A convergent synthesis of 6-o-branched β-Glucan oligosaccharides

Article abstract of DOI:10.1002/ejoc.201500229

β-Glucans are important carbohydrate antigens that are found on the surface of fungal cells, and they could be useful for the development of antifungal vaccines. This paper describes a highly convergent and efficient strategy for the synthesis of structur

Full text of DOI:10.1002/ejoc.201500229

FULL PAPER
DOI: 10.1002/ejoc.201500229
A Convergent Synthesis of 6-O-Branched β-Glucan Oligosaccharides
Guochao Liao,[a] Srinivas Burgula,[a] Zhifang Zhou,[a] and Zhongwu Guo*[a]
Keywords: Medicinal chemistry / Synthesis design / Vaccine design / Carbohydrates / Glycosylation / Glycosides
β-Glucans are important carbohydrate antigens that are
found on the surface of fungal cells, and they could be useful β-glucan oligosaccharides consisting of
ides as glycosyl donors. It was used to successfully prepare
β-1,3-linked
a
for the development of antifungal vaccines. This paper
describes a highly convergent and efficient strategy for the
nonaglucan backbone linked to a β-1,6-glucotetraose, a β-
1,3-glucodiose, or a β-1,3-glucotetraose branch at the 6-O-
synthesis of structurally defined branched β-glucan oligosac- position of the nonaglucan central sugar unit. The structure
charides that can be used for detailed studies of β-glucans
and for the design of β-glucan-based vaccines. The strategy
was highlighted by assembling three target compounds
through preactivation-based glycosylation with thioglycos-
and size of the glycosyl donors and acceptors used in the
syntheses did not significantly affect the efficiency of the
glycosylation, which suggests that this strategy can be gen-
erally useful for the synthesis of more complex structures.
based vaccines, it is essential to have access to homogeneous
and structurally defined β-glucan oligosaccharides. Al-
though several linear β-glucan oligosaccharides, and an
oligosaccharide with monosaccharide branches, have pre-
viously been prepared by the hydrolysis of natural β-glucans
or by chemical synthesis,[8] current strategies have draw-
backs that have limited their synthetic efficiency and appli-
cability, especially in the synthesis of complex structures.
There is a clear need for an efficient and generally appli-
cable method for β-glucan oligosaccharide synthesis. Re-
cently, we described the efficient synthesis of linear β-glucan
oligosaccharides[10] through preactivation-based glycosyl-
ation.[11] In this paper, we report a highly convergent,
efficient, and potentially generally applicable strategy for
the synthesis of branched β-glucan oligosaccharides.
Introduction
With the drastic increase in fungal infections and anti-
fungal-drug resistance in the past two decades,[1] antifungal
vaccines are in urgent demand.[2] The unique polysac-
charides,[3] especially β-1,3-linked glucans (known as β-
glucans),[4] on the surface of fungal cells are attractive anti-
gens for the development of antifungal vaccines.[5] Studies
have shown that β-glucans are not only exposed, but are
also consistently expressed and highly conserved on the cell
surfaces of all pathogenic fungi.[6] It has also been shown
that β-glucans can induce strong immune responses,[4] and
that a vaccine composed of natural β-glucans can engender
effective protection against Candida albicans and
Aspergillus fumigatus infections in mouse.[7] In addition,
CRM197 protein conjugates of β-glucan oligosaccharides
have been shown to elicit immune responses comparable to
that induced by conjugates of the natural β-glucans.[8] This
demonstrates that oligosaccharide analogs of natural β-
glucans are useful for the development of antifungal vac-
cines.
Results and Discussion
Our synthetic targets were 1–3 (Scheme 1). These mol-
ecules have a β-1,3-linked nonaglucan backbone with
branches, including a β-1,6-glucotetraose (1), a β-1,3-gluco-
diose (2), and a β-1,3-glucotetraose (3), attached to the 6-
O-position of the central sugar unit of the nona-β-glucan.
These three compounds were supposed to span different
structural properties and immunologically determinant
epitopes of natural β-glucans.[12] Moreover, we planned to
attach a free aminoethyl group to the reducing end of the
oligosaccharides to facilitate their conjugation with various
biomolecules and tags, such as carrier proteins, to be useful
for biological studies and for the development of conjugate
vaccines.
The structure of β-glucans has been well established.[3,6]
Their main carbohydrate chain is composed of approxi-
mately 1500 β-1,3-linked glucose units, with ca. 40–50 ad-
ditional short β-1,6- or β-1,3-glucans attached to the main-
chain glucose 6-O-positions as branches.[9] However, the
functions of the branches in β-glucans and their influence
on the immunological properties of β-glucans have not been
clarified. To study these issues, and to develop β-glucan-
[a] Department of Chemistry, Wayne State University,
5101 Cass Avenue, Detroit, MI, 48202, USA
E-mail: zwguo@chem.wayne.edu
For the synthesis of target molecules 1–3, we were inter-
ested in a strategy built on preactivation-based iterative
one-pot glycosylation using thioglycosides as glycosyl do-
http://chem.wayne.edu/faculty/guo/index.html
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201500229.
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Synthesis of 6-O-Branched β-Glucan Oligosaccharides
Scheme 1. Structures of target β-glucan oligosaccharides 1–3 with β-1,6-tetraglucose (1), β-1,3-diglucose (2), and β-1,3-tetraglucose (3)
branches attached to the 6-O-position of the central glucose unit of a nonasaccharide; and retrosynthetic analysis of these molecules.
nors. This synthetic strategy has been shown to be simple
Our synthesis began with the preparation of 13 accord-
and efficient as multiple intermediate conversion and sepa- ing to a reported procedure.[14] Regioselective removal of
ration steps can be avoided.[11] Accordingly, our synthetic the NAP group at the 3-O-position in 13 with DDQ, fol-
plan (Scheme 1) was to separately prepare branches 4–6 as lowed by 3-O-benzoylation, and then regioselective re-
glycosyl donors and backbone nonasaccharide 7 as glycosyl ductive ring opening of the benzylidene acetal in 8 using
acceptor, and then to stitch them together to give the fully BH3·THF and trimethylsilyl trifluoromethanesulfonate
protected oligosaccharides, which would finally be depro- (TMSOTf) gave 9 (Scheme 2). On the other hand, reductive
tected. Such a synthetic strategy could be applied more ring opening of the benzylidene acetal in 13, followed by
widely by using different donors to give different branches, protection of the exposed 6-O-position with a levulinoyl
and/or different backbone acceptors that could also have (Lev) group through reaction with levulinic acid and N-(3-
varied branching sites. In turn, oligosaccharides 4–6 could dimethylaminopropyl)-NЈ-ethylcarbodiimide hydrochloride
be prepared from monosaccharides 8, 9, and 10 through (EDC·HCl), and then deprotection of the 3-O-position with
iterative one-pot glycosylation. Key intermediate 7 could be DDQ produced 11. Consequently, all of the required mono-
also constructed through one-pot glycosylation using 6, 11, saccharide building blocks were readily synthesized from 13
and 12. The synthesis of 12 would be similar to that of 6, in excellent overall yields.
but using 13 and 10 as building blocks. In 13, the 3-O-
For the synthesis of disaccharide building block 5, we
position would be temporarily protected with a 2-naphth- used the preactivation glycosylation protocol.[11a] First,
ylmethyl (NAP) group, which can be removed with 2,3- glycosyl donor 8 was treated with the promoter p-tolu-
dichloro-5,6-dicyano-1,4-benzoquinone (DDQ),[13] but enesulfenyl triflate (pTolSOTf; 1.0 equiv.), formed in situ by
which is more acid-stable than the comparable p-meth- the reaction of p-toluenesulfenyl chloride (pTolSCl) with sil-
oxybenzyl group. As a result, this position would be selec- ver triflate (AgOTf), at –78 °C for 10 min, and then glycosyl
tively exposed for elongation of the sugar chain later on. In acceptor 10 (0.9 equiv.) was added for the glycosylation.
addition, the 2-O-positions of all of the glycosyl donors The reaction was β-specific, and 5 was formed in 95% yield.
were protected with benzoyl (Bz) groups to ensure β-selec- Starting from 8, tetrasaccharide building blocks 4 and 6
tive glycosylations as a result of neighboring-group partici- were prepared through preactivation-based iterative one-
pation.
pot glycosylation using 9 and 10 as glycosyl donors, respec-
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G. Liao, S. Burgula, Z. Zhou, Z. Guo
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Scheme 2. Synthesis of the mono-, di-, and tetrasaccharide building blocks.
tively (Scheme 2).[11] Preactivation of the thioglycosyl glycosylation step gave an average of Ͼ75% yield, and that
donors with pTolSOTf was carried out at –78 °C for 10 min the overall yields were not significantly different for β-1,6-
in a mixture of dichloromethane and acetonitrile. After the and β-1,3-linked tetrasaccharides. Tetrasaccharide 15 was
donor had been completely consumed (ca. 5 min at –78 °C, then transformed into building block 12 by glycosylation
as shown by TLC), an acceptor (0.9 equiv.) was added, to- with 2-azidoethanol in the presence of pTolSCl/AgOTf, and
gether with 2,4,6-tri-tert-butylpyrimidine (TTBP), which then removal of the 2-NAP protecting group with DDQ.
was used to neutralize the trifluoromethanesulfonic acid All of the glycosylation reactions were β-specific, as con-
1
formed in the glycosylation reaction. The reaction was firmed by the H NMR spectra of 4, 5, 6, and 12, which
warmed to room temperature and stirred for ca. 20 min to showed coupling constants in the range 6.2–10.1 Hz for all
guarantee complete consumption of the acceptor, as indi- of the anomeric protons.
cated by TLC. Then, the mixture was cooled to –78 °C for
The preactivation-based one-pot glycosylation protocol
another round of preactivation and glycosylation by the was also used to prepare protected nonasaccharide 16 from
same protocol. After the third round of glycosylation, and 6, 11, and 12 (Scheme 3). We were pleased to find that these
then work-up, 4 and 6 were obtained in 45% and 43% iso- reactions gave an excellent overall yield (80%), despite the
lated yields, respectively. Similarly, tetrasaccharide 15 was fact that they involved rather complex glycosyl donors and
prepared from 13 and 10 by iterative one-pot glycosylation acceptors. Thereafter, the Lev group at the 6-O-position of
in an overall yield of 42%. These results suggest that each the central sugar residue in 16 was selectively removed with
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Synthesis of 6-O-Branched β-Glucan Oligosaccharides
hydrazine to give 7. Glycosylation of 7 with 4, 5, and 6 in the mixture was heated at 60 °C to remove all of the benzyl-
the presence of pTolSCl/AgOTf to install the branches was idene groups. Finally, the benzoyl groups were removed by
smooth, and gave fully protected target molecules 17, 18, treatment with sodium hydroxide in tert-butanol and water
and 19, respectively, in very good yields. Global deprotec- (4:1) to give the desired products 1, 2, and 3, which were
tion of 17–19 was carried out by a stepwise protocol to deal purified with a Sephadex-G25 size-exclusion column, and
with the solubility problems associated with the various fully characterized with 1D and 2D NMR spectroscopy, as
partially deprotected reaction intermediates. Thus, 17–19 well as HRMS. Although the intermediates in the global
were first treated with Zn and acetic acid in dichlorometh- deprotection process were not purified, the reactions were
ane to reduce the azide group. After filtration to remove carefully monitored by MS to make sure that each step was
solids and concentration to remove solvents, the crude complete. This was critical for the successful and clean
product was dissolved in acetic acid and water (5:1), and global deprotection, and for product purification.
Scheme 3. Synthesis of target oligosaccharides 1–3.
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G. Liao, S. Burgula, Z. Zhou, Z. Guo
FULL PAPER
purified by silica gel column chromatography (toluene/ethyl acetate,
Conclusions
15:1 to 10:1) to give 10 (5.48 g, 90%) as a white solid. 1H NMR
Research towards β-glucan-based antifungal vaccines has (600 MHz, CDCl3): δ = 8.14–8.02 (m, 2 H), 7.60 (t, J = 7.4 Hz, 1
H), 7.52–7.42 (m, 4 H), 7.36 (m, 5 H), 7.28–7.06 (m, 3 H), 5.55 (s,
1 H), 5.17–5.05 (t, J = 9.12 Hz, 1 H), 4.83 (d, J = 10.0 Hz, 1 H, 1-
H), 4.41 (dd, J = 10.6, 4.9 Hz, 1 H), 4.04 (m, 1 H), 3.81 (t, J =
10.2 Hz, 1 H), 3.66–3.50 (m, 2 H), 2.76 (d, J = 3.2 Hz, 1 H), 2.34
(d, J = 8.8 Hz, 3 H) ppm. 13C NMR (150 MHz, CDCl3): δ =
165.88, 138.72, 136.79, 133.76, 133.43, 130.03, 129.73, 129.50,
129.33, 128.46, 128.34, 127.83, 126.26, 101.91, 86.76 (C-1), 80.61,
77.21, 77.00, 76.79, 73.73, 73.24, 70.39, 68.50, 21.18 ppm. MS (ESI
TOF): calcd. for C27H26NaO6S [M + Na]+ 501.1; found 501.1.
made promising progress in recent decades, but access to
complex, structurally well-defined β-glucan oligosac-
charides that could be used for detailed structure–activity
relationship studies and for the preparation of glycoconjug-
ate vaccines is a significant hindrance in this area. In this
paper, we have described a new, highly convergent, and
efficient strategy for the synthesis of branched β-glucan
oligosaccharides. The strategy was demonstrated by the
application of preactivation-based iterative one-pot glyc-
osylation to the efficient construction of the intermediate
oligosaccharide fragments and the target molecules. This
approach can remarkably decrease the number of synthetic
and purification steps compared to reported methods.[8b,15]
Three complex β-glucan oligosaccharides with β-1,3- and
β-1,6-oligoglucose branches were efficiently assembled by
the new strategy. We observed that the structure and the
size of the glycosyl donors and acceptors used for the
preactivation-based glycosylation had little influence on the
efficiency of the reactions, which suggests that this synthetic
strategy may be broadly applicable for the construction of
more complex oligosaccharides in good yields. For example,
oligosaccharides with significantly larger branches and
backbones could be readily assembled by the procedures
shown in Scheme 2, and used to construct larger oligosac-
charides by the procedures shown in Scheme 3. The syn-
thetic targets were designed to bear a free amino group at
their reducing end, which will facilitate their conjugation
with other molecules, such as carrier proteins, through bi-
functional linkers. The resulting glycoconjugates could then
be used to investigate the functions of branches in β-glu-
cans, gain insights into structure–activity relationships, and
so on.
p-Tolyl
anoside (8):[16] Benzoyl chloride (3.7 mL, 31.37 mmol) was added
to solution of 10 (10.00 g, 20.09 mmol), Et3N (17.9 mL,
2,3-Di-O-Benzoyl-4,6-O-benzylidene-1-thio-β-D-glucopyr-
a
127.27 mmol), and DMAP (catalytic amount) in anhydrous
CH2Cl2 (160 mL) at 0 °C. The reaction mixture was stirred for 12 h,
then the mixture was washed with saturated aq. NaHCO3 solution
and brine, dried with Na2SO4, and concentrated under vacuum.
The residue was purified by silica gel column chromatography
(ethyl acetate/toluene, 1:20) to give 8 (11.44 g, 94%) as a white so-
lid. 1H NMR (600 MHz, CDCl3): δ = 7.98 (d, J = 7.3 Hz, 2 H,
Ph), 7.93 (d, J = 7.3 Hz, 2 H, Ph), 7.52 (t, J = 7.4 Hz, 1 H, Ph),
7.47 (t, J = 7.4 Hz, 1 H, Ph), 7.43–7.21 (m, 11 H, Ph), 7.12 (d, J
= 7.9 Hz, 2 H, Ph), 5.78 (t, J = 9.5 Hz, 1 H, 3-H), 5.53 (s, 1 H,
CHPh), 5.45 (t, J = 9.6 Hz, 1 H, 2-H), 4.96 (d, J = 10.0 Hz, 1 H,
1-H), 4.45 (dd, J = 10.6, 4.9 Hz, 1 H, 6-H), 3.94–3.82 (m, 2 H, 4-
H, 5-H), 3.74 (dd, J = 9.6, 4.9 Hz, 1 H, 6-H), 2.35 (s, 3 H, CH3)
ppm. 13C NMR (150 MHz, CDCl3): δ = 165.56, 165.15, 138.74,
136.71, 133.73, 133.27, 133.05, 129.86, 129.77, 129.76, 129.37,
129.25, 129.01, 128.37, 128.26, 128.16, 127.90, 126.10, 101.44,
87.25 (C-1), 78.57, 77.22, 77.01, 76.80, 73.34, 71.07, 70.91, 68.53,
21.19 ppm. HRMS (ESI TOF): calcd. for C34H31O7S [M + H]+
583.1790; found 583.1799.
p-Tolyl
2,3-Di-O-Benzoyl-4-O-benzyl-1-thio-β-D-glucopyranoside
(9): A mixture of 8 (3.50 g, 6.00 mmol) and molecular sieves (4 Å;
8 g) in anhydrous THF (120 mL) was stirred at room temp. for 1 h,
and then it was cooled to –40 °C. BH3·THF (1 m solution in THF;
29.7 mL, 30.00 mmol) was added. The mixture was stirred for
15 min, then TMSOTf (1.41 mL, 7.80 mmol) was added, and the
mixture was stirred at –40 °C for a further 1 h. The reaction mix-
ture was slowly warmed to room temp. and stirred for 24 h. Then,
saturated aq. NaHCO3 solution (50 mL) was added at 0 °C to
quench the reaction, and the reaction mixture was diluted with
CH2Cl2 (400 mL). The mixture was filtered to remove insoluble
materials, and the two phases were separated. The organic layer
was washed with saturated aq. NaHCO3 solution and brine, dried
with Na2SO4, and concentrated under reduced pressure. The resi-
due was purified by column chromatography (ethyl acetate/toluene,
1:25) to give 9 (3.26 g, 93%) as a white solid. 1H NMR (600 MHz,
CDCl3): δ = 7.95 (dd, J = 8.2, 1.0 Hz, 2 H, Ph), 7.90 (dd, J = 8.2,
1.0 Hz, 2 H, Ph), 7.53–7.46 (m, 2 H, Ph), 7.40–7.32 (m, 6 H, Ph),
7.20–7.07 (m, 7 H, Ph), 5.72 (t, J = 9.4 Hz, 1 H, 3-H), 5.33 (t, J =
9.8 Hz, 1 H, 2-H), 4.89 (d, J = 10.0 Hz, 1 H, 1-H), 4.57 (s, 2 H,
CH2Ph), 4.00–3.94 (m, 1 H, 6-H), 3.89 (t, J = 9.5 Hz, 1 H, 4-H),
3.79 (m, 1 H, 6-H), 3.65–3.56 (m, 1 H, 5-H), 2.33 (s, 3 H), 2.08–
1.95 (br., 1 H, OH) ppm. 13C NMR (150 MHz, CDCl3): δ = 165.65,
165.29, 138.61, 137.08, 133.41, 133.22, 133.17, 129.85, 129.78,
129.71, 129.33, 129.26, 128.35, 128.16, 127.96, 86.29 (C-1), 79.53,
76.23, 75.33, 74.84, 70.88, 61.73, 21.17 ppm. HRMS (ESI TOF):
calcd. for C34H33O7S [M + H]+ 585.1947; found 585.1941.
Experimental Section
General Methods: Chemicals and materials were obtained from
commercial suppliers, and were used as received without additional
purification unless otherwise noted. Molecular sieves (4 Å) were
flame-dried under high vacuum, and were used immediately, after
cooling under a N2 atmosphere. Analytical TLC was carried out
on silica gel 60 Å F254 plates, with detection by UV light and/or by
charring with H2SO4 (15% v/v in EtOH). NMR spectra were re-
corded with a 400, 500, or 600 MHz spectrometer. Chemical shifts
are reported in ppm (δ) downfield from tetramethylsilane, which
was used as an internal reference. Mass spectrometry (MS) was
carried out with either a Bruker Daltonics Ultraflex MALDI TOF
mass spectrometer or a Waters LCT Premier XE high-resolution
ESI TOF mass spectrometer.
p-Tolyl 2-O-Benzoyl-4,6-O-benzylidene-1-thio-β-D-glucopyranoside
(10):[13] DDQ (5.78 g, 25.47 mmol) was added to a stirred solution
of 13 (7.88 g, 12.73 mmol) in CH2Cl2 (400 mL) and water (22 mL)
at room temperature. The reaction was stirred at room temp. for
8 h, then saturated aq. NaHCO3 solution was added, and the mix-
ture was extracted with CH2Cl2. The combined organic extracts
were washed with saturated aq. NaHCO3 solution, and dried with
Na2SO4, and the solvent was evaporated in vacuo. The residue was
p-Tolyl
thio-β-
2-O-Benzoyl-4-O-benzyl-3-O-(naphthalen-2-ylmethyl)-1-
-glucopyranoside (14):[13] Compound 14 (1.84 g, 92%) was
D
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Synthesis of 6-O-Branched β-Glucan Oligosaccharides
prepared from 13 (2.00 g, 3.23 mmol) by the same procedure de-
scribed for 9. 1H NMR (500 MHz, CDCl3): δ = 7.99 (d, J =
19 H), 7.08 (d, J = 7.9 Hz, 2 H), 5.69–5.56 (m, 2 H), 5.44 (t, J =
7.9 Hz, 1 H), 5.34 (dd, J = 17.9, 8.7 Hz, 2 H), 5.05 (d, J = 7.2 Hz,
7.32 Hz, 2 H, Ph), 7.70–7.67 (m, 1 H, Ph), 7.63–7.60 (m, 1 H, Ph), 1 H, 1-HЈЈ), 4.82 (d, J = 10.0 Hz, 1 H, 1-HЈ), 4.44 (dd, J = 10.5,
7.57–7.51 (m, 3 H, Ph), 7.42–7.29 (m, 11 H, Ph), 7.27–7.24 (m, 1
H, Ph), 7.10 (d, J = 7.63 Hz, 2 H, Ph), 5.28 (dd, J = 9.77, 8.55 Hz,
4.7 Hz, 1 H), 4.33–4.22 (m, 2 H), 3.95 (t, J = 9.5 Hz, 1 H), 3.90–
3.82 (m, 2 H), 3.78 (t, J = 10.2 Hz, 1 H), 3.68–3.53 (m, 2 H), 2.34
1 H, 2-H), 4.93 (d, J = 11.60 Hz, 1 H, 1/2 CH2Ar), 4.90 (d, J = (s, 3 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 165.54, 164.90,
11.29 Hz, 1 H, 1/2 CH2Ar), 4.82 (d, J = 11.29 Hz, 1 H, 1/2 CH2Ar), 164.64, 138.50, 137.12, 136.84, 133.37, 133.07, 132.75, 129.83,
4.76 (d, J = 10.07 Hz, 1 H, 1-H), 4.71 (d, J = 10.68 Hz, 1 H, 1/2
CH2Ar), 3.96–3.90 (m, 2 H, 3-H, 6-H), 3.78–3.71 (m, 2 H, 4-H, 6- 128.25, 128.15, 128.11, 126.15, 126.11, 101.52, 101.30, 100.72,
H), 3.53–3.49 (m, 1 H, 5-H), 2.33 (s, 3 H, CH3), 1.93 (br. s, 1 H, 87.63, 79.56, 79.32, 78.30, 72.90, 72.35, 72.07, 70.75, 68.68, 68.60,
129.72, 129.41, 129.33, 129.08, 128.99, 128.51, 128.40, 128.37,
OH) ppm. 13C NMR (125 MHz, CDCl3): δ = 165.20, 138.43, 66.27, 21.19 ppm. HRMS (ESI TOF): calcd. for C54H49NO13S [M
137.73, 135.10, 133.32, 133.21, 133.07, 132.90, 129.78, 129.73,
128.56, 128.49, 128.36, 128.16, 128.10, 128.04, 127.85, 127.64,
126.90, 126.07, 125.94, 125.81, 86.36 (C-1), 83.91, 79.54, 77.62,
75.39, 75.23, 72.42, 62.04, 21.17 ppm. HRMS (ESI TOF): calcd.
for C38H36NaO6S [M + Na]+ 643.2130; found 643.2139.
+ H]+ 937.2894; found 937.2864.
p-Tolyl [2,3-Di-O-Benzoyl-4,6-O-benzylidene-β-D-glucopyranosyl]-
(1Ǟ6)-[2,3-di-O-benzoyl-4-O-benzyl-β-
[2,3-di-O-benzoyl-4-O-benzyl-β- -glucopyranosyl]-(1Ǟ6)-2,3-di-O-
benzoyl-4-O-benzyl-1-thio-β- -glucopyranoside (4): A mixture of
D-glucopyranosyl]-(1Ǟ6)-
D
D
donor 8 (349.8 mg, 0.60 mmol) and activated molecular sieves (4 Å)
in CH2Cl2 (8 mL) was stirred at room temp. for 1 h, and then it
was cooled to –78 °C. A solution of AgOTf (462.5 mg, 1.80 mmol)
in dry acetonitrile (1.5 mL) was added, and then after 10 min
pTolSCl (86 μL, 0.60 mmol) was added by microsyringe. The mix-
ture was stirred for a further 15 min, after which time TLC showed
that 8 had been completely consumed. Then, a solution of acceptor
9 (316.2 mg, 0.54 mmol) and TTBP (122.1 mg, 0.54 mmol) in
CH2Cl2 (2 mL) was added. The mixture was warmed to room temp.
slowly over 1 h, and stirred at room temp. for a further 20 min. The
mixture was then cooled to –78 °C for another round of glycosyl-
ation with 9 (288.1 mg, 0.49 mmol) as the glycosyl acceptor by the
same protocol. This was followed by a third round of glycosylation,
also with 9 (262.3 mg, 0.45 mmol) as the glycosyl acceptor. Finally,
the reaction mixture was warmed to room temp., stirred for 20 min,
and then quenched with saturated aq. NaHCO3 solution. The mix-
ture was filtered to remove insoluble materials, and the organic
layer was washed with saturated aq. NaHCO3 solution and brine,
dried with Na2SO4, and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (ethyl
acetate/toluene, 1:12) to give 4 (395.7 mg, 45%) as a white solid.
1H NMR (600 MHz, CDCl3): δ = 8.03 (d, J = 7.9 Hz, 2 H), 8.01–
7.89 (m, 11 H), 7.85 (d, J = 8.0 Hz, 2 H), 7.60–7.04 (m, 40 H), 7.01
(t, J = 7.4 Hz, 1 H), 6.99–6.91 (m, 4 H), 6.88 (t, J = 7.5 Hz, 2 H),
6.73 (d, J = 7.7 Hz, 2 H), 5.85 (t, J = 9.6 Hz, 1 H), 5.78 (t, J =
9.5 Hz, 1 H), 5.71–5.60 (m, 3 H), 5.47 (dd, J = 10.7, 7.0 Hz, 3 H),
5.30 (t, J = 9.7 Hz, 1 H), 4.98–4.87 (m, 3 H, anomeric), 4.58 (d, J
= 7.9 Hz, 1 H, anomeric), 4.35 (dt, J = 10.5, 5.2 Hz, 4 H), 4.19 (m,
5 H), 4.06 (d, J = 11.3 Hz, 1 H), 3.91 (m, 5 H), 3.74 (m, 4 H), 3.63
(d, J = 9.5 Hz, 1 H), 3.54 (dd, J = 11.5, 6.3 Hz, 1 H), 2.39 (s, 3 H)
ppm. 13C NMR (150 MHz, CDCl3): δ = 165.79, 165.68, 165.64,
165.60, 165.29, 165.23, 165.10, 164.81, 138.08, 137.14, 137.06,
136.92, 134.31, 133.30, 133.16, 133.09, 133.01, 132.97, 132.88,
132.77, 130.02, 129.89, 129.82, 129.78, 129.73, 129.67, 129.62,
129.56, 129.49, 129.46, 129.39, 129.33, 128.95, 128.93, 128.58,
128.50, 128.40, 128.29, 128.26, 128.22, 128.19, 128.14, 127.98,
127.92, 127.88, 127.80, 127.54, 126.08, 102.93, 101.70, 101.10,
101.06, 85.45, 79.17, 78.38, 77.83, 76.69, 76.62, 75.61, 75.15, 74.96,
74.89, 74.86, 74.77, 73.66, 72.94, 72.48, 72.15, 71.99, 70.75, 69.75,
69.39, 68.88, 68.36, 66.82, 21.30 ppm. HRMS (ESI TOF): calcd.
for C115H106NO28S [M + NH4]+ 1980.6622; found 1980.6481.
p-Tolyl 2-O-Benzoyl-4-O-benzyl-6-O-levulinoyl-1-thio-β-D-glucopyr-
anoside (11): A mixture of 14 (3.72 g, 6.00 mmol), levulinic acid
(0.84 g, 7.23 mmol), and EDC·HCl (1.38 g, 7.20 mmol) in CH2Cl2
(50 mL) was stirred at room temp. for 4 h. The reaction mixture
was washed with water and brine, dried with Na2SO4, and concen-
trated under vacuum.
The residue was dissolved in a mixture of CH2Cl2 (100 mL) and
water (1.5 mL) at room temp., and then DDQ (2.72 g, 12.00 mmol)
was added. The resulting mixture was stirred at room temp. for 6 h,
then it was washed with saturated aq. NaHCO3 solution and brine,
dried with Na2SO4, and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (ethyl
1
acetate/toluene, 1:10) to give 11 (3.10 g, 89%) as a foamy solid. H
NMR (500 MHz, CDCl3): δ = 8.16–8.06 (m, 2 H, Ph), 7.66–7.56
(m, 1 H, Ph), 7.49 (t, J = 7.8 Hz, 2 H, Ph), 7.39–7.21 (m, 7 H, Ph),
7.10 (d, J = 7.9 Hz, 2 H, Ph), 4.98 (t, J = 9.5 Hz, 1 H, 3-H), 4.87
(d, J = 11.2 Hz, 1 H, 1-H), 4.72 (dd, J = 15.6, 10.6 Hz, 2 H,
CH2Ph), 4.46 (dd, J = 11.8, 2.0 Hz, 1 H, 6-H), 4.28 (dd, J = 11.8,
5.2 Hz, 1 H, 6-H), 3.96 (td, J = 8.9, 2.6 Hz, 1 H, 3-H), 3.61 (m, 1
H, 5-H), 3.56–3.48 (m, 1 H, 4-H), 2.88 (d, J = 3.4 Hz, 1 H, OH),
2.77 (t, J = 6.6 Hz, 2 H, CH2CO2), 2.69–2.56 (m, 2 H, CH2CO),
2.34 (s, 3 H, CH3), 2.21 (s, 3 H, CH3) ppm. 13C NMR (125 MHz,
CDCl3): δ = 206.50, 172.43, 166.43, 138.44, 137.74, 133.53, 133.48,
130.05, 129.63, 129.45, 128.59, 128.51, 128.26, 128.08, 85.70, 77.69,
77.59, 76.86, 74.97, 73.34, 63.28, 37.91, 29.92, 27.88, 21.19 ppm.
HRMS (ESI TOF): calcd. for C32H35O8S [M + H]+ 579.2053;
found 579.2051.
p-Tolyl [2,3-Di-O-Benzoyl-4,6-O-benzylidene-β-D-glucopyranosyl]-
(1Ǟ3)-2-O-benzoyl-4,6-O-benzylidene-1-thio-β-
D
-glucopyranoside
(5): A mixture of glycosyl donor 8 (300.0 mg, 0.52 mmol) and mo-
lecular sieves (4 Å; 1.50 g) in anhydrous CH2Cl2 (10 mL) was
stirred at room temp. for 1 h, and then it was cooled to –78 °C. A
solution of AgOTf (397.0 mg, 1.55 mmol) in dry acetonitrile (3 mL)
was added, and then after 10 min p-TolSCl (74 μL, 0.52 mmol) was
added by microsyringe. The mixture was stirred at –78 °C for a
further 15 min, after which time TLC showed that 8 had been com-
pletely consumed. A solution of acceptor 10 (221.8 mg, 0.46 mmol)
and TTBP (127.9 mg, 0.52 mmol) in CH2Cl2 (3 mL) was added.
The mixture was stirred at –78 °C for 20 min, and then it was
warmed to room temp., followed by filtration to remove the molec-
ular sieves (4 Å). The filtrate was washed with saturated aq.
NaHCO3 solution and brine, dried with Na2SO4, and concentrated
under vacuum. The residue was purified by silica gel column
p-Tolyl [2,3-Di-O-Benzoyl-4,6-O-benzylidene-β-
(1Ǟ3)-[2-O-benzoyl-4,6-O-benzylidene-β- -glucopyranosyl]-(1Ǟ3)-
[2-O-benzoyl-4,6-O-benzylidene-β- -glucopyranosyl]-(1Ǟ3)-2-O-
benzoyl-4,6-O-benzylidene-1-thio-β- -glucopyranoside (6): Com-
D-glucopyranosyl]-
D
D
chromatography (ethyl acetate/toluene, 1:30) to give 5 (411.4 mg,
D
1
95%). H NMR (500 MHz, CDCl3): δ = 7.89 (d, J = 7.6 Hz, 2 H), pound 6 (415.3 mg, 43 %) was prepared from 8 (475.0 mg,
7.81 (d, J = 7.6 Hz, 2 H), 7.58 (t, J = 6.5 Hz, 4 H), 7.48–7.19 (m,
0.82 mmol) and 10 (1st glycosylation: 341.5 mg, 0.72 mmol; 2nd
Eur. J. Org. Chem. 2015, 2942–2951
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2947

G. Liao, S. Burgula, Z. Zhou, Z. Guo
FULL PAPER
glycosylation: 310.8 mg, 0.65 mmol; 3rd glycosylation: 282.8 mg,
0.59 mmol) after three rounds of glycosylation reactions by the pro-
tocol described for 4, and was purified by silica gel column
chromatography (ethyl acetate/toluene, 1:12). 1H NMR (600 MHz,
0.18 mmol) by the same protocol described for 5 gave a crude tri-
saccharide intermediate. This material was dissolved in CH2Cl2
(10 mL) and water (0.5 mL), and treated with DDQ (82.5 mg,
0.36 mmol). The reaction mixture was stirred at room temp. for
CDCl3): δ = 7.90 (t, J = 7.6 Hz, 4 H), 7.81 (d, J = 7.4 Hz, 2 H), 6 h, then it was washed with saturated aq. NaHCO3 solution and
7.70 (d, J = 7.5 Hz, 2 H), 7.67 (d, J = 7.4 Hz, 2 H), 7.60 (t, J =
7.4 Hz, 1 H), 7.54 (d, J = 7.4 Hz, 2 H), 7.52–7.11 (m, 34 H), 7.07
(d, J = 8.0 Hz, 2 H), 5.67 (t, J = 9.2 Hz, 1 H), 5.48 (dd, J = 10.4,
5.8 Hz, 2 H), 5.39 (s, 1 H), 5.27 (t, J = 6.5 Hz, 1 H), 5.16 (s, 1 H),
5.12 (d, J = 7.5 Hz, 1 H, anomeric), 5.07 (d, J = 6.4 Hz, 1 H,
anomeric), 4.89–4.81 (m, 2 H, anomeric), 4.76 (t, J = 9.3 Hz, 1 H),
4.66 (d, J = 10.1 Hz, 1 H, anomeric), 4.50 (s, 1 H), 4.34 (dd, J =
10.5, 4.9 Hz, 1 H), 4.27 (dd, J = 10.5, 4.9 Hz, 1 H), 4.22–4.15 (m,
2 H), 4.14–4.07 (m, 2 H), 3.94 (ddd, J = 18.8, 15.5, 9.3 Hz, 3 H),
brine, dried with Na2SO4, and concentrated under reduced pres-
sure. The residue was purified by silica gel column chromatography
(ethyl acetate/toluene, 1:8) to give 12 (213.6 mg, 78%). 1H NMR
(500 MHz, CDCl3): δ = 7.95 (d, J = 7.8 Hz, 2 H), 7.90 (d, J =
7.9 Hz, 2 H), 7.82 (d, J = 7.8 Hz, 2 H), 7.70 (d, J = 7.9 Hz, 2 H),
7.62–7.16 (m, 32 H), 5.57 (s, 1 H), 5.44 (s, 1 H), 5.21 (dd, J = 9.3,
6.6 Hz, 2 H, anomeric), 5.08 (d, J = 7.4 Hz, 1 H, anomeric), 5.03
(d, J = 5.2 Hz, 1 H, anomeric), 4.97 (t, J = 8.1 Hz, 1 H), 4.91–4.85
(m, 2 H), 4.59 (d, J = 7.6 Hz, 1 H, anomeric), 4.37 (dd, J = 10.4,
3.76 (dt, J = 14.0, 9.6 Hz, 2 H), 3.71–3.57 (m, 4 H), 3.53–3.37 (m, 4.8 Hz, 1 H), 4.23 (dd, J = 10.4, 4.9 Hz, 1 H), 4.14 (m, 3 H), 3.98
3 H), 3.12 (t, J = 9.5 Hz, 1 H), 2.32 (s, 3 H) ppm. 13C NMR
(150 MHz, CDCl3): δ = 165.48, 165.08, 164.86, 164.57, 164.54,
(m, 3 H), 3.89 (dd, J = 10.4, 4.8 Hz, 1 H), 3.80–3.38 (m, 11 H),
3.33–3.22 (m, 2 H), 2.71 (br. s, 1 H) ppm. 13C NMR (125 MHz,
138.23, 137.30, 137.09, 136.88, 133.60, 133.17, 132.98, 132.73, CDCl3): δ = 165.82, 164.68, 164.64, 164.60, 137.34, 137.26, 137.13,
130.16, 129.82, 129.76, 129.70, 129.66, 129.45, 129.39, 129.11, 137.06, 133.62, 133.41, 133.16, 133.11, 129.91, 129.77, 129.72,
129.08, 129.04, 129.01, 128.94, 128.77, 128.47, 128.39, 128.34, 129.68, 129.46, 129.41, 129.31, 129.25, 129.11, 129.07, 128.94,
128.27, 128.23, 128.18, 128.12, 127.92, 126.47, 126.18, 126.14, 128.66, 128.59, 128.40, 128.37, 128.33, 128.29, 128.26, 128.07,
126.05, 125.31, 101.99, 101.31, 101.00, 100.62, 99.74, 97.62, 97.11, 126.42, 126.33, 126.11, 125.34, 101.88, 101.76, 101.28, 101.16,
87.89, 78.81, 78.60, 78.45, 77.74, 77.19, 75.15, 74.71, 73.19, 72.87,
100.79, 98.85, 98.42, 97.06, 80.81, 78.74, 78.30, 77.52, 76.94, 75.08,
72.78, 72.63, 72.29, 70.68, 68.69, 68.49, 66.21, 65.86, 65.26, 74.88, 74.43, 73.78, 73.50, 72.65, 72.49, 68.71, 68.65, 67.89, 66.56,
21.13 ppm. HRMS (ESI TOF): calcd. for C94H88NO25S [M + NH4]
1662.5366; found 1662.5244.
66.04, 65.58, 50.68 ppm. HRMS (ESI TOF): calcd. for
C82H81N4O25 [M + NH4]+ 1521.5190; found 1521.5090.
p-Tolyl [2-O-Benzoyl-4,6-O-benzylidene-3-O-(naphthalen-2-yl-
2-Azidoethyl [2,3-Di-O-Benzoyl-4,6-O-benzylidene-β-
anosyl]-(1Ǟ3)-[2-O-benzoyl-4,6-O-benzylidene-β- -glucopyranosyl]-
(1Ǟ3)-[2-O-benzoyl-4,6-O-benzylidene-β- -glucopyranosyl]-(1Ǟ3)-
[2-O-benzoyl-4,6-O-benzylidene-β- -glucopyranosyl]-(1Ǟ3)-[2-O-
benzoyl-4-O-benzyl-6-O-levulinoyl-β- -glucopyranosyl]-(1Ǟ3)-[2-O-
benzoyl-4,6-O-benzylidene-β- -glucopyranosyl]-(1Ǟ3)-[2-O-benz-
oyl-4,6-O-benzylidene-β- -glucopyranosyl]-(1Ǟ3)-[2-O-benzoyl-4,6-
O-benzylidene-β- -glucopyranosyl]-(1Ǟ3)-2-O-benzoyl-4,6-O-benz-
ylidene-β- -glucopyranoside (16): A mixture of 6 (329.1 mg,
D-glucopyr-
methyl)-β-
ene-β- -glucopyranosyl]-(1Ǟ3)-[2-O-benzoyl-4,6-O-benzylidene-β-
-glucopyranosyl]-(1Ǟ3)-2-O-benzoyl-4,6-O-benzylidene-1-thio-β-
-glucopyranoside (15): Compound 15 (312.5 mg, 42%) was pre-
D-glucopyranosyl]-(1Ǟ3)-[2-O-benzoyl-4,6-O-benzylid-
D
D
D
D
D
D
D
pared from 13 (475.0 mg, 0.82 mmol) and 10 (1st glycosylation:
258.4 mg, 0.54 mmol; 2nd glycosylation: 235.2 mg, 0.49 mmol; 3rd
glycosylation: 214.4 mg, 0.45 mmol) after three rounds of glycosyl-
ation reactions by the same protocol described for 4, and was puri-
fied by silica gel column chromatography (ethyl acetate/toluene,
D
D
D
D
0.20 mmol) and activated molecular sieves (4 Å) in CH2Cl2 (4 mL)
was stirred at room temp. for 1 h, and then it was cooled to –78 °C.
A solution of AgOTf (154.2 mg, 0.60 mmol) in dry acetonitrile
(1.5 mL) was added, and then after 10 min pTolSCl (29 μL,
0.20 mmol) was added by microsyringe. The mixture was stirred for
a further 15 min, after which time TLC indicated that donor 6 had
been completely consumed. A solution of 11 (104.2 mg, 0.18 mmol)
and TTBP (44.7 mg, 0.18 mmol) in CH2Cl2 (1.5 mL) was added,
and the mixture was slowly warmed to room temp. over 1 h. The
mixture was stirred at room temp. for a further 20 min, then it was
cooled to –78 °C for glycosylation with 12 (246.4 mg, 0.16 mmol)
by the same protocol using AgOTf (138.7 mg, 0.54 mmol in 1 mL
acetonitrile), pTolSCl (26 μL, 0.18 mmol), and TTBP (40.7 mg,
0.16 mmol). The reaction was finally quenched with saturated aq.
NaHCO3 solution, and filtered to remove insoluble materials. The
organic layer was washed with saturated aq. NaHCO3 solution and
brine, dried with Na2SO4, and concentrated under reduced pres-
sure. The residue was purified by silica gel column chromatography
1
1:12). H NMR (600 MHz, CDCl3): δ = 7.86 (d, J = 7.3 Hz, 2 H),
7.79 (d, J = 7.3 Hz, 2 H), 7.75 (d, J = 7.4 Hz, 2 H), 7.69 (d, J =
8.0 Hz, 1 H), 7.62 (d, J = 7.3 Hz, 2 H), 7.59–7.14 (m, 40 H), 7.06
(d, J = 8.0 Hz, 2 H), 5.53 (s, 1 H), 5.44 (s, 1 H), 5.32 (t, J = 7.8 Hz,
1 H), 5.17 (t, J = 5.8 Hz, 1 H), 5.00–4.94 (m, 3 H), 4.90 (d, J =
12.4 Hz, 1 H), 4.85–4.75 (m, 4 H), 4.66 (d, J = 10.1 Hz, 1 H, anom-
eric), 4.61 (s, 1 H), 4.35 (dd, J = 10.5, 4.9 Hz, 1 H), 4.20 (dd, J =
10.4, 5.0 Hz, 1 H), 4.16–4.04 (m, 4 H), 3.95 (t, J = 8.8 Hz, 1 H),
3.91–3.86 (m, 2 H), 3.83 (t, J = 8.7 Hz, 1 H), 3.72 (td, J = 10.2,
7.4 Hz, 2 H), 3.58 (t, J = 9.1 Hz, 3 H), 3.49–3.37 (m, 4 H), 3.24 (t,
J = 9.4 Hz, 1 H), 2.32 (s, 3 H) ppm. 13C NMR (150 MHz, CDCl3):
δ = 164.99, 164.82, 164.54 (2 C), 138.25, 137.33, 137.29, 137.24,
137.07, 135.38, 133.50, 133.26, 133.17, 133.10, 133.03, 132.83,
132.74, 129.81, 129.78, 129.75, 129.70, 129.63, 129.56, 129.44,
129.41, 129.37, 129.04, 129.01, 128.96, 128.87, 128.63, 128.49,
128.39, 128.36, 128.24, 128.07, 127.97, 127.90, 127.88, 127.56,
126.67, 126.42, 126.24, 126.09, 126.00, 125.77, 125.62, 125.30,
101.93, 101.14, 100.87, 100.84, 99.30, 97.99, 96.93, 87.93, 81.22,
78.57, 78.49, 78.17, 77.10, 75.69, 74.29, 73.72, 73.45, 73.20, 73.01,
72.66, 70.67, 68.72, 68.51, 66.01, 65.67, 65.38, 21.15 ppm. HRMS
(ESI TOF): calcd. for C98H92NO24S [M + NH4]+ 1698.5730; found
1698.5598.
1
(ethyl acetate/toluene, 1:10) to give 16 (455.3 mg, 80%). H NMR
(600 MHz, CDCl3): δ = 7.89 (d, J = 8.2 Hz, 2 H), 7.86 (d, J =
8.1 Hz, 2 H), 7.74 (m, 10 H), 7.62 (m, 8 H), 7.55–7.13 (m, 73 H),
5.65 (t, J = 9.2 Hz, 1 H), 5.53 (s, 1 H), 5.46–5.42 (m, 1 H), 5.38 (s,
1 H), 5.30 (s, 1 H), 5.13 (dd, J = 9.7, 6.6 Hz, 2 H), 4.98–4.73 (m,
17 H), 4.73–4.68 (m, 2 H), 4.55 (d, J = 7.6 Hz, 1 H), 4.33 (dd, J =
10.3, 4.8 Hz, 1 H), 4.25 (td, J = 11.0, 4.6 Hz, 2 H), 4.20–4.01 (m,
11 H), 4.00–3.84 (m, 8 H), 3.78–3.67 (m, 3 H), 3.63–3.34 (m, 20
H), 3.26 (ddd, J = 13.9, 13.2, 7.1 Hz, 5 H), 2.59 (dd, J = 11.3,
2-Azidoethyl [2-O-Benzoyl-4,6-O-benzylidene-β-
(1Ǟ3)-[2-O-benzoyl-4,6-O-benzylidene-β- -glucopyranosyl]-(1Ǟ3)-
[2-O-benzoyl-4,6-O-benzylidene-β- -glucopyranosyl]-(1Ǟ3)-2-O-
benzoyl-4,6-O-benzylidene-β- -glucopyranoside (12): Glycosylation
D-glucopyranosyl]-
D
D
D
of azidoethanol (15.8 mg, 0.18 mmol) with 15 (305.5 mg, 6.3 Hz, 2 H), 2.44 (dd, J = 10.9, 6.3 Hz, 2 H), 2.10 (s, 3 H) ppm.
2948
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© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2015, 2942–2951

Synthesis of 6-O-Branched β-Glucan Oligosaccharides
13C NMR (150 MHz, CDCl3): δ = 206.42, 172.46, 170.62, 165.47,
165.11, 164.69, 164.55, 164.48, 138.18, 137.86, 137.46, 137.30, syl]-(1Ǟ3)-[2-O-benzoyl-4,6-O-benzylidene-β-
137.24, 137.18, 136.91, 133.67, 133.58, 133.48, 133.35, 133.18, (1Ǟ3)-[2-O-benzoyl-4,6-O-benzylidene-β-
133.00, 129.88, 129.77, 129.72, 129.64, 129.54, 129.48, 129.42, 2-O-benzoyl-4,6-O-benzylidene-β-
pyranosyl]-(1Ǟ3)-[2-O-benzoyl-4,6-O-benzylidene-β-
-glucopyranosyl]-
-glucopyranosyl]-(1Ǟ3)-
-glucopyranoside (17): Com-
D-glucopyrano-
D
D
D
129.33, 129.17, 129.07, 128.96, 128.65, 128.53, 128.47, 128.42, pound 17 (155.0 mg, 83 %) was prepared from 4 (77.4 mg,
128.30, 128.26, 128.11, 127.73, 126.48, 126.41, 126.34, 126.15, 39.4 μmol) and 7 (120.0 mg, 35.5 μmol) by the same protocol de-
126.08, 125.34, 101.98, 101.50, 101.31, 101.25, 101.21, 101.16, scribed for 5, and was purified by silica gel column chromatography
101.08, 100.80, 99.19, 99.11, 98.40, 98.01, 97.46, 97.11, 96.79, (ethyl acetate/toluene, 1:12). 1H NMR (600 MHz, CDCl3): δ =
96.70, 79.31, 78.78, 78.75, 78.67, 78.36, 77.94, 77.91, 77.58, 77.27,
75.26, 75.02, 74.96, 74.70, 74.60, 74.45, 74.15, 73.79, 73.64, 73.56,
73.23, 73.15, 73.02, 72.57, 72.49, 72.31, 72.05, 68.72, 68.65, 67.94,
66.49, 66.39, 66.27, 65.62, 65.53, 63.75, 50.66, 37.87, 29.85, 29.53,
27.90 ppm. HRMS (ESI TOF): calcd. for C194H181N3O58 [M +
2H]2+ 1740.0653; found 1740.0428.
8.37–6.69 (m, 155 H), 5.82 (d, J = 9.6 Hz, 1 H), 5.75 (m, 2 H),
5.69–5.64 (m, 2 H), 5.61–5.56 (m, 2 H), 5.49–5.46 (m, 1 H), 5.45–
5.38 (m, 3 H), 5.31 (s, 1 H), 5.21 (d, J = 7.6 Hz, 1 H), 5.14 (m, 1
H), 5.09 (m, 1 H), 5.04–4.67 (m, 20 H), 4.65–4.57 (m, 4 H), 4.51
(m, 1 H), 4.41 (d, J = 10.8 Hz, 2 H), 4.36 (d, J = 6.2 Hz, 2 H),
4.31–4.24 (m, 4 H), 4.23–4.04 (m, 15 H), 4.03–3.74 (m, 19 H), 3.72–
3.33 (m, 22 H), 3.32–3.22 (m, 5 H), 3.17 (d, J = 8.5 Hz, 3 H) ppm.
13C NMR (150 MHz, CDCl3): δ = 165.75, 165.72, 165.69, 165.40,
165.30, 165.07, 164.88, 164.81, 164.72, 164.62, 164.57, 164.50,
164.42, 163.91, 138.83, 137.74, 137.39, 137.35, 137.29, 137.21,
137.18, 137.12, 136.91, 134.45, 133.57, 133.22, 133.11, 133.07,
132.99, 132.93, 132.82, 132.64, 132.58, 130.08, 129.92, 129.74,
129.70, 129.52, 129.45, 129.40, 129.32, 129.22, 129.18, 129.10,
129.00, 128.92, 128.84, 128.77, 128.69, 128.48, 128.44, 128.36,
128.26, 128.21, 128.16, 128.12, 128.06, 127.99, 127.86, 127.80,
127.65, 127.49, 127.39, 126.87, 126.38, 126.35, 126.25, 126.17,
126.12, 126.07, 103.55, 102.49, 101.81, 101.63, 101.42, 101.30,
101.25, 101.15, 100.89, 100.80, 100.72, 100.66, 100.60, 98.73, 98.57,
97.91, 97.81, 97.57, 97.31, 96.68, 78.91, 78.83, 78.77, 78.22, 78.15,
78.03, 77.95, 77.53, 76.38, 75.89, 75.64, 75.53, 75.36, 75.11, 75.02,
74.87, 74.81, 74.63, 74.25, 73.98, 73.61, 73.30, 73.21, 73.10, 72.94,
72.64, 72.37, 72.33, 72.01, 68.65, 68.01, 67.84, 66.83, 66.51, 66.25,
65.63, 65.37, 60.03, 50.65 ppm. MS (MALDI TOF): calcd. for
2-Azidoethyl [2,3-Di-O-Benzoyl-4,6-O-benzylidene-β-
anosyl]-(1Ǟ3)-[2-O-benzoyl-4,6-O-benzylidene-β- -glucopyranosyl]-
(1Ǟ3)-[2-O-benzoyl-4,6-O-benzylidene-β- -glucopyranosyl]-(1Ǟ3)-
[2-O-benzoyl-4,6-O-benzylidene-β- -glucopyranosyl]-(1Ǟ3)-[2-O-
benzoyl-4-O-benzyl-β- -glucopyranosyl]-(1Ǟ3)-[2-O-benzoyl-4,6-O-
benzylidene-β- -glucopyranosyl]-(1Ǟ3)-[2-O-benzoyl-4,6-O-benzyl-
idene-β- -glucopyranosyl]-(1Ǟ3)-[2-O-benzoyl-4,6-O-benzylidene-β-
-glucopyranosyl]-(1Ǟ3)-2-O-benzoyl-4,6-O-benzylidene-β- -gluco-
D-glucopyr-
D
D
D
D
D
D
D
D
pyranoside (7): A mixture of 16 (420.0 mg, 120.7 μmol) and hydraz-
ine [0.5 m solution in pyridine/acetic acid (4:1) buffer; 10 mL] was
stirred under an Ar atmosphere at room temp. for 1 h. Then 2,4-
pentanedione (1 mL) was added, and the stirring was continued for
a further 20 min. The mixture was diluted with CH2Cl2, washed
sequentially with saturated aq. NaHCO3, CuSO4, and NH4Cl solu-
tions, dried with Na2SO4, and concentrated under vacuum. The
residue was purified by silica gel column chromatography (ethyl
acetate/toluene, 1:8) to give 7 (380.6 mg, 93%) as a white foamy
C
297H267KN3O84 [M + K]+ 5257.6; found 5257.9.
1
solid. H NMR (600 MHz, CDCl3): δ = 7.90 (d, J = 7.6 Hz, 2 H),
7.86 (d, J = 7.6 Hz, 2 H), 7.77 (d, J = 7.7 Hz, 2 H), 7.73–7.57 (m,
16 H), 7.55–7.42 (m, 11 H), 7.42–7.19 (m, 62 H), 5.66 (t, J = 9.2 Hz,
1 H), 5.52 (s, 1 H), 5.48–5.43 (m, 1 H), 5.39 (s, 1 H), 5.27 (s, 1 H),
5.13 (dd, J = 9.5, 4.2 Hz, 2 H), 4.93–4.79 (m, 14 H), 4.78–4.70 (m,
5 H), 4.55 (d, J = 7.6 Hz, 1 H), 4.34 (dd, J = 10.4, 4.7 Hz, 1 H),
4.25 (dt, J = 10.7, 5.3 Hz, 2 H), 4.18–4.08 (m, 7 H), 4.06–4.01 (m,
2 H), 3.99–3.85 (m, 8 H), 3.76–3.68 (m, 2 H), 3.66–3.58 (m, 3 H),
3.57–3.36 (m, 20 H), 3.34–3.21 (m, 5 H) ppm. 13C NMR (151 MHz,
CDCl3): δ = 165.43, 165.07, 164.66, 164.62, 164.60, 164.57, 164.54,
164.52, 164.47, 164.40, 138.30, 137.29, 137.20, 137.17, 137.13,
136.87, 133.51, 133.44, 133.36, 133.32, 133.10, 132.96, 129.81,
129.75, 129.72, 129.62, 129.47, 129.39, 129.29, 129.24, 129.17,
129.08, 129.02, 128.95, 128.62, 128.57, 128.54, 128.47, 128.43,
128.38, 128.35, 128.29, 128.26, 128.21, 128.16, 128.11, 128.10,
128.07, 127.86, 127.69, 126.43, 126.34, 126.32, 126.29, 126.28,
126.26, 126.12, 126.04, 101.94, 101.44, 101.30, 101.24, 101.14,
101.11, 101.09, 100.78, 99.13, 99.06, 98.27, 98.23, 97.61, 97.44,
97.35, 96.87, 78.89, 78.69, 78.67, 78.34, 78.04, 77.95, 77.58, 77.46,
77.22, 75.50, 75.24, 75.02, 74.86, 74.83, 74.66, 74.34, 74.22, 73.78,
73.56, 73.53, 73.30, 73.26, 73.12, 72.94, 72.53, 72.45, 72.27, 68.69,
68.61, 67.91, 66.49, 66.29, 66.23, 65.62, 65.58, 65.54, 65.49, 62.13,
50.66 ppm. HRMS (ESI TOF): calcd. for C189H181N5O56 [M +
2NH4]2+ 1708.0734; found 1708.0634.
2-Azidoethyl [2,3-Di-O-Benzoyl-4,6-O-benzylidene-β-
anosyl]-(1Ǟ3)-[2-O-benzoyl-4,6-O-benzylidene-β- -glucopyranosyl]-
(1Ǟ3)-[2-O-benzoyl-4,6-O-benzylidene-β- -glucopyranosyl]-(1Ǟ3)-
[2-O-benzoyl-4,6-O-benzylidene-β- -glucopyranosyl]-(1Ǟ3)-[2-O-
benzoyl-4-O-benzyl-6-O-{[2,3-di-O-benzoyl-4,6-O-benzylidene-β-
glucopyranosyl]-(1Ǟ3)-2-O-benzoyl-4,6-O-benzylidene-β-
glucopyranosyl}-β-
benzylidene-β- -glucopyranosyl]-(1Ǟ3)-[2-O-benzoyl-4,6-O-benzyl-
idene-β- -glucopyranosyl]-(1Ǟ3)-[2-O-benzoyl-4,6-O-benzylidene-β-
-glucopyranosyl]-(1Ǟ3)-2-O-benzoyl-4,6-O-benzylidene-β- -gluco-
D-glucopyr-
D
D
D
D-
D
-
D
-glucopyranosyl]-(1Ǟ3)-[2-O-benzoyl-4,6-O-
D
D
D
D
pyranoside (18): Compound 18 (126.6 mg, 85%) was prepared from
5 (36.9 mg, 39.4 μmol) and 7 (120.0 mg, 35.5 μmol) by the protocol
described for 5, and was purified by silica gel column chromatog-
1
raphy (ethyl acetate/toluene, 1:15). H NMR (600 MHz, CDCl3): δ
= 7.87 (m, 6 H), 7.76 (m, 3 H), 7.73–6.98 (m, 111 H), 5.67–5.63
(m, 1 H), 5.61–5.58 (m, 1 H), 5.55–5.49 (m, 2 H), 5.47–5.40 (m, 2
H), 5.37 (d, J = 7.5 Hz, 2 H), 5.21 (dd, J = 11.5, 5.1 Hz, 2 H),
5.15–5.09 (m, 2 H), 4.99–4.93 (m, 2 H), 4.91 (d, J = 5.3 Hz, 2 H),
4.87–4.69 (m, 13 H), 4.64 (d, J = 6.0 Hz, 2 H), 4.55 (dd, J = 14.0,
8.2 Hz, 2 H), 4.45 (d, J = 7.9 Hz, 1 H), 4.33 (s, 2 H), 4.24 (s, 2 H),
4.14 (d, J = 7.1 Hz, 3 H), 4.11–4.04 (m, 5 H), 4.00–3.81 (m, 11 H),
3.74 (dt, J = 18.7, 9.7 Hz, 5 H), 3.67–3.16 (m, 32 H) ppm. 13C
NMR (150 MHz, CDCl3): δ = 165.50, 165.43, 165.07, 164.86,
164.63, 164.59, 164.53, 164.20, 138.50, 137.57, 137.33, 137.28,
137.16, 136.87, 136.84, 133.42, 133.35, 133.29, 133.21, 133.14,
133.06, 132.98, 132.94, 132.68, 129.79, 129.70, 129.67, 129.61,
2-Azidoethyl [2,3-Di-O-Benzoyl-4,6-O-benzylidene-β-
anosyl]-(1Ǟ3)-[2-O-benzoyl-4,6-O-benzylidene-β- -glucopyranosyl]-
(1Ǟ3)-[2-O-benzoyl-4,6-O-benzylidene-β- -glucopyranosyl]-(1Ǟ3)-
[2-O-benzoyl-4,6-O-benzylidene-β-
benzoyl-4-O-benzyl-6-O-{[2,3-di-O-benzoyl-4,6-O-benzylidene-β-
glucopyranosyl]-(1Ǟ6)-[2,3-di-O-benzoyl-4-O-benzyl-β- -glucopyr-
anosyl]-(1Ǟ6)-[2,3-di-O-benzoyl-4-O-benzyl-β- -glucopyranosyl]-
-glucopyranosyl}-β- -gluco-
D-glucopyr-
D
D
D
-glucopyranosyl]-(1Ǟ3)-[2-O- 129.52, 129.48, 129.38, 129.36, 129.32, 129.21, 129.07, 129.01,
D-
128.98, 128.93, 128.58, 128.48, 128.44, 128.39, 128.36, 128.25,
128.20, 128.14, 128.11, 128.06, 127.85, 127.68, 127.59, 127.43,
126.53, 126.43, 126.39, 126.29, 126.27, 126.11, 126.04, 101.90,
101.62, 101.43, 101.29, 101.22, 101.10, 100.91, 100.77, 100.66,
D
D
(1Ǟ6)-2,3-di-O-benzoyl-4-O-benzyl-β-
Eur. J. Org. Chem. 2015, 2942–2951
D
D
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2949

G. Liao, S. Burgula, Z. Zhou, Z. Guo
FULL PAPER
99.09, 98.54, 98.46, 98.20, 97.40, 96.93, 96.87, 96.64, 79.27, 78.78,
78.68, 78.41, 78.32, 78.12, 77.85, 77.56, 75.32, 75.26, 74.75, 74.66,
74.55, 73.87, 73.66, 73.55, 73.51, 73.43, 73.36, 72.99, 72.93, 72.81,
72.74, 72.57, 72.50, 72.44, 72.33, 72.28, 68.64, 68.60, 68.21, 67.87,
66.47, 66.31, 66.22, 66.16, 65.62, 65.55, 65.40, 60.03, 50.64 ppm.
MS (MALDI TOF): calcd. for C236H213KN3O69 [M + K]+ 4231.3;
found 4231.5.
the solvents were removed by lyophilization. The residue was dis-
solved in water, and the solution was neutralized with HCl (0.25 n),
and then lyophilized. The crude product was purified on a Se-
phadex G-25 gel filtration column with water as the eluent. Lyophi-
lization gave 1 (7.1 mg, 87%) as a white fluffy solid. 1H NMR
(600 MHz, D2O): δ = 4.58 (m, 8 H), 4.39–4.33 (m, 5 H), 4.08–4.02
(m, 4 H), 3.97–3.94 (m, 1 H), 3.77 (m, 10 H), 3.69 (m, 5 H), 3.65–
3.50 (m, 18 H), 3.50–3.26 (m, 34 H), 3.26–3.11 (m, 9 H), 3.10 (t, J
= 4.8 Hz, 1 H) ppm. HRMS (ESI TOF): calcd. for C80H138NNaO66
[M + H + Na]2+ 1095.8686; found 1095.8658.
2-Azidoethyl [2,3-Di-O-Benzoyl-4,6-O-benzylidene-β-
anosyl]-(1Ǟ3)-[2-O-benzoyl-4,6-O-benzylidene-β- -glucopyranosyl]-
(1Ǟ3)-[2-O-benzoyl-4,6-O-benzylidene-β- -glucopyranosyl]-(1Ǟ3)-
[2-O-benzoyl-4,6-O-benzylidene-β- -glucopyranosyl]-(1Ǟ3)-[2-O-
benzoyl-4-O-benzyl-6-O-{[2,3-di-O-benzoyl-4,6-O-benzylidene-β-
glucopyranosyl]-(1Ǟ3)-[2-O-benzoyl-4,6-O-benzylidene-β- -gluco-
pyranosyl]-(1Ǟ3)-[2-O-benzoyl-4,6-O-benzylidene-β- -glucopyran-
osyl]-(1Ǟ3)-2-O-benzoyl-4,6-O-benzylidene-β- -glucopyranosyl}-β-
-glucopyranosyl]-(1Ǟ3)-[2-O-benzoyl-4,6-O-benzylidene-β-
glucopyranosyl]-(1Ǟ3)-[2-O-benzoyl-4,6-O-benzylidene-β- -gluco-
pyranosyl]-(1Ǟ3)-[2-O-benzoyl-4,6-O-benzylidene-β- -glucopyran-
osyl]-(1Ǟ3)-2-O-benzoyl-4,6-O-benzylidene-β- -glucopyranoside
D-glucopyr-
D
D
D
2-Aminoethyl β-
(1Ǟ3)-β- -glucopyranosyl-(1Ǟ3)-β-
[β- -glucopyranosyl-(1Ǟ3)-β-
glucopyranosyl}-(1Ǟ3)-β- -glucopyranosyl-(1Ǟ3)-β-
syl-(1Ǟ3)-β- -glucopyranosyl-(1Ǟ3)-β- -glucopyranoside (2):
D
-Glucopyranosyl-(1Ǟ3)-β-
-glucopyranosyl-(1Ǟ3)-{6-O-
-glucopyranosyl]-β-
-glucopyrano-
D-glucopyranosyl-
D-
D
D
D
D
D
D -
D
D
D
D
D
D
D
D-
Compound 2 (5.8 mg, 92 %) was prepared from 18 (15.0 mg,
3.6 μmol) by the same protocol described for 1. 1H NMR
(600 MHz, D2O): δ = 4.58 (m, 9 H), 4.40 (d, J = 8.2 Hz, 2 H), 4.06
(d, J = 10.7 Hz, 1 H), 4.00–3.95 (m, 1 H), 3.83–3.71 (m, 12 H),
3.58 (m, 20 H), 3.41 (m, 30 H), 3.26–3.23 (m, 2 H), 3.22–3.18 (m,
2 H), 3.12 (d, J = 4.7 Hz, 2 H) ppm. HRMS (ESI TOF): calcd. for
C68H118NO56 [M + H]+ 1844.6416; found 1844.6464.
D
D
D
(19): Compound 19 (135.8 mg, 78 %) was prepared from 6
(64.8 mg, 39.4 μmol) and 7 (120.0 mg, 35.5 μmol) by the same pro-
tocol described for 5, and was purified by silica gel column
chromatography (ethyl acetate/toluene, 1:12). 1H NMR (600 MHz,
CDCl3): δ = 8.05–6.90 (m, 140 H), 5.64 (m, 2 H), 5.51 (s, 1 H),
5.44 (m, 3 H), 5.36 (d, J = 14.3 Hz, 3 H), 5.27 (m, 2 H), 5.18 (m,
1 H), 5.15–5.08 (m, 3 H), 5.00 (d, J = 5.5 Hz, 2 H), 4.94–4.64 (m,
22 H), 4.54 (d, J = 7.6 Hz, 1 H), 4.35–4.30 (m, 2 H), 4.22 (dd, J =
10.1, 4.6 Hz, 4 H), 4.16–4.06 (m, 8 H), 4.03 (d, J = 8.3 Hz, 1 H),
3.99–3.80 (m, 13 H), 3.70 (ddd, J = 33.2, 21.1, 11.0 Hz, 6 H), 3.62–
3.21 (m, 33 H), 3.15 (d, J = 8.8 Hz, 1 H), 3.06–3.01 (m, 1 H) ppm.
13C NMR (150 MHz, CDCl3): δ = 165.44, 165.05, 165.00, 164.95,
164.78, 164.65, 164.61, 164.55, 164.51, 164.48, 164.30, 164.19,
138.57, 137.60, 137.30, 137.19, 137.16, 136.88, 133.47, 133.39,
133.23, 133.15, 133.10, 132.96, 129.83, 129.75, 129.71, 129.67,
129.47, 129.43, 129.39, 129.29, 129.20, 129.06, 129.01, 128.92,
128.62, 128.56, 128.54, 128.50, 128.41, 128.37, 128.26, 128.21,
128.11, 128.07, 128.01, 127.65, 127.54, 126.64, 126.50, 126.42,
126.29, 126.12, 126.04, 101.95, 101.73, 101.55, 101.42, 101.30,
101.23, 101.15, 100.98, 100.91, 100.76, 100.65, 99.64, 99.27, 99.07,
98.93, 98.83, 98.42, 98.23, 97.47, 97.02, 96.79, 96.66, 78.74, 78.68,
78.53, 78.49, 78.30, 77.96, 77.88, 77.66, 77.60, 77.55, 77.40, 75.67,
75.36, 75.13, 74.91, 74.66, 74.50, 74.16, 74.02, 73.88, 73.73, 73.60,
73.49, 73.41, 73.18, 73.11, 73.06, 72.76, 72.66, 72.51, 72.44, 72.28,
68.65, 68.43, 68.24, 67.91, 66.47, 66.23, 65.65, 65.58, 65.53, 65.45,
60.39, 60.03, 50.65 ppm. MS (MALDI TOF): calcd. for
2-Aminoethyl β-
(1Ǟ3)-β- -glucopyranosyl-(1Ǟ3)-β-
[β- -glucopyranosyl-(1Ǟ3)-β- -glucopyranosyl-(1Ǟ3)-β-
anosyl-(1Ǟ3)-β- -glucopyranosyl]-β- -glucopyranosyl}-(1Ǟ3)-β-
glucopyranosyl-(1Ǟ3)-β- -glucopyranosyl-(1Ǟ3)-β- -glucopyran-
osyl-(1Ǟ3)-β- -glucopyranoside (3): Compound 3 (6.0 mg, 85%)
D
-Glucopyranosyl-(1Ǟ3)-β-
-glucopyranosyl-(1Ǟ3)-{6-O-
-glucopyr-
D-glucopyranosyl-
D
D
D
D
D
D
D
D-
D
D
D
was prepared from 19 (15.0 mg, 3.1 μmol) by the same protocol
described for 1. 1H NMR (600 MHz, D2O): δ = 4.60 (m, 11 H),
4.39 (d, J = 7.8 Hz, 2 H), 4.06 (d, J = 12.1 Hz, 1 H), 3.98–3.93 (m,
1 H), 3.77 (m, 14 H), 3.58 (m, 25 H), 3.49–3.28 (m, 35 H), 3.25 (t,
J = 8.0 Hz, 2 H), 3.20 (t, J = 8.6 Hz, 2 H), 3.09 (s, 2 H) ppm.
HRMS (ESI TOF): calcd. for C80H138NNaO66 [M + H + Na]2+
1095.8686; found 1095.8641.
Acknowledgments
This work was supported in part by the National Institutes of
Health (NIH)/NCI (grant number R01 CA95142). The authors
thank Dr. B. Ksebati, Department of Chemistry at Wayne State
University, for some 2D NMR measurements. The 600 MHz NMR
spectrometer used in this research was supported by a National
Science Foundation (NSF) (grant number CHE-0840413).
C
276H249KN3O81 [M + K]+ 4939.5; found 4939.4.
2-Aminoethyl β- -Glucopyranosyl-(1Ǟ3)-β- -glucopyranosyl-
(1Ǟ3)-β- -glucopyranosyl-(1Ǟ3)-β- -glucopyranosyl-(1Ǟ3)-{6-O-
[β- -glucopyranosyl-(1Ǟ6)-β- -glucopyranosyl-(1Ǟ6)-β- -glucopyr-
anosyl-(1Ǟ6)-β- -glucopyranosyl]-β- -glucopyranosyl}-(1Ǟ3)-β-
glucopyranosyl-(1Ǟ3)-β- -glucopyranosyl-(1Ǟ3)-β- -glucopyran-
osyl-(1Ǟ3)-β- -glucopyranoside (1): Acetic acid (4 drops) and zinc
D
D
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D
D
D
D
D
D
D
D-
D
D
D
powder (20 mg) were added to a solution of 17 (20.0 mg, 3.8 μmol)
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Received: February 16, 2015
Published Online: March 24, 2015
Eur. J. Org. Chem. 2015, 2942–2951
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2951