Synthesis of heparin oligosaccharides and their interaction with eosinophil-derived neurotoxin

Article abstract of DOI:10.1039/c1ob06415k

A convenient route for the synthesis of heparin oligosaccharides involving regioselective protection of d-glucosamine and a concise preparation of rare l-ido sugars from diacetone α-d-glucose is described. Stereoselective coupling of a d-glucosamine-derived trichloroacetimidate with a 1,6-anhydro-β-l-idopyranosyl 4-alcohol gave the desired α-linked disaccharide, which was used as repeating unit for dual chain elongation and termination. Stepwise assembly from the reducing to the non-reducing end with a d-glucosamine-derived monosaccharide as starting unit furnished the oligosaccharide skeletons having different chain lengths. A series of functional group transformations afforded the expected heparin oligosaccharides with 3, 5 and 7 sugar units. Interaction of these oligosaccharides with eosinophil-derived neurotoxin (EDN), a cationic ribonuclease and a mediator produced by human eosinophils, was further investigated. The results revealed that at 5 μg mL^-1, the heptasaccharide has sufficiently strong interference to block EDN binding to Beas-2B cells. The tri- and pentasaccharides have moderate inhibitory properties at 50 μg mL^-1 concentration, but no inhibition has been observed at 10 μg mL^-1. The IC₅₀ values of the tri-, penta- and heptasaccharides are 69.4, 47.2 and 0.225 μg mL^-1, respectively.

Full text of DOI:10.1039/c1ob06415k

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PAPER
Synthesis of heparin oligosaccharides and their interaction with
eosinophil-derived neurotoxin†
Shang-Cheng Hung,*a,b Xin-An Lu,c Jinq-Chyi Lee,c Margaret Dah-Tsyr Chang,*d Shun-lung Fang,d
Tan-chi Fan,d Medel Manuel L. Zuluetaa and Yong-Qing Zhonga,c
Received 18th August 2011, Accepted 3rd November 2011
DOI: 10.1039/c1ob06415k
A convenient route for the synthesis of heparin oligosaccharides involving regioselective protection of
D-glucosamine and a concise preparation of rare L-ido sugars from diacetone a-D-glucose is described.
Stereoselective coupling of a D-glucosamine-derived trichloroacetimidate with a 1,6-anhydro-b-L-
idopyranosyl 4-alcohol gave the desired a-linked disaccharide, which was used as repeating unit for
dual chain elongation and termination. Stepwise assembly from the reducing to the non-reducing end
with a D-glucosamine-derived monosaccharide as starting unit furnished the oligosaccharide skeletons
having different chain lengths. A series of functional group transformations afforded the expected
heparin oligosaccharides with 3, 5 and 7 sugar units. Interaction of these oligosaccharides with
eosinophil-derived neurotoxin (EDN), a cationic ribonuclease and a mediator produced by human
eosinophils, was further investigated. The results revealed that at 5 mg mL-1, the heptasaccharide has
sufficiently strong interference to block EDN binding to Beas-2B cells. The tri- and pentasaccharides
have moderate inhibitory properties at 50 mg mL-1 concentration, but no inhibition has been observed
at 10 mg mL-1. The IC50 values of the tri-, penta- and heptasaccharides are 69.4, 47.2 and 0.225 mg mL-1,
respectively.
loop 3 of ECP, which, interestingly, shares common features with
Introduction
the corresponding loop 3 of EDN.5 EDN is less cationic and
Human eosinophils are white blood cells mobilised from the
less toxic, but has ribonuclease activity that is one hundred-fold
stronger than ECP.6 It has antiviral7 and chemotactic activity8 and
is also responsible, in part, for the anti-HIV-1 activity of mixed
lymphocyte cultures.9 Nevertheless, many of its physiological
functions and mechanism of action still remain unclear.
bone marrow in response to stimuli commonly caused by allergic
inflammation (e.g., asthma) and parasitic helminth infection.1
Degranulation of the leukocyte at the site of action releases
four major cationic proteins, of which, two—eosinophil-derived
neurotoxin (EDN) and eosinophil cationic protein (ECP)—have
ribonuclease activities.2 Both proteins belong to the ribonuclease
A superfamily and share around a 67% identity of their amino acid
sequences.3 Recent reports revealed that the uptake mechanism of
ECP in human bronchial epithelial Beas-2B cells requires initial
interaction with cell surface heparan sulfate (HS) proteoglycans4
and an unusual and novel binding site for HS was located in
HS and heparin (HP) are structurally related linear polysulfated
polysaccharides that belong to the glycosaminoglycan family.
These polyanionic sugars consist of a uronic acid (b-D-glucuronic
acid or a-L-iduronic acid) and a-D-glucosamine alternately linked
in a 1 → 4 fashion.10 Their multifaceted roles in biological
processes are reflected by the continually increasing number of
heparin-binding proteins being identified.11 Regulation of the
biological activity of several proteins in the coagulation cascade12
along with many processes of biomedical importance including
growth factor interactions,13 viral entry14 and angiogenesis15 are
just a few of their currently acknowledged functions. HS and HP
proteoglycans share a similar biosynthetic pathway. It initially in-
volves prior assemblage of a core protein with the tetrasaccharide
linkage region (4GlcAb1 → 3Galb1 → 3Galb1 → 4Xylb1→)
followed by successive attachment of monomers producing
the disaccharide repeating unit, 4GlcAb1 → 4GlcNAca1 →.
The polysaccharide backbone is subsequently modified to dif-
ferent extents through a series of enzymatic reactions, includ-
ing N-deacetylation, N-sulfonation, 5-C-epimerisation and O-
sulfonation,16 generating variable substitution patterns resulting
aGenomics Research Center, Academia Sinica, 128, Section 2, Academia
Road, Taipei 115, Taiwan. E-mail: schung@gate.sinica.edu.tw; Fax: (+886)
2-2789-8771
bDepartment of Applied Chemistry, National Chiao Tung University, 1001,
Ta-Hsueh Road, Hsinchu 300, Taiwan
cDepartment of Chemistry, National Tsing Hua University, 101, Section 2,
Kuang-Fu Road, Hsinchu 300, Taiwan
dInstitute of Molecular and Cellular Biology and Department of Life Science,
National Tsing Hua University, 101, Section 2, Kuang-Fu Road, Hsinchu
300, Taiwan. E-mail: lscmdt@life.nthu.edu.tw; Fax: (+886) 3-571-5934
† Electronic supplementary information (ESI) available: Additional exper-
imental data and copies of 1H and 13C and DEPT NMR spectra of relevant
compounds as well as the HRMS of the target oligosaccharides. See DOI:
10.1039/c1ob06415k
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Scheme 1 Retrosynthesis of the target heparin oligosaccharides 1–3.
in microheterogeneity. HS has a more complex fine structure
Retrosynthesis of heparin oligosaccharides
compared to HP and often contains heparin-like domains with
substantial amounts of L-iduronic acid, as well as high sul-
fation. There is increasing interest in the characterisation of
heparin–protein interactions because they serve as a model
for the binding of proteins with the highly sulfated regions
of HS.
The preparation of HP sugars has attracted great attention from
chemists as documented in the literature.19 The discrete challenges
for HP synthesis include the preparation of the rare L-idopyranosyl
derivatives,20 the distinction of all hydroxyl groups in the L-idose
and D-glucosamine subunits, the choice of appropriate protecting
groups, the stereocontrol of all glycosidic bonds, the elongation
to various chain lengths, the transformation of multi-functional
groups and the cleavage of multi-protecting groups. Scheme 1 il-
lustrates the retrosynthetic analysis of our approach to addressing
these points. The disaccharide 4 and the D-glucosamine-derived
4-alcohol 5 were utilized as building blocks. The former serves
as an elongation unit as well as a termination unit that masks
the non-reducing end of the growing chain, whereas the latter
functions as the starting unit by acting as the acceptor in the initial
glycosylation. Coupling of the glycosyl donor 4 with the alcohol 5
followed by an iterative deprotection–glycosylation protocol could
furnish various lengths of oligosaccharide skeletons that could
be subjected to a series of functional group transformations to
yield the target molecules 1–3. Since both 4 and 5 have the D-
glucosamine unit, the trichloroacetimidate 6 was designed as a
common synthon to couple with methanol and 1,6-anhydro-2-O-
benzoyl-3-O-benzyl-b-L-idopyranose (7), individually, followed by
conversion into the desired building blocks. The 4-alcohol 7 serves
as an excellent glycosyl acceptor because its rigid conformation
secures the orientation of the 4-C-hydroxyl group towards the
equatorial position that is favourable for sugar glycosylation. The
synthesis of L-ido compounds would be carried out through the
5-C-epimerisation of the mesylate 8, which could be prepared
from diacetone a-D-glucose 9. On the other hand, compound 6
would be acquired from per-O-silylated D-glucosamine deriva-
tive 10 through the regioselective one-pot protection strategy
developed by us.21 Compound 10 could be obtained from D-
glucosamine hydrochloride 11 via a combination of amino–azido
transformation and per-O-trimethylsilylation. Thus, the whole
synthesis of HP oligosaccharides could be executed starting from
two commercially available materials 9 and 11.
HP is found mainly in the secretory granules of mast cells
in contrast to the ubiquitous HS on the cell surface. It is a
widely used anticoagulant in the treatment of thromboembolic
disease.17 The major structural component of HP is a disaccharide
repeating unit having a 2-O-sulfonated L-iduronic acid and an
N- and 6-O-sulfonated D-glucosamine (Fig. 1). Structure–activity
relationship studies require procurement of homogeneous HP
oligosaccharides which are, unfortunately, difficult to obtain from
natural sources. The development of efficient synthetic routes
could offer a dependable solution for the growing demand for
chemically defined HP compounds. In 2004, we successfully
developed a procedure for the synthesis of regular heparin
oligosaccharides.18 However, the preparation of monosaccharide
building blocks, D-glucosamine in particular, were tedious, and the
yield of the final products required improvement. We report herein
our newly optimised synthetic steps for the preparation of each
building block leading to the synthesis of HP oligosaccharides. The
interaction of the target sugars with EDN has also been investiga-
ted.
The nature of the oligosaccharide assembly and the functional
group pattern of the target molecules require a careful selection
of protecting groups. To block the 4¢-C-hydroxyl of 4, the 2-
naphthylmethyl (2-NAP) group22 is used for two reasons. During
chain elongation, it allows chemoselective deprotection under
Fig. 1 Structure of heparin showing its major disaccharide repeating
component.
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essentially neutral conditions without affecting other protecting
groups. In the final transformation process, it can be simulta-
neously removed along with permanent benzyl groups under
hydrogenolytic conditions. Benzoyl groups can be selectively re-
moved and, therefore, are used to protect the hydroxyl groups that
would eventually be sulfonated. They can also offer neighbouring
group participation to generate exclusive 1,2-trans-glycosidic
linkages. The 2-C-amino group of glucosamine is typically masked
as an azide due to its non-participation during glycosylation,
mainly resulting in 1,2-cis-linkages, and its ready conversion into
the N-sulfonate group in just two steps. Moreover, an additional
orthogonal protecting group (levulinyl, Lev) is employed to block
the 6-C-hydroxyl of the L-idose unit that would be oxidised to
the corresponding carboxylic acid in one of the intermediate
transformation steps.
corresponding trichloroacetimidate 6 (98%, a/b = 1/1.9), which
could serve as glycosyl donor.
Synthesis of the starting sugar unit
Having acquired the D-glucosamine-derived donor 6, we pro-
ceeded to synthesise the starting unit 5 for oligosaccharide
elongation (Scheme 3). Conceptually, the 4-alcohol 5 should
be accessible after coupling of methanol with the imidate 6
followed by chemoselective removal of 2-NAP group at the 4-
O position. Unfortunately, the TMSOTf-catalysed glycosylation
of methanol failed to provide the desired compound 17. The
reaction only furnished the amide product resulting from the
rearrangement of the imidate functionality. In light of this, the
6-alcohol 14 was evaluated as a possible entry point for the target
compound. Treatment with BF3/OEt2 in methanol at 55 ◦C gave
the methyl a-glycoside 18 and its b-isomer in 62% and 20% yields,
respectively. Subsequent 6-O-benzoylation (BzCl, Et3N, 94%)
and 4-O-denaphthylmethylation mediated by 2,3-dichloro-5,6-
dicyano-1,4-benzoquinone (DDQ, 89%) provided the expected 4-
alcohol 5. With the success of this method, the fully protected
compound 13 was further examined under BF3/OEt2 in methanol
at 45 ◦C. The reaction allowed simultaneous removal of the
naphthylmethylidene acetal and formation of the methyl glycosidic
bond,26 furnishing the diol 19 and its b-isomer in 42% and
5% yields, respectively. Regioselective benzoylation at the less-
hindered 6-O position of 19 using BzCl and pyridine generated
the desired product 5 in 88% yield. Compound 13 is, therefore,
a suitable common intermediate for the preparation of the two
synthons 5 and 6.
Results and discussion
Synthesis of D-glucosamine synthons
Compound 12 was acquired from per-O-silylated D-glucosamine
derivative 10 in 87% overall yield via regioselective one-pot
protection that includes 4,6-O-naphthylmethylidenation and re-
gioselective 1-O-benzoylation (Scheme 2).21 Treatment of 12 with
benzyl bromide and silver(I) oxide gave the corresponding ether
derivative 13 in 83% yield. A sequential one-pot process, involving
regioselective 6-O-ring opening of the naphthylmethylidene acetal,
6-O-benzoylation and anomeric debenzoylation, was carried out
to synthesise the hemiacetal 16 in 72% yield. Here, a TMSOTf-
catalysed borane-reductive 6-O-ring opening of the naphthyl-
methylidene acetal in 13 led to the 6-alcohol 14.21a,23 To destroy
the excess borane in the reaction mixture, a quantitative amount
of methanol was added. Then, without further workup, Et3N
and Bz2O were consecutively added to the same flask to provide
the dibenzoate 15,24 which was subsequently treated with a
saturated ammonia solution in THF/MeOH to eliminate the
anomeric benzoyl group.25 Reaction of the 1-alcohol 16 with
trichloroacetonitrile and potassium carbonate resulted in the
Scheme 3 Synthesis of the starting unit 5.
Synthesis of the 1,6-anhydro-b-L-idopyranose
Procurement of the rare L-idose synthon is especially demanding
and involves synthesis either from developed de novo approaches27
or by chemical manipulation of D-glucose.19a,28 The utility of
1,6-anhydro-b-L-idopyranoses as key building blocks was first
developed by us19i,29 and later applied by others.19r,19u,30 The 1,6-
anhydro ring not only reduces the number of protecting groups
that need to be installed, but could also be readily cleaved for
further functionalization. Unlike the typical pyranose form of L-
idose, the rigid structure, which often leads to easy crystallization
of the sugar derivatives, allows for only one anomeric isomer
Scheme 2 Synthesis of the D-glucosamine-derived hemiacetal 16.
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Table 1 The one-pot synthesis of 1,6-anhydro-3-O-benzyl-b-L-
idopyranose (27) from compound 8
bypassing the time-consuming purification and identification of
the a- and b-anomers. In addition, the [3.2.1]-bicyclic skeleton
forces the functional groups at 2-C, 3-C and 4-C positions to
be equatorially oriented, essentially enhancing their reactivities.
Here, we report, in detail, the newly optimised conditions for
the preparation of 1,6-anhydro-b-L-idopyranosyl sugars with
improved yields.
Entry
x
Solvent
T
t
Yield (%)
As described in Scheme 4, the diol 20, generated from diacetone
a-D-glucose (9) in 88% overall yield via typical 3-O-benzylation
and regioselective removal of the 5,6-O-isopropylidene ring,
underwent regioselective 6-O-acetylation and subsequent 5-O-
mesylation to furnish the 5-OMs-6-OAc product 21 and the
5,6-diacetate derivative in 63% and 34% yields, respectively. To
improve the regioselectivity, a one-pot 6-O-benzoylation and 5-
O-mesylation of the diol 20 was tried. The desired 5-OMs-6-
OBz compound 8 was, therein, obtained as a pure solid in 81%
yield after recrystallisation from ethanol. Through intramolecular
SN2 substitution involving a 6-alkoxide intermediate, the 5-C-
epimerised L-ido epoxide 23 was predicted to be obtained from
the benzoate 8. However, the unexpected 6-alcohol 22 was
observed under the NaOMe/MeOH conditions. The relatively
higher basicity of the 6-alkoxide as compared to methoxide, thus
favouring proton abstraction from the solvent (MeOH), is believed
to cause this outcome. As expected, treatment of compound 8 with
the more basic tBuOK in tBuOH and CH2Cl2 at 0 ◦C19a gave the
desired product 23 in an excellent 89% yield.
1
2
3
4
5
6
0.2
0.2
0.2
0.2
0.2
1
H2O
H2O
120
120
160
160
160
120
16
16
16
8
16
16
0
0
0
48
52
61
HOCH2CH2OH/H2O (1/1)
Diglyme/H2O (1/1)
Diglyme/H2O (2/1)
1,4-Dioxane/H2O (2/1)
Scheme 5 Synthesis of the 1,6-anhydro-b-L-idopyranosyl sugar 27.
generation of freely equilibrating mixtures of the tetraols 24 and 25
(entries 1 and 2). In entry 3, aqueous H2SO4 diluted with ethylene
glycol was added to the reaction flask to facilitate a higher reaction
temperature of 160 ◦C, but the expected product was not obtained.
Alternatively, with the high boiling and stable solvent diglyme to
heat the epoxide 23 and H2SO4 at elevated temperature (160 ◦C),
the target molecule 27 was finally isolated in 48% (8 h, entry 4)
and 52% (16 h, entry 5) yields. Faced with the difficult removal
of diglyme, 1,4-dioxane was used instead and a better yield (61%,
entry 6) was acquired. As a result, the preparation of the rare L-ido
building block 27 was obtained from diacetone a-D-glucose 9 in
only four purification stages and in 43% overall yield.
Synthesis of HP disaccharide synthons
Scheme 4 Synthesis of the epoxide 23.
Because the glycosidic bonds of HP oligosaccharides are all 1 → 4
linked, the next encountered problem is the differentiation of the
two hydroxyls in the 1,6-anhydro sugar 27. The 2-OH group of
compound 27 needs to be selectively protected as an ester, leaving
the 4-OH group available for further coupling. Esterification of
compound 27 was carried out by slow addition of benzoyl chloride
to the reaction flask in the presence of pyridine as the base
at 0 ◦C, providing the 2-O-benzoylated 4-alcohol 7 in a highly
regioselective manner and in 85% yield after recrystallisation from
ethanol. The small amount of 2,4-di-O-benzoylated derivative
isolated from the reaction mixture was transformed back to the
2,4-diol 27 by sodium methoxide in methanol.
With both compounds 6 and 7 in hand, their coupling reactions
were studied and the results are disclosed in Table 2. Three
activators, AgOTf (entry 1), BF3/Et2O (entry 2) and TMSOTf
(entry 3),32 were examined using CH2Cl2 as the solvent, and the
yields and ratios of the expected a-linked disaccharide 28a18 and
its b-isomer 28b were obtained in 77% (1.7/1), 16% (1/1) and 78%
(6.1/1), respectively. In order to improve the a-selectivity, the less
The transformation of the L-ido epoxide 23 into the 1,6-anhydro-
b-L-idopyranosyl sugar 27 is illustrated in Scheme 5. Hydrolysis
of 23 in acidic medium (1 N H2SO4(aq) in 1,4-dioxane/H2O)
furnished the L-idofuranose 24 through a combination of epoxide
ring opening and isopropylidene ring cleavage.30,31 Equilibrium
between 24 and the two L-idopyranosyl forms—the 1C4 conformer
25 and the C1 conformer 26—followed by the elimination of a
water molecule at reflux temperature yielded the 2,4-diol 27 in
71% yield.
To reduce the synthetic steps, we further investigated the one-
pot preparation of the diol 27 from compound 8. The conditions
and results are outlined in Table 1. Treatment of compound 8
with tBuOK in tBuOH/CH2Cl2 followed by acidic hydrolysis was
considered to lead to compound 27 in the same flask. However,
early studies wherein the aqueous acid was solely added to the
mixture after epoxide formation and evaporation of the solvents
showed no formation of the 1,6-anhydro sugar 27 despite the
4
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Scheme 6 Synthesis of the heparin oligosaccharide skeletons.
Table 2 Coupling of the imidate donor 6 with the 1,6-anhydro-b-L-
idopyranosyl acceptor 7
group that can be differentiated from the benzoyl moiety should be
installed at the 6-O position of the L-idose residue. Levulinyl (Lev)
ester, which could be selectively deprotected by hydrazinolysis
and form an orthogonal set with other ester type groups, was
selected for this purpose. To facilitate levulinolysis, 28a was
treated with freshly prepared levulinic anhydride (Lev2O) in the
presence of Cu(OTf)2 as the catalyst.33 This reaction, however,
did not yield the desired dilevulinyl product 31. To tackle this
problem, we resorted to the typical acetolysis of 28a using acetic
anhydride and a catalytic amount of Cu(OTf)2, which provided
the 1,6-diacetate 29 in 88% yield. Full deacetylation of compound
29 with p-toluenesulfonic acid (PTSA) in methanol gave the
corresponding 1,6-diol 30 (81%).34 Reprotection of this diol with
Lev2O in the presence of N,N-dimethyl-4-aminopyridine (DMAP)
produced the dilevulinate 31 in 97% yield (Scheme 6). Owing
to the inductive effect of two oxygen atoms at the anomeric
centre, the regioselective anomeric delevulinylation of 31 was
accomplished with saturated ammonia in tetrahydrofuran (THF)
to afford the hemiacetal 32 in 62% yield together with some 1,6-
diol 30 (29%), which was recovered for reuse. The disaccharide
donor 4 was generated from the hemiacetal 32 by treatment
with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and CCl3CN in
89% yield, and subsequently, its coupling with the starting sugar
unit 5, promoted by TMSOTf, allowed the construction of the
trisaccharide 33. The newly formed trans-glycosidic bond is likely
induced by the neighbouring group participation of the 2-O-
benzoyl group. No orthoester side product was observed during
the coupling stage. Next, the chain-elongation cycle included
DDQ-mediated selective removal of the 2-NAP group to supply
the acceptor for glycosylation with the disaccharide synthon 4.
Following this procedure, the trisaccharide acceptor 34 (82%) and
the pentasaccharide acceptor 36 (79%) were acquired. By repeating
the modified coupling conditions (40 mol% TMSOTf, -40 ◦C), the
pentasaccharide 35 and the heptasaccharide 37 were assembled in
81% and 66% yields, respectively.
Entry Activator
Solvent
T
t
Yield (%)
28a/28b
1
2
3
4
5
AgOTf
CH2Cl2
CH2Cl2
CH2Cl2
Et2O
-40
0
-78
-78
-78
0.5
1
1
2
2
77
16
78
61
68
1.7/1
1/1
6.1/1
2.5/1
1.3/1
BF3/Et2O
TMSOTf
TMSOTf
TMSOTf
PhCH3
polar diethyl ether (entry 4) and toluene (entry 5), were utilized
as solvents in the reactions. Regrettably, low a-selectivities with
slight decrease in yields were observed under these conditions.
The diastereoisomers 28a and 28b could be purified by column
chromatography on silica gel. The a-form product 28a gave a 3J1¢,2¢
value of 3.7 Hz in its 1H NMR spectrum whereas its corresponding
b-isomer 28b showed a 7.8 Hz splitting (trans-diaxial coupling).
Synthesis of HP oligosaccharide skeletons
To assemble the HP oligosaccharide chain, the generation of
a disaccharide repeating building block for skeleton elongation
requires the opening of the 1,6-anhydro ring of the disaccharide
28a followed by transformation into the corresponding glycosyl
donor. Considering the protecting group pattern of 28a, a suitable
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Scheme 7 Synthesis of the target HP oligosaccharides 1–3.
Synthesis of HP oligosaccharides
to be better than our original method.18 The structures of
compounds 1–3 were confirmed and characterised by NMR
and high resolution electrospray ionisation mass spectroscopy
(see the ESI†).
Scheme 7 illustrates the transformations leading to the target HP
oligosaccharides 1–3. The Lev groups in compounds 33, 35 and 37
were removed by hydrazinolysis in a mixture of ethanol/toluene
(2/1) at room temperature to provide compounds 38 (97%), 39
(94%) and 40 (92%), respectively. Subsequent oxidation of the
resultant primary alcohols using 2,2,6,6-tetramethylpiperidine-1-
oxyl (TEMPO) free radical in the presence of sodium hypochlo-
rite and tetra-n-butylammonium chloride35 gave the individual
Competitive inhibition of EDN binding to cells by synthetic HP
oligosaccharides
The synthetic HP oligosaccharides 1–3 were tested for their
abilities to interfere with EDN binding to human bronchial
epithelial Beas-2B cells.5 The cells were incubated with maltose-
binding protein (MBP)-conjugated EDN together with 0.5, 1, 2.5,
5, 7.5, 10 and 50 mg mL-1 of compounds 1–3. The level of bound
MBP-EDN was assessed by enzyme-linked immunosorbent assay
(ELISA) and the relative amounts of bound protein in the presence
and absence of the oligosaccharides are shown in Fig. 2. The
binding percentage of MBP-EDN to the cell surface decreased
in an oligosaccharide concentration-dependent manner. The data
indicated that, at £10 mg mL-1 concentration, the trisaccharide
1 and pentasaccharide 2 did not block EDN binding to the
cells, but at 50 mg mL-1, these oligosaccharides could inhibit
around 40–50% of the interaction. For the heptasaccharide
3, 10 mg mL-1 concentration was capable of inhibiting over
90% of EDN binding. The degree of inhibition increased with
increasing oligosaccharide length. Relative binding affinities of
EDN with compounds 1–3 were evaluated by the determination
of IC50 values, defined as the concentration of competitor that
inhibits 50% of EDN binding to Beas-2B cells. As described
in Table 3, the data obtained for compounds 1–3 through the
GraphPad Prism 5 calculations are 69.4, 47.2 and 0.225 mg
mL-1, respectively. In these experiments, the trisaccharide 1 and
pentasaccharides 2 showed moderate inhibition properties, but the
heptasaccharide 3 exhibited significant interference in the interac-
tion between EDN and Beas-2B cells, indicating that longer HP
oligosaccharides possessed stronger EDN binding and inhibitory
abilities.
1
carboxylic acids 41–43, but the corresponding H NMR spec-
tra were difficult to obtain in good resolution after column
purification. Alternatively, the combination of TEMPO and
bis(acetoxy)iodobenzene (BAIB)36 was employed to oxidise the
primary alcohols. The reactions were carried out in a shorter
time period and the resulting mixtures were easier to work up,
yielding the pure compounds 41–43 in 85%, 71% and 67% yields,
respectively. Typically, the ester functionality could be cleaved
using a catalytic amount of NaOMe in methanol. Here, removal
of all benzoyl groups in compounds 41–43 required quantitative
NaOMe furnishing the alcohols 44 (88%), 45 (86%) and 46 (75%).
Sulfonation of the hydroxyl groups using SO3/Et3N complex
in DMF at 55 ◦C delivered the products 47–49 in 78%, 76%
and 73% yields, respectively. In the NMR spectroscopic analysis,
downfield shifts were observed for protons geminal to the sulfate
groups.
Finally, a two-step protocol was used to convert compounds
47–49 into the fully functionalized HP oligosaccharides 1–3.
Cleavage of the ether-type protecting groups (Bn and 2-NAP)
in 47–49 with concomitant reduction of the azido groups under
hydrogenolytic conditions furnished the amino-alcohols 50–52.
Further N-sulfonations (SO3/Pyr. complex, H2O, pH 9.5)19b,19f
gave the target molecules 1–3 in 67%, 60% and 55% overall
yields, respectively. The HP oligosaccharides were purified using
Sephadex G25 followed by ion-exchange with Dowex 50WX2-
Na+ resin.19c This purification, with an appropriate column
size and gel series, reduced material losses and was found
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in water or ninhydrin and acetic acid solution in n-butanol and
subsequent heating on a hot plate. Melting points were determined
with a Bu¨chi B-540 apparatus and are uncorrected. Optical rota-
tions were measured with a HORIBA Sepa-300 high sensitivity
polarimeter at ambient temperature. [a]D values are given in 10-1
deg cm2 g-1. 1H and 13C NMR spectra were recorded with Bruker
AMX400, 500 MHz and AVANCE-600 instruments. Chemical
shifts are in ppm calibrated using the carbon and residual proton
resonance of the d-solvent. Coupling constants (J) are given in
Hz. Proton peak assignments were performed using 2D NMR
techniques (1H–1H COSY, HMQC and NOESY). IR spectra were
taken with a Perkin–Elmer Paragon 1000 FT-IR spectrometer.
Elemental analyses were measured with a Perkin–Elmer 2400CHN
instrument. Mass spectra were obtained with a FAB JMS-700
double focusing mass spectrometer (JEOL), MALDI Voyager
DE-PRO (Applied Biosystems) and ESI Finnigan LCQ mass
spectrometer (Thermo Finnigan). Gel-filtration chromatography
(SephadexꢀR G25 fine and SephadexꢀR LH20) was used in order
to achieve purification of the final products.
Fig. 2 HP oligosaccharides inhibit EDN binding to Beas-2B cells. The
amount of MBP-EDN bound to cells without HP treatment was set to
100%. Cells incubated with MBP were used as control. The results shown
are the means of triplicate measurements; the error bars are standard
deviations.
Table 3 The IC50 values for inhibition of EDN binding to Beas-2B cells
by HP oligosaccharides
a
Entry
Oligosaccharide
IC50 (mg mL-1)
SDb (mg mL-1)
2.13
2.60
0.006
1
2
3
Trisaccharide 1
Pentasaccharide 2
Heptasaccharide 3
69.4
47.2
0.225
2-Azido-3-O-benzyl-2-deoxy-4,6-O-(2-naphthylmethylidene)-b-
D-glucopyranosyl benzoate (13). Compound 12 (14.0 g, 0.03 mol)
was dissolved in CH2Cl2 (140 mL) at room temperature under N2
atmosphere. Silver(I) oxide (14.5 g, 0.06 mol) was added to the
solution, and the mixture was cooled to 0 ◦C. Benzyl bromide
(5.6 mL, 0.05 mol) was added to the solution, and then the ice
bath was removed. After 2 days, the mixture was filtered, and the
filtrate was concentrated under reduced pressure. The residue was
purified by flash column chromatography (ethyl acetate/hexanes =
1/8) to give 13 (14.0 g, 83%) as a white solid. mp 173–174 ◦C (from
EtOH); [a]2D7 -174.7 (c 0.52 in CHCl3); IR (CHCl3) n 2923, 2106,
1733, 1268, 1087 cm-1; partial characterisation data were reported
previously.19n
a Represented by three independent experiments. b SD: standard deviation.
Conclusions
A concise strategy using cheap D-glucosamine and diacetone a-
D-glucose as starting materials to prepare the 2-azido-2-deoxy-
D-glucosyl donor 6 and the L-idopyranosyl acceptor 7 has been
developed. Coupling of these compounds resulted in the acquisi-
tion of a key disaccharide building block that was utilized as the
chain elongation as well as the termination unit for the assembly
of the oligosaccharide backbones from the reducing to the non-
reducing end. After a series of functional group transformations
that include TEMPO oxidation, debenzoylation, O-sulfonation,
hydrogenolytic reduction and N-sulfonation, chemically defined
HP tri-, penta- and heptasaccharides were obtained. Using these
oligosaccharides, inhibition of EDN binding to the bronchial
epithelial cell surface by HP was demonstrated for the first time.
The synthetic oligosaccharides disrupted the cellular binding, with
specific cell-surface glycosaminoglycan–EDN interaction strongly
blocked by the HP heptasaccharide 3. These results may lead to
protection of bronchial epithelial cells from EDN-induced damage
and facilitate further design of anti-asthma therapeutics. Detailed
analysis of the specific EDN–heparin binding mode remained to
be determined.
2-Azido-6-O-benzoyl-3-O-benzyl-2-deoxy-4-O-(2-naphthyl-
methyl)-D-glucopyranose (16). BH3/THF complex (1 M in THF,
0.66 mL, 0.66 mmol) and TMSOTf (8 mL, 0.044 mmol) were
sequentially added to a solution of compound 13 (118 mg,
0.22 mmol) in CH2Cl2 (1.2 mL) at 0 ◦C under N2 atmosphere. After
stirring for 4 h, methanol (26.7 mL, 0.66 mmol) was slowly added
to destroy the excess borane. The ice bath was removed, benzoic
anhydride (978 mg, 4.4 mmol) and Et3N (612 mL, 4.4 mmol)
were consecutively added to the solution, and the mixture was
continuously stirred at room temperature for 16 h. The reaction
flask was immersed in an ice bath, and a mixed solvent of
MeOH/THF (1/5, 2.4 mL) was added, ammonia gas was bubbled
through the solution for 20 min, and the mixture was then kept
◦
stirring for 8 h at 0 C. The solution was concentrated in vacuo,
and the residue was purified by flash column chromatography on
silica gel (ethyl acetate/hex◦anes = 1/4) to yield 16 (85 mg, 72%) as
a white solid. mp 101–102 C (from EtOH); [a]2D8 +67.4 (c 0.56 in
CHCl3); IR (CHCl3) n 3435, 2928, 2108, 1729, 1260, 1068 cm-1; 1H
NMR (400 MHz, CDCl3) d 7.94–7.91 (3.6H, m, Bz-H), 7.80–7.66
(10.3H, m, Ar-H), 7.54–7.50 (3.6H, m, Ar-H), 7.46–7.31 (21.9H,
m, Ar-H), 5.33 (1.0H, d, J 3.4), 4.98–4.91 (3.0H, m), 4.87–4.79
(2.5H, m), 4.69–4.62 (2.5H, m), 4.50–4.44 (1.7H, m), 4.28 (1.0H,
dt, J 9.8, 2.8), 4.13 (0.9H, t, J 9.8), 3.79–3.72 (4.3H, m), 3.69–3.66
(0.6H, m), 3.55 (0.8H, t, J 9.8), 3.49–3.43 (2.2H, m); 13C NMR (100
MHz, CDCl3) d 166.3 (C), 137.6 (C), 134.8 (C), 134.7 (CH), 133.1
(C), 129.5 (CH), 128.5 (CH), 128.3 (CH), 128.2 (CH), 128.0 (CH),
Experimental
Chemical synthesis
General procedures. Anhydrous DMF, methanol and pyridine
were purchased from Aldrich and directly used for the reactions.
CH2Cl2 and THF were purified and dried in a safe purification
system filled with anhydrous Al2O3. Flash column chromatogra-
phy was carried out on Silica Gel 60 (230–400 mesh, E. Merk).
TLC was performed on pre-coated glass plates of Silica Gel 60
F254 (0.25 mm, E. Merck); detection was executed by spraying
with a solution of Ce(NH4)2(NO3)6, (NH4)6Mo7O24, and H2SO4
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127.8 (CH), 127.6 (CH), 126.9 (CH), 126.8 (CH), 126.1 (CH),
126.0 (CH), 125.7 (CH), 96.2 (CH), 92.0 (CH), 83.2 (CH), 80.3
(CH), 77.8 (CH), 77.3 (CH), 75.7 (CH2), 75.1 (CH2), 73.3 (CH),
69.3 (CH), 67.5 (CH), 64.0 (CH), 63.0 (CH2); HRMS (FAB, [M]+)
m/z calc. for C31H29N3O6 539.2056, found 539.2050.
(CH), 69.9 (CH), 63.4 (CH2), 63.0 (CH), 55.1 (CH3); HRMS (ESI,
[M + Na]+) m/z calc. for C21H23N3O6Na 436.1476, found 436.1485;
characterisation data were reported previously.19r
6-O-Benzoyl-3-O-benzyl-1,2-O-isopropylidene-5-O-methanesul-
fonyl-a-D-glucofuranose (8). Compound 20 (1.3 g, 4.2 mmol) was
dissolved in CH2Cl2 (13 mL) under N2 atmosphere, the reaction
flask was immersed in an ice bath, pyridine (3.4 mL, 42 mmol) and
benzoyl chloride (0.39 mL, 4.4 mmol) were sequentially added to
the solution, and the mixture was stirred at the same temperature
for 2 h. Mesyl chloride (0.49 mL, 6.1 mmol) was added to the
solution, the ice bath was removed, and, then, the mixture was
stirred at room temperature for 16 h. The reaction was quenched
with water (5 mL), and the crude target material was extracted
with ethyl acetate (3 ¥ 15 mL). The combined organic layer was
sequentially washed with 1 N HCl(aq), saturated NaHCO3(aq) and
brine. The organic phase was dried over MgSO4, filtered and
concentrated in vacuo to get a residue, which was purified by flash
column chromatography on silica gel (ethyl acetate/hexanes = 1/3)
to provide 8 (1.65 g, 81%) as a white solid. mp 92–93 ◦C (from
EtOH); [a]1D9 -29.1 (c 9.1 in CHCl3); characterisation data were
reported previously.18
Methyl 2-azido-3-O-benzyl-2-deoxy-a-D-glucopyranoside (19).
A mixture of compound 13 (202 mg, 0.37 mmol) in methanol
(4 mL) was cooled down to 0 ◦C under N2 atmosphere. BF3/OEt2
(950 mL, 7.70 mmol) was added to the solution, the ice bath was
removed, and the mixture was warmed up and kept stirring at
45 ◦C for 14 h. The solution was cooled down to room temperature,
and CH2Cl2 (15 mL) was added to the mixture. The reaction flask
was immersed in an ice bath, and the mixture was neutralised with
saturated NaHCO3(aq). The crude target material was extracted
with CH2Cl2 (2 ¥ 10 mL), and the combined organic layer was dried
over MgSO4, filtered and concentrated in vacuo. The residue was
purified by flash column chromatography (ethyl acetate/hexanes =
1/1) on silica gel to furnish the 4,6-diol 19 (47 mg, 42%). [a]2D1 +76.5
1
(c 4.0 in CHCl3); IR (CHCl3) n 3401, 2933, 2111, 1051 cm-1; H
NMR (400 MHz, CDCl3) d 7.38–7.28 (5H, m, Ar-H), 4.87, 4.77
(2H, ABq, J 11.2, CH2Ph), 4.71 (1H, d, J 3.6, 1-H), 3.79–3.73
(3H, m, 3-H, 6-Ha, 6-Hb), 3.63–3.52 (2H, m, 4-H, 5-H), 3.36 (3H,
s, OCH3), 3.28 (1H, dd, J 10.0, 3.6, 2-H), 2.87 (1H, br s, OH), 2.52
(1H, br s, OH); 13C NMR (100 MHz, CDCl3) d 137.9 (C), 128.6
(CH), 128.1 (CH ¥ 2), 98.8 (CH), 80.1 (CH), 75.1 (CH2), 71.2 (CH),
70.6 (CH), 63.1 (CH), 61.8 (CH2), 55.3 (CH3); HRMS (ESI, [M
+ Na]+) m/z calc. for C14H19N3O5Na 332.1222, found 332.1223;
partial characterisation data were reported previously.19w
3-O-Benzyl-5,6-O-epoxyl-1,2-O-isopropylidene-b-L-idopyranose
(23). Compound 8 (6.9 g, 14 mmol) was dissolved in tert-butanol
(35 mL) and CH2Cl2 (35 mL), the reaction flask was immersed
in an ice bath, and potassium tert-butoxide (3.5 g, 30.8 mmol)
was added to the mixture. After stirring for 16 h, water (3 mL)
was added to quench the reaction, and the solvent mixture was
evaporated under reduced pressure. The residue was diluted with
ethyl acetate (30 mL), and the resulting solution was washed
with brine (2 ¥ 20 mL). The mixture was dried over MgSO4,
filtered and concentrated in vacuo to give a residue, which was
purified by flash column chromatography on silica gel (ethyl
acetate/hexanes = 1/4) to yield the epoxide 23 (3.6 g, 89%). [a]D26
-47.2 (c 0.5, CHCl3); IR (CHCl3) n 3524, 2989, 2934, 1075, 1028
Methyl 2-azido-6-O-benzoyl-3-O-benzyl-2-deoxy-a-D-glucopy-
ranoside (5). Method 1: DDQ (123 mg, 0.54 mmol) was added
to a solution of compound 17 (100 mg, 0.18 mmol) in CH2Cl2
(4.7 mL) and water (0.3 mL) in three equal portions at half-hour
intervals. After stirring for 5 h, the reaction was quenched by
addition of saturated NaHCO3(aq) (10 mL). The organic solution
was washed with saturated NaHCO3(aq) (2 ¥ 10 mL), dried
over MgSO4, filtered and concentrated in vacuo. The residue
was purified by column chromatography on silica gel (ethyl
acetate/hexanes = 2/5) to give the 4-alcohol 5 (66 mg, 89%).
Method 2: A solution of compound 19 (3.63 g, 12 mmol) and
pyridine (4.8 mL, 60 mmol) in CH2Cl2 (33 mL) was cooled down
to 0 ◦C under N2 atmosphere. Benzoyl chloride (1.65 mL, 14 mmol)
was slowly added to the solution, the ice bath was removed, and
the mixture was stirred at room temperature for 2 h. The reaction
was sequentially washed with 1 N HCl(aq), saturated NaHCO3(aq)
and finally with brine. The organic portion was dried over MgSO4,
filtered and concentrated in vacuo. Purification of this residue via
flash column chromatography (ethyl acetate/hexanes = 1/3) led
to the product 5 (4.21 g, 88%). [a]2D4 +73.8 (c 1.0 in CHCl3); IR
1
cm-1; H NMR (400 MHz, CDCl3) d 7.36–7.25 (1H, m, Ar-H),
5.97 (1H, d, J 3.8, 1-H), 4.71 (1H, d, J 12.2, CH2Ph), 4.62 (1H,
d, J 3.8, 2-H), 4.49 (1H, d, J 12.2, CH2Ph), 3.94 (1H, d, J 3.5,
3-H), 3.78 (1H, dd, J 6.2, 3.5, 4-H), 3.25 (1H, ddd, J 6.2, 4.5, 2.7,
5-H), 2.73 (1H, t, J 4.5, 6-Ha), 2.51 (1H, dd, J 4.5, 2.7, 6-Hb), 1.42
(3H, s, CH3), 1.29 (3H, s, CH3); 13C NMR (100 MHz, CDCl3) d
137.1 (C), 128.4 (CH), 127.9 (CH), 127.5 (CH), 111.8 (C), 105.3
(CH), 82.5 (CH), 82.3 (CH), 82.0 (CH), 71.8 (CH2), 50.0 (CH),
43.0 (CH2), 26.7 (CH3), 26.2 (CH3); HRMS (ESI, [M + H]+) m/z
calc. for C16H21O5 293.1389, found 293.1395.
1,6-Anhydro-3-O-benzyl-b-L-idopyranose (27). Method 1:
Compound 23 (740 mg, 2.5 mmol) was dissolved in 1,4-dioxane
(1.5 mL), 2 N H2SO4(aq) (1.5 mL) was added to the solution,
and the mixture was refluxed for 22 h. The reaction flask was
immersed in an ice bath, and 3 N NaOH(aq) (1 mL) was added
to neutralize the solution. Water (10 mL) was added and the
crude target material was extracted with ethyl acetate (3 ¥
10 mL). The combined organic layer was washed with brine, dried
over MgSO4, filtered and concentrated in vacuo. Purification
of this residue through flash column chromatography (ethyl
acetate/hexanes = 1/1) on silica gel provided the 2,4-diol 27
(452 mg, 71%). Method 2: Potassium tert-butoxide (232 mg,
2.1 mmol) was added to a solution of compound 8 (463 mg,
1
(CHCl3) n 3484, 2913, 2106, 1720, 1277, 1056 cm-1; H NMR
(400 MHz, CDCl3) d 8.02 (2H, dd, J 8.4, 1.3, Bz-H), 7.61–7.51
(1H, m, Ar-H), 7.48–7.26 (7H, m, Ar-H), 4.92 (1H, d, J 11.0,
CH2Ph), 4.81 (1H, d, J 11.0, CH2Ph), 4.80 (1H, d, J 3.6, 1-H),
4.73 (1H, dd, J 12.2, 4.2, 6-Ha), 4.45 (1H, dd, J 12.2, 2.1, 6-Hb),
3.86 (1H, ddd, J 12.2, 4.2, 2.1, 5-H), 3.83 (1H, dd, J 10.1, 9.2,
3-H), 3.56 (1H, t, J 9.2, 4-H), 3.43 (1H, s, CH3), 3.36 (1H, dd, J
10.1, 3.6, 2-H); 13C NMR (100 MHz, CDCl3) d 166.9 (C), 137.7
(C), 129.9 (C), 129.6 (CH), 129.4 (C), 128.5 (CH), 128.3 (CH),
128.1 (CH), 127.9 (CH), 98.6 (CH), 79.7 (CH), 75.2 (CH2), 70.7
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˚
0.94 mmol) in a mixed solvent of CH2Cl2/tert-butanol (1/1,
9.3 mL) at 0 ◦C under nitrogen atmosphere. After stirring for 16 h,
the reaction was neutralised with 0.6 N H2SO4(aq) (ca. 4.5 mL), and
the flask was equipped with a simple distillation head to evaporate
CH2Cl2 and tert-butanol under reduced pressure. 1,4-Dioxane
(15.5 mL) and 3 N H2SO4(aq) (7.7 mL) were sequentially added to
the resulting solution, and the mixture was kept stirring at 120 ◦C
for another 16 h. After cooling to room temperature, the reaction
was neutralised with 10 N NaOH(aq) (4.6 mL), and the solvent
was removed under reduced pressure. Water (10 mL) was added
to the mass, and the mixture was extracted with ethyl acetate (3
¥ 10 mL). The combined organic layers were washed with brine,
dried over MgSO4, filtered and concentrated in vacuo to get a
residue, which was purified by flash column chromatography on
silica gel (ethyl acetate/hexanes = 1/1) to give 27 (145 mg, 61%)
6.96 mmol) and fleshly dried 4 A molecular sieves (2 g) in CH2Cl2
(65 mL) was stirred at room temperature for 1 h under N2 atmo-
sphere. The reaction flask was cooled to -78 ◦C, TMSOTf (116 mL,
1.2 mmol) was added to the solution, and the resulting mixture was
gradually warmed up to room temperature. After stirring for 6 h,
Et3N (100 mL) was added to quench the reaction, the mixture was
filtered through Celite, the solid was washed with CH2Cl2, and the
filtrate was concentrated in vacuo. The residue was purified by flash
column chromatography on silica gel (ethyl acetate/hexanes = 1/4)
to afford 28a (3.93 g, 68%) and its b-isomer 28b (0.648 g, 11%).
28a: [a]2D7 +123.9 (c 0.52 in CHCl3); Elemental analysis calc. (%)
for C51H47N3O11: C 69.77, H 5.40, N 4.79; found: C 69.65, H 5.39,
N 4.79; IR (CHCl3) n 2928, 2108, 1729, 1260, 1068 cm-1; partial
characterisation data were reported previously.18 28b: [a]D24 +70.5
(c 5.0 in CHCl3); IR (CHCl3) n 3435, 2928, 2108, 1729, 1260, 1068
cm-1; partial characterisation data were reported previously.18
◦
as a white solid. mp 158–159 C (from CH2Cl2/hexanes); [a]D27
+69.2 (c 1.0 in MeOH); Elemental analysis calc. (%) for C13H16O5:
C 61.90, H 6.39; found: C 61.65, H 6.35; IR (CHCl3) n 3468,
3306, 2954, 2889, 1026 cm-1; 1H NMR (400 MHz, CDCl3) d
7.37–7.26 (5H, m, Ar-H), 5.27 (1H, d, J 2.0, 1-H), 4.93 (1H, d,
J 11.7, CH2Ph), 4.71 (1H, d, J 11.7, CH2Ph), 4.41 (1H, t, J 4.4,
5-H), 4.01 (1H, d, J 8.2, 6-Ha), 3.85 (1H, dd, J 8.0, 4.4, 4-H), 3.70
(1H, dd, J 8.2, 4.4, 6-Hb), 3.67–3.60 (1H, m, 2-H), 3.37 (1H, t, J
8.0, 3-H), 2.17 (1H, br s, 4-OH), 1.97 (1H, br s, 2-OH); 13C NMR
(100 MHz, CD3OD) d 140.4 (C), 129.3 (CH), 129.0 (CH), 128.6
(CH), 103.6 (CH), 84.9 (CH), 77.2 (CH), 76.4 (CH), 76.0 (CH2),
72.7 (CH), 65.9 (CH2); HRMS (ESI, [M + Na]+) m/z calc. for
C13H16O5Na 275.0895, found 275.0891.
6-O-Acetyl-4-O-[2-azido-6-O-benzoyl-3-O-benzyl-2-deoxy-4-
O-(2-naphthylmethyl)-a-D-glucopyranosyl]-2-O-benzoyl-3-O-ben-
zyl-L-idopyranosyl acetate (29). Cu(OTf)2 (6 mg, 0.015 mmol)
was added to a solution of compound 28a (100 mg, 0.11 mmol) in
Ac2O (1.0 mL) at room temperature under N2 atmosphere. After
stirring for 2 days, the reaction was quenched with MeOH, and
the solvent was evaporated under reduced pressure. Water (5 mL)
was added to the mass, and the crude target material was extracted
with ethyl acetate (3 ¥ 5 mL). The combined organic layers were
sequentially washed with saturated NaHCO3(aq) and brine, dried
over MgSO4, filtered and concentrated in vacuo. Purification of
this residue via flash column chromatography on silica gel (ethyl
acetate/hexanes = 1/4) furni◦shed the 1,6-diacetate 29 (98 mg, 88%)
as a white solid. mp 57–58 C (from EtOH); Elemental analysis
calc. (%) for C55H52N3O14: C 67.41, H 5.45, N 4.29; found: C 67.21,
H 5.47, N 3.98; IR (CHCl3) n 3435, 2928, 2108, 1729, 1260, 1068
cm-1; 1H NMR (400 MHz, CDCl3) d 8.18–8.10 (3.4H, m, Bz-H),
7.93–7.88 (3.4 H, m, Bz-H), 7.80–7.75 (3.4H, m, Ar-H), 7.75–7.70
(1.7H, m, Ar-H), 7.62 (1.7H, s, Ar-H), 7.55–7.47 (1.7H, m, Ar-H),
7.47–7.41 (3.4H, m, Ar-H), 7.40–7.24 (23.8H, m, Ar-H), 7.21–7.14
(3.4H, m, Ar-H), 6.25 (1H, s), 6.22 (0.7H, d, J 2.0), 5.22 (0.7H,
dd, J 4.6, 2.1), 5.14 (1H, s), 4.92–4.81 (3.4H, m), 4.78–4.71 (3.7H,
m), 4.66 (1H, d, J 3.6), 4.64–4.58 (1.7H, m), 4.52–4.46 (2.7H, m),
4.46–4.39 (1.7H, m), 4.46–4.31 (3.4H, m), 4.30–4.21 (1.7H, m),
4.20–4.16 (1H, m), 4.16–4.05 (3.4H, m), 3.75–3.65 (3.4H, m), 3.60
(1.7H, dd, J 9.4, 1.4), 3.34 (1.7H, dd, J 10.1, 3.6), 2.08 (5.1H, s),
2.07–2.04 (5.1H, m); 13C NMR (100 MHz, CDCl3) d 170.5 (C),
170.4 (C), 168.9 (C), 168.7 (C), 165.8 (C), 165.5 (C), 137.34 (C),
137.29 (C), 137.0 (C), 134.7 (C), 133.3 (CH), 133.2 (CH), 133.04
(C), 132.95 (CH), 129.8 (CH), 129.5 (C), 129.4 (CH), 128.6 (CH),
128.4 (CH), 128.34 (CH), 128.31 (CH), 128.26 (CH), 128.1 (CH),
128.0 (CH), 127.9 (CH), 127.8 (CH), 127.6 (CH), 126.84 (CH),
126.79 (CH), 126.1 (CH), 126.0 (CH), 125.7 (CH), 102.3 (CH),
99.3 (CH), 98.8 (CH), 91.8 (CH), 90.5 (CH), 80.7 (CH), 80.6
(CH), 77.5 (CH), 75.1 (CH2), 74.6 (CH), 73.6 (CH), 73.4 (CH2),
73.3 (CH), 72.4 (CH2), 71.6 (CH), 70.5 (CH), 68.5 (CH), 67.2
(CH), 67.0 (CH), 63.8 (CH), 63.7 (CH), 63.3 (CH2), 63.0 (CH2),
62.8 (CH2), 20.9 (CH3), 20.7 (CH3); HRMS (ESI, [M + Na]+) m/z
calc. for C55H53N3O14Na 1002.3425, found 1002.3430.
1,6-Anhydro-2-O-benzoyl-3-O-benzyl-b-L-idopyranose
(7).
Benzoyl chloride (9.7 mL, 0.083 mol) was slowly added to a
solution of compound 27 (20 g, 0.08 mol) and pyridine (32.4 mL,
0.4 mol) in CH2Cl2 (200 mL) at 0 ◦C under N2 atmosphere.
After stirring for 2 h, water (5 mL) was added to quench the
reaction, and the organic layer was sequentially washed with 1
N HCl(aq), saturated NaHCO3(aq) and water. The organic portion
was dried over MgSO4, filtered and concentrated in vacuo. The
residue was purified by flash column chromatography on silica
gel (ethyl acetate/hexane◦s = 1/3) to yield 7 (24.1 g, 85%) as a
white solid. mp 140–141 C (from EtOH); [a]2D6 +118.8 (c 1.7 in
CHCl3); IR (CHCl3) n 3474, 2904, 1722, 1274, 1071 cm-1; partial
characterisation data were reported previously.19n
1,6-Anhydro-4-O-[2-azido-6-O-benzoyl-3-O-benzyl-2-deoxy-4-
O-(2-naphthylmethyl)-a-D-glucopyranosyl]-2-O-benzoyl-3-O-ben-
zyl-b-L-idopyranose (28a) and 1,6-anhydro-4-O-[2-azido-6-O-
benzoyl-3-O-benzyl-2-deoxy-4-O-(2-naphthylmethyl)-b-D-glucopy-
ranosyl]-2-O-benzoyl-3-O-benzyl-b-L-idopyranose
(28b). A
solution of compound 6 (509 mg, 0.94 mmol) in CH2Cl2
(5 mL) was cooled in an ice bath under N2 atmosphere.
Anhydrous potassium carbonate (260 mg, 1.88 mmol) and
trichloroacetonitrile (0.95 mL, 9.47 mmol) were sequentially
added to the reaction solution, the ice bath was removed, and
the mixture was kept stirring for 16 h. The resulting solution
was filtered through Celite, the solid was washed with CH2Cl2,
and the filtrate was concentrated in vacuo to afford the crude
trichloroacetimidate 6 (635 mg, 98%, a/b = 1/1.9), which was
directly used for the next reaction.
4-O-[2-Azido-6-O-benzoyl-3-O-benzyl-2-deoxy-4-O-(2-naphth-
ylmethyl)-a-D-glucopyranosyl]-2-O-benzoyl-3-O-benzyl-L-idopyra-
nose (30). PTSA (113 mg, 0.60 mmol) was added to a solution
A solution of the crude trichloroacetimidate 6 (3.97 g,
5.8 mmol), the 1,6-anhydro-b-L-idopyranosyl 4-alcohol 7 (2.48 g,
768 | Org. Biomol. Chem., 2012, 10, 760–772
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of compound 29 (292 mg, 0.30 mmol) in a mixed solvent
[CH2Cl2/MeOH (2/1), 3 mL] at room temperature under N2
atmosphere. After stirring for 48 h, Et3N (0.5 mL) was added to
quench the reaction, and the whole mixture was evaporated under
reduced pressure. Water (5 mL) was added to this mass, and the
mixture was extracted with ethyl acetate (3 ¥ 5 mL). The combined
organic layer was sequentially washed with saturated NaHCO3(aq)
and brine, dried over MgSO4, filtered and concentrated in vacuo.
The residue was purified by column chromatography (ethyl
acetate/hexane = 2/3) to get the desired 1,6-diol 30 (217 mg,
81%). [a]2D4 +9.4 (c 0.285 in CHCl3); IR (CHCl3) n 3429, 2929,
2108, 1710, 1646, 1453, 1274, 1023, 748, 709 cm-1; 1H NMR (400
MHz, CDCl3) d 8.23–8.11 (2H, m, Bz-H), 7.96–7.86 (2H, m,
Bz-H), 7.81–7.67 (3H, m, Ar-H), 7.62 (1H, s, Ar-H), 7.55–7.47
(1H, m, Ar-H), 7.47–7.41 (2H, m, Ar-H), 7.41–7.25 (15H, m,
Ar-H), 7.18–7.09 (1H, m, Ar-H), 5.32–5.21 (1H, m), 5.07 (1H,
dd, J 2.9, 1.9), 4.94–4.80 (2H, m), 4.78–4.69 (2H, m), 4.65–4.55
(2H, m), 4.54–4.37 (2H, m), 4.32–4.20 (2H, m), 4.14–4.07 (2H,
m), 4.04–3.89 (2H, m), 3.83–3.73 (1H, m), 3.68–3.50 (3H, m),
3.38–3.28 (1H, m); 13C NMR (100 MHz, CDCl3) d 166.0 (C),
165.6 (C), 137.3 (C), 137.1 (C), 136.6 (C), 134.6 (C), 133.0 (CH),
132.9 (C), 129.8 (C), 129.73 (CH), 129.66 (CH), 129.4 (CH),
128.4 (CH), 128.3 (CH), 128.2 (CH), 128.1 (CH), 128.0 (CH),
127.9 (CH), 127.7 (CH), 127.5 (CH), 126.84 (CH), 126.81 (CH),
126.0 (CH), 125.9 (CH), 125.7 (CH), 99.0 (CH), 98.9 (CH), 92.7
(CH), 91.9 (CH), 80.6 (CH), 77.4 (CH), 75.1 (CH), 75.0 (CH2),
74.9 (CH2), 73.3 (CH), 72.84 (CH2), 72.76 (CH2), 72.2 (CH), 70.3
(CH), 69.8 (CH), 68.6 (CH), 67.1 (CH), 63.7 (CH), 63.0 (CH2),
61.8 (CH2), 61.6 (CH2); HRMS (ESI, [M + Na]+) m/z calc. for
C51H49N3O12Na 918.3214, found 918.3210.
128.3 (CH), 128.07 (CH), 128.01 (CH), 127.9 (CH), 127.85 (CH),
127.6 (CH), 126.9 (CH), 126.1 (CH), 126.0 (CH), 125.87 (CH),
125.83 (CH), 99.4 (CH), 98.9 (CH), 91.9 (CH), 90.8 (CH), 80.8
(CH), 80.6 (CH), 77.6 (CH), 77.5 (CH), 75.1 (CH2), 74.9 (CH),
74.6 (CH), 73.7 (CH), 73.6 (CH2), 73.2 (CH), 72.4 (CH2), 71.4
(CH), 70.54 (CH), 70.50 (CH), 68.7 (CH), 67.2 (CH), 68.8 (CH),
63.9 (CH), 63.8 (CH), 63.4 (CH2), 63.1 (CH2), 62.8 (CH2), 37.83
(CH2), 37.75 (CH2), 37.63 (CH2), 37.56 (CH2), 29.6 (CH3), 28.1
(CH2), 27.9 (CH2), 27.79 (CH2), 27.76 (CH2); HRMS (ESI, [M +
Na]+) m/z calcd for C61H61N3O16Na 1114.3950, found 1114.3961.
4-O-[2-Azido-6-O-benzoyl-3-O-benzyl-2-deoxy-4-O-(2-naphth-
ylmethyl)-a-D-glucopyranosyl]-2-O-benzoyl-3-O-benzyl-6-O-levu-
linyl-L-idopyranose (32). Ammonia gas was passed through a
solution of compound 31 (2.58 g, 2.36 mmol) in THF (260 mL) at
0 ◦C for 10 min. The reaction, kept stirring at 0 ◦C, was monitored
by TLC until the full consumption of the starting material (ca. 56
h). The solvent was concentrated under reduced pressure, and
the residue was diluted with ethyl acetate (50 mL) and water
(50 mL). The crude target material was extracted with ethyl acetate
(2 ¥ 50 mL) and the combined organic layers were sequentially
washed with saturated NaHCO3(aq) and brine, dried over MgSO4,
filtered and concentrated in vacuo. The residue was purified by
column chromatography (ethyl acetate/hexanes = 2/3) to provide
compound 32 (1.6 g, 62%) and the 1,6-diol 30 (607 mg, 29%).
[a]2D5 +19.1 (c 0.41 in CHCl3); IR (CHCl3) n 3448, 2108, 1716,
1
1447, 1267, 1068, 748, 709 cm-1; H NMR (400 MHz, CDCl3)
d 8.20–8.10 (2H, m, Bz-H), 7.94–7.85 (2H, m, Bz-H), 7.80–7.68
(3H, m, Ar-H), 7.61 (1H, s, Ar-H), 7.53–7.47 (1H, m, Ar-H), 7.47–
7.40 (2H, m, Ar-H), 7.39–7.25 (14H, m, Ar-H), 7.17–7.09 (2H, m,
Ar-H), 5.26 (1H, t, J 7.8), 5.07–5.04 (1H, m), 4.91–4.80 (2H, m),
4.80–4.67 (2H, m), 4.65–4.58 (1H, m), 4.58–4.53 (1H, m), 4.53–
4.38 (3H, m), 4.33–4.20 (3H, m), 4.01–3.91 (2H, m), 3.74–3.53
(3H, m), 3.38–3.26 (1H, m), 2.81–2.65 (2H, m), 2.65–2.50 (2H,
m), 2.12 (1.5H, s), 2.11 (1.5H, s); 13C NMR (100 MHz, CDCl3)
d 207.1 (C), 206.8 (C), 172.4 (C), 172.3 (C), 166.0 (C), 165.8 (C),
137.4 (C), 137.2 (C), 136.6 (C), 134.8 (C), 133.3 (CH), 133.2 (CH),
133.1 (C), 133.06 (CH), 133.02 (C), 129.86 (CH), 129.82 (CH),
129.79 (CH), 129.7 (C), 129.5 (CH), 128.7 (CH), 128.61 (CH),
128.58 (CH), 128.5 (CH), 128.4 (CH), 128.36 (CH), 128.32 (CH),
128.1 (CH), 128.0 (CH), 127.91 (CH), 127.86 (CH), 127.7 (CH),
126.9 (CH), 126.2 (CH), 126.1 (CH), 125.8 (CH), 99.2 (CH), 93.0
(CH), 92.0 (CH), 80.83 (CH), 80.77 (CH), 77.49 (CH), 77.45 (CH),
75.14 (CH), 75.11 (CH2), 75.07 (CH2), 74.9 (CH), 73.3 (CH2),
73.1 (CH), 72.23 (CH), 72.17 (CH), 70.5 (CH2), 69. 9 (CH), 68.2
(CH), 64.5 (CH), 63.89 (CH), 63.85 (CH), 63.7 (CH2), 63.3 (CH2),
62.84 (CH2), 62.80 (CH2), 38.0 (CH2), 37.9 (CH2), 29.8 (CH3), 29.7
(CH3), 27.93 (CH2), 27.85 (CH2); HRMS (ESI, [M + Na]+) m/z
calc. for C56H55N3O14Na 1016.3582, found 1016.3586.
4-O-[2-Azido-6-O-benzoyl-3-O-benzyl-2-deoxy-4-O-(2-naphth-
ylmethyl)-a-D-glucopyranosyl]-2-O-benzoyl-3-O-benzyl-6-O-levu-
linyl-L-idopyranosyl levulinate (31). DMAP (0.9 mg, 7.36 mmol)
and (Lev)2O (38 mg 0.18 mmol) were consecutively added to a
solution of the 1,6-diol 30 (63 mg, 0.07 mmol) in pyridine (0.6 mL)
at 0 ◦C under N2 atmosphere. The reaction flask was gradually
warmed up to room temperature, and the solution was kept stirring
for another 1.5 h. The reaction was quenched by addition of
cold water, and the mixture was extracted with ethyl acetate (3
¥ 5 mL). The combined organic layer was sequentially washed by
1 N HCl(aq), saturated NaHCO3(aq) and brine, dried over MgSO4,
filtered and concentrated in vacuo. The residue was purified by
flash column chromatography (ethyl acetate/hexanes = 1/1) to
provide 31 (75 mg, 97%). IR (CHCl3) n 3028, 2925, 2110, 1720,
1
1272, 753, 714 cm-1; H NMR (400 MHz, CDCl3) d 8.18–8.10
(3.4H, m, Bz-H), 7.93–7.87 (3.4H, m, Bz-H) 7.80–7.74 (3.4H, m,
Ar-H), 7.74–7.69 (1.7H, m, Ar-H), 7.61 (1.7H, s, Ar-H), 7.54–7.25
(28.9H, m, Ar-H), 7.22–7.17 (1.7H, m, Ar-H), 7.17–7.10 (1.7H,
m, Ar-H), 6.25 (0.7H, s), 6.21 (1H, d, J 2.2), 5.22 (1H, dd, J 4.9,
2.3), 5.1 (0.7H, s), 4.93–4.69 (8.5H, m), 4.65 (0.7H, d, J 3.7), 4.63–
4.55 (1H, m), 4.54–4.23 (10.2H, m), 4.21–4.15 (1H, m), 4.13–3.99
(2.4H, m), 3.78–3.56 (5.1H, m), 3.38–3.31 (1.7H, m), 2.74–2.53
(13.6H, m), 2.10 (4.2H, s), 2.07 (6H, s); 13C NMR (100 MHz,
CDCl3) d 206.43 (C), 206.38 (C), 206.0 (C), 205.9 (C), 172.4 (C),
172.3 (C), 170.7 (C), 170.6 (C), 166.0 (C), 165.9 (C), 165.6 (C),
137.44 (C), 137.36 (C), 137.1 (C), 134.8 (C), 133.3 (CH), 133.2
(CH), 133.11 (C), 133.06 (CH), 133.02 (CH), 129.84 (CH), 129.77
(C), 129.6 (C), 129.5 (CH), 128.6 (CH), 128.43 (CH), 128.38 (CH),
4-O-[2-Azido-6-O-benzoyl-3-O-benzyl-2-deoxy-4-O-(2-naphth-
ylmethyl)-a-D-glucopyranosyl]-2-O-benzoyl-3-O-benzyl-6-O-levu-
linyl-L-idopyranosyl trichloroacetimidate (4). DBU (36 mL,
0.24 mmol) and trichloroacetonitrile (146 mL, 1.46 mmol) were
sequentially added to a solution of the hemiacetal 32 (242 mg,
0.24 mmol) in CH2Cl2 (2.5 mL) at 0 ◦C under N2 atmosphere. After
stirring for 3 h, the solvent was removed under reduced pressure.
The residue was purified by flash column chromatography (ethyl
acetate/hexanes = 1/2) to give 4 (246 mg, 89%). IR (CHCl3) n
3333, 2922, 2109, 1720, 1268, 1069, 714 cm-1; 1H NMR (400 MHz,
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˚
CDCl3) d 8.79 (1H, s, NH), 8.65 (0.8H, s, NH), 8.23 (1.8H, dd, J
7.3, 1.8, Bz-H), 8.13 (1.8H, dd, J 6.7, 1.6, Bz-H), 8.05–7.93 (3.6H,
m, Ar-H), 7.85–7.73 (5.4H, m, Ar-H), 7.68 (1.8H, s, Ar-H), 7.58–
7.13 (34.2H, m, Ar-H), 6.59 (0.8H, d, J 2.8), 6.46 (1H, s), 5.44
(0.8H, dd, J 6.5, 2.9), 5.40 (1H, s), 5.10 (0.8H, s), 5.04–4.88 (4.6H,
m), 4.87–4.65 (7.2H, m), 4.65–4.24 (10.8H, m), 4.23–4.04 (2.8H,
m), 3.98 (0.8H, t, J 9.5 Hz), 3.85 (1H, s), 3.81–3.63 (2.6H, m),
3.44 (1.8H, dt, J 9.8, 3.5), 2.72–2.49 (7.2H, m), 2.10 (3H, s), 2.02
(2.4H, s); 13C NMR (100 MHz, CDCl3) d 205.93 (C), 205.86 (C),
172.1 (C), 171.9 (C), 165.73 (C), 165.65 (C), 165.5 (C), 165.2 (C),
160.20 (C), 160.1 (C), 137.4 (C), 137.2 (C), 137.1 (C), 134.61 (C),
134.55 (C), 133.1 (CH), 132.9 (C), 132.8 (CH), 129.6 (CH), 129.5
(CH), 129.4 (C), 129.33 (CH), 129.28 (CH), 129.2 (C), 128.4 (CH),
128.3 (CH), 128.22 (CH), 128.17 (CH), 128.1 (CH), 128.0 (CH),
127.8 (CH), 127.7 (CH), 127.6 (CH), 127.4 (CH), 126.7 (CH),
125.9 (CH), 125.8 (CH), 125.72 (CH), 125.67 (CH), 99.1 (CH),
98.8 (CH), 95.3 (CH), 94.6 (CH), 90.8 (C), 90.4 (C), 80.6 (CH),
80.2 (CH), 77.5 (CH), 75.1 (CH2), 74.9 (CH2), 74.8 (CH2), 74.6
(CH), 74.1 (CH), 73.6 (CH), 71.9 (CH2), 71.0 (CH), 70.4 (CH),
70.1 (CH), 69.9 (CH), 66.8 (CH), 66.3 (CH), 63.9 (CH2), 63.7
(CH), 63.5 (CH), 63.1 (CH2), 62.8 (CH2), 62.6 (CH2), 37.5 (CH2),
37.3 (CH2), 29.4 (CH3), 29.3 (CH3), 27.5 (CH2); HRMS (ESI,
[M + Na]+) m/z calc. for C58H55N4O14Cl3Na 1159.2678, found
1159.2672.
34 (646 mg, 0.52 mmol) and 4 A molecular sieves (1.2 g) in
CH2Cl2 (30 mL) was stirred at room temperature for 1 h under N2
atmosphere. The reaction flask was cooled to -40 C, TMSOTf
◦
(47 mL, 0.26 mmol) was added to the solution, and the mixture
was continuously stirred for 1.5 h. A solution of the imidate
donor 4 (592 mg, 0.52 mmol) in CH2Cl2 (6 mL) and TMSOTf (47
mL, 0.26 mmol) were sequentially added to the mixture, and the
reaction solution was kept stirring for another 3 h. The reaction
was gradually warmed up to room temperature, and Et3N (100
mL) was added to quench the reaction. The resulting mixture
was filtered through Celite, the solid was washed with CH2Cl2,
and the filtrate was concentrated in vacuo. Purification of this
residue by flash column chromatography (ethyl acetate/hexanes =
1/2) yielded the pentasaccharide 35 (937 mg, 81%). Reaction
of compound 36 (217 mg, 0.10 mmol) following the above
procedure yielded the heptasaccharide 37 (202 mg, 66%, eluent:
ethyl acetate/hexanes = 2/3).
General procedure for the cleavage of levulinyl esters (33→38,
35→39, 37→40). Hydrazine acetate (193 mg, 2.1 mmol, 5 equiv.
per OLev) was added to a solution of compound 33 (583 mg,
420 mmol) in a mixed solvent [ethanol/toluene (2/1), 12 mL]
at room temperature under N2 atmosphere. After stirring for 2
h, the reaction mixture was evaporated under reduced pressure,
water was added to the residue, and the crude target material was
extracted with ethyl acetate thrice. The combined organic layer was
washed with saturated NaHCO3(aq) and brine, dried over MgSO4,
filtered and concentrated in vacuo. The residue was purified by
flash column chromatography (ethyl acetate/hexanes = 1/3) to
provide the product 38 (520 mg, 97%). Reactions of compounds
35 (378 mg, 0.17 mmol) and 37 (107 mg, 0.03 mmol) following the
above procedure afforded the products 39 (330 mg, 94%, eluent:
ethyl acetate/hexanes = 1/2) and 40 (89 mg, 92%, eluent: ethyl
acetate/hexanes = 1/1.8), respectively.
Methyl [2-azido-6-O-benzoyl-3-O-benzyl-2-deoxy-4-O-(2-naph-
thylmethyl)-a-D-glucopyranosyl]-(1 → 4)-(2-O-benzoyl-3-O-ben-
zyl-6-O-levulinyl-a-L-idopyranosyl)-(1 → 4)-2-azido-6-O-benzoyl-
3-O-benzyl-2-deoxy-a-D-glucopyranoside (33). A solution of 4
˚
(1.4 g, 12 mmol), 5 (409 mg, 10 mmol) and freshly dried 4 A
molecular sieves (1.5 g) in CH2Cl2 (35 mL) was stirred at room
temperature for 1 h under N2 atmosphere. The reaction flask was
cooled to -78 ◦C, TMSOTf (36 mL, 2 mmol) was added, and the
resulting mixture was gradually warmed up to room temperature
and kept stirring for 3 h. The reaction was quenched with Et3N
(8 mL), the mixture was filtered through Celite, the filtrate was
concentrated in vacuo, and the residue was purified by flash column
chromatography (ethyl acetate/hexanes = 1/3) to afford 33 (1.48 g,
89%). [a]2D4 +48.5 (c 0.28 in CHCl3); IR (CHCl3) n 2916, 2101, 1716,
1446, 1267, 1107, 1023, 748, 703 cm-1; HRMS (ESI, [M+Na]+)
m/z calc. for C77H76N6O19Na 1411.5063, found: 1411.5076; partial
characterisation data were reported previously.18
General procedure for TEMPO oxidation (38→41, 39→42,
40→43). BAIB (90 mg, 0.28 mmol, 2.5 equiv. per OH group)
and TEMPO (3.5 mg, 22.4 mmol) were sequentially added to a
solution of the alcohol 36 (143 mg, 0.11 mmol) in a mixed solvent
[H2O/CH2Cl2 (1/2), 2.25 mL] at room temperature. After stirring
overnight, 10% Na2S2O3(aq) was added to quench the reaction, and
the crude target material was extracted with ethyl acetate thrice.
The combined organic layers were dried over MgSO4, filtered and
concentrated in vacuo to give a residue, which was purified by flash
column chromatography on silica gel (ethyl acetate/hexanes =
2/3) to yield the corresponding carboxylic acid 41 (123 mg, 85%).
Reactions of compounds 39 (140 mg, 0.07 mmol) and 40 (230 mg,
0.08 mmol) following the above procedure provided the products
42 (102 mg, 71%, eluent: ethyl acetate/hexanes = 1/1) and 43
(156 mg, 67%, eluent: ethyl acetate/hexanes = 2/1), respectively.
General procedure for DDQ-mediated cleavage of the 2-
naphthylmethyl group (33→34, 35→36). DDQ (195 mg,
2.58 mmol) was added to a solution of the starting material
33 (1.2 g, 0.86 mmol) in a mixed solvent [CH2Cl2/H2O (18/1),
60 mL] in three equal portions at 30 min intervals at room
temperature. After stirring for 4 h, the reaction was quenched
with saturated NaHCO3(aq), and the organic layer was washed with
saturated NaHCO3(aq) and brine, dried over MgSO4, filtered and
concentrated in vacuo. The residue was purified by flash column
chromatography (ethyl acetate/hexanes = 2/3) on silica gel to
afford the desired alcohol 34 (0.88 g, 82%). Reaction of compound
35 (855 mg, 0.38 mmol) following the above procedure yielded the
product 36 (630 mg, 79%, eluent: ethyl acetate/hexanes = 2/3).
General procedure for cleavage of the benzoyl esters (41→44,
42→45, 43→46). Sodium methoxide (18 mg, 0.34 mmol, 2 equiv.
per Bz group) was added to a solution of the starting material 41
(73 mg, 0.057 mmol) in MeOH (0.7 mL) at room temperature
under N2 atmosphere. After stirring overnight, Dowex 50WX4-
200 ion-exchange resin was added to neutralize the reaction, the
whole mixture was filtered and the filtrate was concentrated in
vacuo. Purification of the residue by flash column chromatography
(MeOH/CHCl3 = 1/10) led to the debenzoylated product 44
General procedure for chain elongation: the synthesis of the
pentasaccharide (35) and the heptasaccharide (37). A solution
of the imidate donor 4 (592 mg, 0.52 mmol), the glycosyl acceptor
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(50 mg, 88%). Reactions of compounds 42 (60 mg, 0.029 mmol)
and 43 (114 mg, 0.04 mmol) following the above procedure
furnished the products 45 (39 mg, 86%, eluent: MeOH/CHCl3 =
1/15) and 46 (62 mg, 75%, eluent: MeOH/CHCl3 = 1/15),
respectively.
previously described.5 Briefly, confluent monolayers of Beas-2B
cells in 96-well plates were pre-treated with various concentrations
of oligosaccharides in serum-free RPMI 1640 medium at 4 ◦◦C
for 30 min before incubation with 5 mg ml-1 MBP-EDN at 4 C
for 1 h. The cells were then washed with ice-cold phosphate-
buffered saline (PBS) and fixed with 2% paraformaldehyde at
room temperature for 15 min prior to blocking with 2% bovine
serum albumin in PBS at room temperature for 90 min. The
level of bound MBP-EDN was quantified by ELISA analysis.
MBP-EDN was detected using mouse monoclonal anti-MBP and
goat anti-mouse HRP-conjugated secondary antibody, followed
by enhanced chemiluminescence detection system. The amount of
MBP-EDN bound to cells without oligosaccharide treatment was
set to 100%.
General procedure for O-sulfonation (44→47, 45→48, 46→49).
A solution of the starting material 44 (30 mg, 0.03 mmol) and
SO3/Et3N (113 mg, 0.63 mmol, 7 equiv. per OH group) in DMF
(1 mL) was stirred at 55 ◦C for 48 h. After cooling down to
room temperature, 1 M NaHCO3(aq) (1.0 mL per 0.01 mmol of
SO3/Et3N) was added to the solution, and the mixture was kept
stirring at the same temperature for another 16 h. The solvent
was coevaporated with ethanol under reduced pressure, and the
resulting mass was washed with a mixed solvent (CH2Cl2/MeOH =
1/1), filtered and concentrated in vacuo. The residue was purified
by flash column chromatography on silica gel (MeOH/CHCl3 =
1/5) followed by elution on Sephadex LH20 column (MeOH)
to afford the desired product 47 (29 mg, 78%). Reactions of
compounds 45 (28 mg, 0.018 mmol) and 46 (20 mg, 9.6 mmol)
following the above procedure yielded the products 48 (28 mg,
76%) and 49 (19 mg, 73%), respectively.
Data analysis. Data analyses of IC50 were performed using
GraphPad Prism.
Acknowledgements
This work was supported by the National Science Council
(NSC 97-2113-M-001-033-MY3, NSC 98-2119-M-001-008-MY2,
NSC 98-3112-B-007-005, NSC 98-2627-B-007-008) and Academia
Sinica.
General procedure for hydrogenolysis and N-sulfonation
(47→50→1, 48→51→2, 49→52→3). Hydrogen gas was passed
through a mixture of the starting material 47 (15 mg, 12 mmol),
10% Pd/C (15 mg) and AcOH (2 mL) in a mixed solvent
[water/methanol (1/9), 1.5 mL] at room temperature for 20 min.
The reaction flask was equipped with 50 psi of hydrogen gas for 2
days. The mixture was filtered through Celite, and the filtrate was
concentrated in vacuo to give the crude amino-alcohol 50.
The crude amino-alcohol 50 was dissolved in water (1 mL), and
the pH value of the solution was adjusted to 9.5 by slow addition
of 0.2 N NaOH(aq). SO3/Pyr. (57 mg, 0.36 mmol, 15 equiv. per
NH2) was added in five equal portions at half-hour intervals at
room temperature while keeping the pH at 9.5. After stirring
for 3 h, the reaction mixture was filtered, and the filtrate was
concentrated under reduced pressure. The residue was dissolved
in water (1 mL) and was purified by a Sephadex G25 column
eluted with double distilled water. The resulting compound was
concentrated in vacuo to provide a residue, which was passed
through the Dowex 50WX2-Na+ resin using double distilled water
as eluent. The water was removed by lyophilisation, and the desired
target molecule 1 (8 mg, 67% in two steps) was obtained as a
white solid. Reactions of compounds 48 (21 mg, 10 mmol) and
49 (19 mg, 6.6 mmol) following the above procedure afforded the
products 2 (10 mg, 60% in two steps) and 3 (9 mg, 55% in two
steps), respectively.
Notes and references
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