, respectively, whereas the literature data
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