Design and synthesis of unnatural heparosan and chondroitin building blocks

Article abstract of DOI:10.1021/jo200076z

Triazole linked heparosan and chondroitin disaccharide and tetrasaccharide building blocks were synthesized in a stereoselective manner by applying a very efficient copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction of appropriately substituted

Full text of DOI:10.1021/jo200076z

ARTICLE
pubs.acs.org/joc
Design and Synthesis of Unnatural Heparosan and Chondroitin
Building Blocks
Smritilekha Bera and Robert J. Linhardt*
Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic
Institute, Troy, New York 12180, United States
S Supporting Information
b
ABSTRACT: Triazole linked heparosan and chondroitin dis-
accharide and tetrasaccharide building blocks were synthesized
in a stereoselective manner by applying a very efficient copper
catalyzed azideꢀalkyne cycloaddition (CuAAC) reaction of
appropriately substituted azido-glucuronic acid and propargy-
luted N-acetyl glucosamine and N-acetyl galactosamine deriva-
tive, respectively. The resulting suitably substituted tetrasaccharide
analogues can be easily converted into azide and alkyne unit for
further synthesis of higher oligosaccharide analogues.
’ INTRODUCTION
The glycan chains of proteoglycans,1 glycosaminoglycans
(GAGs), correspond to the most abundant class of heteropoly-
saccharides in the body. These molecules control a wide range of
physiological and pathological events2ꢀ7 including cellꢀcell
communication,6 growth factor receptor and enzyme inhibition,7
cell proliferation, angiogenesis and inflammatory processes,
wound repair and healing,8 and viral invasion.6,9,10 GAGs are
linear, highly sulfated anionic polysaccharides having repeating
disaccharide motifs and are divided into families based on
variations in stereochemistry and patterns of sulfation. It is
through discrete structural features that they interact with
GAG-binding proteins11 to exhibit their biological effects.
GAG structural diversity creates an enormous number of protein
binding motifs and poses a daunting challenge to define the
structural and functional properties of GAGs. Based on the
difference the repeating disaccharide units comprising GAGs,
they are classified into four main families: heparin/heparan
sulfate, chondroitin sulfate/dermatan sulfate, hyaluronan, and
keratan sulfate (Figure 1).1 Sulfated GAGs are naturally found in
all animals, and GAGs without sulfo group substitution, includ-
ing heparosan, chondrotin, and hyaluronan, are also found in
many bacteria.12 GAGs are implicated in many diseases and are
used therapeutically as drugs.6ꢀ10
Figure 1. Structures of different glycosaminoglycans. Heparan sulfate con-
tains primarily glucuronic acid, whereas heparin contains primarily iduronic
acid and N-sulfoglucosamine. Heparosan has the same basic structure as
heparan sulfate with R = H, X = COCH3 ,and chondroitin has the same
basic structure as chondroitin sulfate with R = H. For hyaluronan R = H.
glycosidic linkage might be finely tuned to be inert to subsequent
synthetic transformations.
Although GAGs are generally isolated by extraction from
animal tissues, synthetic carbohydrate chemistry can offer a
potentially more reliable route to homogeneous naturally occur-
ring GAGs or for incorporating unnatural structures, such as
enzymatically stable glycosidic linkages, into GAGs. One benefit
of the latter approach is that an unnatural or “artificial” GAG may
retain the same geometric and spatial characteristics of the native
GAG while exhibiting stability toward hydrolase activity
and might even potentially inhibit these enzymes, thus suggest-
ing novel therapeutic applications. In addition, an unnatural
The goal of this study was to develop novel synthetic
approaches, with precise control over length, stereochemistry,
linkage, and sulfation patterns, that lead to unnatural GAG chains
as discovery tools to understand the biological roles of GAGs and
explore their utility in treating diseases. Our initial targets are
structural analogues of the disaccharide and tetrasaccharide
Received: January 12, 2011
Published: March 25, 2011
r
2011 American Chemical Society
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backbones of heparosan and chondroitin. The synthetic ap-
proach should be useful in preparing larger and sulfated oligo-
saccharides for use in protein interaction studies.
In the current synthesis, the natural glycosidic linkage of these
building blocks was replaced with a 1,2,3-triazole linkage. This
synthesis relies on click chemistry,13 a Cu(I)-catalyzed 1,
3-dipolar cycloaddition reaction (1,3-DCR) that affords superior
regioselectivity, high tolerance of other functionalities, and al-
most quantitative transformation under mild conditions. The
stereospecific (R or β) construction of glycosidic bonds is a
critical challenge in carbohydrate chemistry and is often low-
yielding, generating the undesired side products. The click reaction
has been previously used in the synthesis of neoglycoconjugates14
and the bioconjugation study of glycosides.15 The 1,3-DCR utilizes
relatively low-cost, nontoxic reagents and solvents alleviating the
need for expensive and highly toxic glycosylation and coupling
reagents. Finally, product purification is simple using precipitation
or liquidꢀliquid extraction and typically not requiring chromatog-
raphy to obtain pure products.
Herein, we report the synthesis of triazole linked tetrasac-
chaide analogue of heparosan and chondroitin. Heparosan is
a repeating disaccharide unit of D-glucuronic (GlcA) acid and
N-acetyl-D-glucosamine (GlcNAc) residues with a (1f4) link-
age, [f4) β-GlcUA (1f4) R-GlcNAc 1f]n (Figure 1). It is a
nonsulfated member of the heparin/heparan sulfate GAG family
and is found in the capsule of certain pathogenic bacteria as well
as the precursor in the biosynthesis of animal heparin and
heparan sulfate. Heparin is a particularly important anticoagulant
drug that has also been recently exploited in treating cancers and
suppressing the inflammatory response.1,2
Chondroitin is composed of an alternating sequence of GlcA
and N-acetyl-D-galactosamine (GalNAc) residues linked through
alternating (β(1f3)) and (R (1f4)) linkages, [f4) β-GlcUA
(1f3) R-GalNAc 1f]n (Figure 1). Chondroitin is a nonsulfated
member of the chondroitin sulfate/dermatan sulfate family of
GAGs and is found in the capsule of certain pathogenic bacteria
as well as the precursor in the biosynthesis of animal chondroitin
sulfate and dermatan sulfate. The chondroitin sulfate/dermatan
sulfate family plays an important role in biological processes, such
as neural development, viral invasion, cancer metastasis, arthritis,
and spinal cord injury.7ꢀ10
Figure 2. Triazole linked heparosan and chondroitin disaccharide and
tetrasaccharide analogues.
either an acceptor by selective deprotection or a glycosyl donor
through aglycone adjustment. Selective deprotection, aglycone
adjustment, and additional glycosylation steps lead to synthesis
of a GAG oligosaccharide. In the second, at first disaccharide
analogue would be synthesized from two monosaccharide unit
with an orthogonally protecting group, and then this disaccharide
analogue would be used as an donor as well as acceptor for the
next tetrasaccharide analogue and so on.
Herein, we report the synthesis of heparosan and chondroitin
disaccharide and tetrasaccharide analogues where GlcA is linked
with GlcNAc/GalNAc through a triazole linkage instead of
glycoside linkage. In this synthesis GlcA, having an azide func-
tional group, will act as an acceptor and GlcNAc/GalNAc, having
an alkyne functional group, will serve as a donor. This disaccha-
ride unit will then be transformed into either an acceptor, having
an azide functional group, or donor, having an alkyne functional
group, through selective protection and deprotection.
Retrosynthetic analysis suggests that heparosan tetrasaccha-
ride analogue 3 will be obtained from a disaccharide building
block 1a through the modification at the anomeric position of the
GlcNAc residue to a glycosyl azide and the propargylation of the
GlcA residue at the C4. Heparosan disaccharide analogue 1a will
be obtained from monosaccharide building blocks through the
modification at the anomeric position of the GlcA residue to a
glycosyl azide 5 and the propargylation of the GlcNAc residue 6
at the C4 (Figure 3).
Retrosynthetic analysis suggests that chondroitin tetrasaccha-
ride analogue 4 will be obtained from a disaccharide building
block 2a through the modification at the anomeric position of the
GlcNAc residue to a glycosyl azide and the propargylation of the
GlcA residue at the C4. Chondroitin disaccharide analogue 2a
will be obtained from monosaccharide building blocks through
the modification at the anomeric position of the GlcA residue to a
glycosyl azide 5 and the propargylation of the GalNAc residue 7
at the C3 (Figure 4).
Our synthetic strategy for preparing the triazole-linked oligo-
saccharide mimetics of heparosan and chondroitin (Figure 2)
begins with the synthesis of suitably protected glucosamine and
glucuronic acid building blocks having appropriately placed azide
and alkyne functional groups.10 This building block will also be
required for further chain extension to prepare future polysac-
charide targets.
Two monosaccharide units that can be easily be transformed
to azido and the propargyluted saccharide building blocks are
required for synthesis of the triazolyl linkages in the GAG
oligosaccharide targets. Solution phase synthesis was selected
and protecting groups and reaction conditions were carefully chosen
to avoid side reactions and maximize the overall yield. The building
blocks used for the synthesis of the heparosan and chondroitin
tetrasaccharide analogues are shown in the retrosynthetic schemes
(Figures 3 and 4). Two iterative 1,3-dipolar cycloaddition reactions
will be used to form the required β-(1,4) or β-(1,3) linkages.
The chemical synthesis of GAGs is typically carried out using
one of two general approaches. In the first, GlcA building blocks
are directly utilized to react with a GlcN or GalN derivative.16
The newly formed disaccharide analogue was transformed into
’ RESULTS AND DISCUSSION
Building Block Synthesis. Retrosynthetic analysis (Figures 3
and 4) shows that GlcA glycosyl azide 5 is common to both
heparosan and chondroitin synthesis. To reach our goal, synthe-
ses begin with properly protected GlcA glycosyl azide 5. The C-1
and C-4 hydroxyl groups require orthogonal protection
to allow the introduction of C-1 azido and a C-4 propargyl
groups. The synthesis starts from commercially available phenyl
4,6-O-benzylidene-1-thio-β-D-glucopyranoside thioglycoside 8.
Thioglycoside 8 was protected using benzoyl chloride in pyridine
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Figure 3. Retrosynthesis of heparosan disaccharide and tetrasaccharide analogues.
Figure 4. Retrosynthesis of chondroitin disaccharide and tetrasaccharide analogues.
to obtain the 2,3-di benzoate ester 9. The neighboring group
participation of benzoyl at C-2 should allow β-directed azidation
of the thiophenyl moiety. The 4,6-O-benzylidene acetal of
compound 9 was selectively reduced by treatment with PhBCl2
and triethylsilane16f at ꢀ78 °C for 1 h to afford the correspond-
ing 4-O-benzyl derivative (10). The remaining primary hydroxyl
group at C-6 was oxidized using a catalytic amount of 2,2,6,6-
tetramethyl-1-piperidinyloxy (TEMPO) and [bis(acetoxy)iodo]-
benzene (BAIB),17 followed by esterification with Cs2CO3 and MeI
affording the completely protected thioglycoside 11. Direct conver-
sion of 11 to azido glycoside18 with NIS, TMSN3 and TMSOTf
failed to give the desired product. Conversion of the thioglycoside to
acetate 12 (R:β/3:7) was accomplished in quantitative yield with
NIS in the presence of acetic acid.19 The β-selectivity is due to the
directing effect of benzoate group at C2 position. Prior to introdu-
cing an azido group at C-1, the reductive removal of C4 benzyl ether
has been attempted under hydrogenation condition using H2/
Pd(OH)2 and H2/PdꢀC in MeOH and catalytic amount of HCl,
but the reaction failed to proceed. We ascribe the failure of this
reaction to steric hindrance at the C-4 position. At this point we
decided to revise Scheme 1 modifying a benzylidine 2,3-dibenzoate
derivative (9). Acid hydrolysis of 9 using p-TSA in MeOH and
CH2Cl2 mixture for 8 h afforded diol 14, which was reprotected with
p-methoxybenzylidene dimethyl acetal at 50 °C in the presence of
catalytic 10-camphorsulfonic acid as p-methoxybenzylidene thio-
glycoside 15.20 The p-methoxy-benzylidene acetal 15 was selectively
reduced to the corresponding 4-O-p-methoxybenzyl derivative (16)
by treatment with PhBCl2 and triethylsilane16 at ꢀ78 °C for 30 min
under anhydrous condition, keeping the C-6 hydroxyl group free.
Oxidation of the C-6 hydroxyl of 16 followed by esterification with
Cs2CO3 and MeI to afford 17 in 75% yield. The thioglycoside was
converted to acetate 18 (R:β/2:8) in 61% yield with NIS in the
presence of acetic acid at 20 °C. The anomeric acetate 18 was
converted to the β-azide derivative 5 in good yield, by treatment
with TMSN3 and SnCl4. During this reaction the in situ removal of
p-methoxybenzyl group at the C-4 position was observed. After
chromatographic separation, the compound was fully characterized
with spectroscopic analysis and HRMS. Disappearance of the
acetate peak at 2.04 ppm in the NMR spectrum confirmed the
complete conversion to the C-1 azide, and the coupling constant at
the anomeric proton at 4.95 ppm as a doublet (J = 8.7 Hz) con-
firmed the stereospecific formation of the β-conformation. The
in situ removal of p-methoxybenzyl group eliminates the need to
modify the disaccharide building block in assembling the tetrasac-
charide analogue.
Next, we undertook the synthesis of the GlcNAc β-benzyl
glycoside derivative. Although several methods are reported for
the synthesis of selective β-O-benzyl glucosamine derivatives,21
we selected an approach that treated GlcNAc (19) with sodium
hydride and benzyl bromide in DMF at room temperature for 3 h
affording benzyl glycoside 20 as a mixture of R:β anomers
(1:4).22 To facilitate separation, the C-4 and C-6 hydroxyl group
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Scheme 1a
a Reagents and conditions: (a) benzoyl chloride, pyridine, rt; (b) PhBCl2, mol sieves powder 4 Å, Et3SiH, CH2Cl2, ꢀ78 °Cꢀrt; (c) BAIB, CH2Cl2, H2O,
TEMPO, rt; (d) MeI, Cs2CO3, DMF; (e) NIS, AcOH, CH2Cl2, 20 °C; (f) H2, 20% Pd(OH)2/C, MeOH, 1 N HCl or (g) H2, 10% Pd/C, MeOH, 1 N
HCl; (i) p-TSA, MeOH, CH2Cl2; (j) p-OMe-PhCH(OMe)2, 10-camphorsulfonic acid, 50 °C; (k) SnCl4, TMSN3, CH2Cl2, 0 °C.
Scheme 2a
a Reagents and conditions: (a) NaH, BnBr, DMF, 0 °Cꢀrt; (b) PhCH(OMe)2, p-TSA, DMF, rt; (c) Ba(OH)2, BaO, BnBr, DMF, 0 °Cꢀrt; (d)
Sc(OTf)3, BnOH, DCE, 90 °C; (e) 0.5 M NaOMe, MeOH, 0 °C; (f) PhCH(OMe)2, p-TSA, DMF, rt; (g) Ba(OH)2, BaO, BnBr, DMF, 0 °Cꢀrt; (h)
Et3SiH, CH2Cl2, BF3 Et2O, 0 °C; (i) Ba(OH)2, BaO, propargyl bromide, DMF, 0 °C.
3
was protected as the benzylidene acetal with benzaldehyde
dimethyl acetal and p-TSA in DMF giving a mixture 21(R:β =
1:4) in quantitative yield. The chromatographic separation in
small scale gave the compound 21β in 25% yield, which was
treated with benzyl bromide, Ba(OH)2, and BaO mixture in
DMF to protect the C3 hydroxyl group as the benzyl ether23
affording compound 22 in 80% yield. We next explored the use of
rare earth metal triflates in the formation of the β-benzyl glyco-
side to enhance the yield of the β-selective compound. Despite
their expense, metal triflates have proven to be a highly valuable
tool in a wide range of Lewis acid mediated reactions. The
advantage of using these metals as catalysts is their high level of
acidity and their stability toward moisture. In our revised
Scheme 2, we began the synthesis with the commercially avail-
able β-D-glucosamine pentaacetate (β-derivative) 23. Refluxing
23 with benzyl alcohol and scandium triflate in dichloroethane at
90 °C provided the β-benzyl glycoside of D-glucosamine tetra-
acetate 24 in good yield and with good selectivity.24 Zemplen
deacetylation reaction with sodium methoxide in MeOH af-
forded triol24 20β, which was treated with benzaldehyde dimethyl
acetal and p-toluenesulfonic acid in DMF at room temperature
afforded the corresponding 4,6-O-benzylidene protected benzyl-
N-acetyl D-glucosamine derivative 21β. The C3-hydroxyl group
was protected as benzyl ether to obtain 22 in good yield. Selective
opening of the 4,6-O-benzylidene ring with triethylsilane in the
presence of catalytic BF3 Et2O furnished the selective 6-O-benzyl
3
protected compound 25 in quantitative yield25 and exposing the
4-OH. Propargylation of the C4 hydroxyl group with propargyl
bromide in the presence of Ba(OH)2 and BaO mixture afforded
monosaccharide building block 6 in good yield.
The synthesis of the 3-O-propargyluted GalNAc β-benzyl
glycoside building block is presented in Scheme 3. The 4,6-O-
benzylidene protected glucosamine intermediate 21β, prepared
in Scheme 2, was used to begin this synthesis. The C3 hydroxyl
group was first protected as propargyl ether using propargyl
bromide in the presence of Ba(OH)2 and BaO affording the
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Scheme 3a
a Reagent and conditions: (a) Ba(OH)2, BaO, Propargyl bromide, DMF, rt; (b) Et3SiH, CH2Cl2, BF3 Et2O, 0 °C; (c) Ac2O, pyridine, rt; (d) (i) Tf2O,
3
pyridine, CH2Cl2, ꢀ18 °C; (ii) NaNO2, DMF, rt.
Scheme 4a
monosaccharide 5 and 6 in the presence of CuI and H€unig’s
base in acetonitrile at room temperature for 2 h. The reaction was
monitored by TLC, and the reaction was quenched with satd
NH4Cl.27 After standard workup, the desired disaccharide ana-
logue 1a was obtained in quantitative yield (Scheme 5). The
structure of 1a was confirmed by 1H NMR, which showed a peak
at 7.80 ppm of triazole proton of the linker and 3.85 ppm of
methyl ester of glucuronic acid and the acetyl group at 1.78
ppm from glucosamine indicating the presence of two mono-
saccharide units in the disaccharide motif. The high-resolution
mass spectrometric data at 971.3717 (M þ H) support the
structure of compound 1a
Synthesis of Chondroitin Disaccharide Analogue. Simi-
larly, a 1,3-dipolar cycloaddition reaction was carried out to
a Reagent and conditions: (a) Sc(OTf)3, BnOH, DCE, 90 °C; (b) 0.5 M
couple 5 and 7 in the presence of CuI and H€unig’s base in
NaOMe, MeOH, 0 °C; (c) PhCH(OMe)2, p-TSA, DMF, rt; (d)
acetonitrile and CH2Cl2 (2:2) mixture at room temperature for
Ba(OH)2, BaO, propargyl bromide, DMF, 0 °Cꢀrt; (e) p-TSA, MeOH,
2 h. After standard workup, the disaccharide analogue 2a was
recovered in 85% yield (Scheme 6). The structure of 2a was
rt; (f) Ac2O, pyridine, rt.
propargylated derivative 26. Selective reductive ring opening
using triethylsilane in the presence of acid catalyst, afforded the
6-O-benzyl protected glucosamine derivative 27.25 Epimeriza-
tion of the C-4 hydroxyl group was accomplished through its
conversion to triflate by treatment with trifluoromethane sulfonic
anhydride followed by overnight stirring at room temperature
with sodium nitrite (NaNO2) affording the galactosamine deri-
vative 28 in 40% yield.26 For better identification, both of the C-4
hydroxyl group of compounds 27 and 28 were acetylated with
confirmed by NMR spectroscopy where the triazole proton
appeared at 7.90 ppm, the methyl proton at 3.89 ppm, and
acetate at 1.96 ppm, which indicates the presence of two
monosaccharide unit. The high resolution mass spectrometric
data support the structure of compound 2a.
Synthesis of Heparosan and Chondroitin Tetrasaccharide
Analogues. After successful synthesis of disaccharide analogues
our next goal was to synthesize the tetrasaccharide analogues. To
synthesize heparosan tetrasaccharide analogue 3, the disacchar-
ide analogue building block 1a first was needed for transforma-
tion to two disaccharide analogue building blocks, one an anomeric
azido derivative and the other a 4-O-propargyl derivative. These will
then be subjected to click reaction to form the protected tetra-
saccharide analogue that would be ready for global deprotection to
provide the desired tetrasaccharide analogue 3. The propargylation
of the C-4 position of 1a was undertaken using several propargyla-
tion conditions including (1) propargyl bromide, silver oxide in
DMF (resulting in a very sluggish and low yielding reaction);28
(2) Ba(OH)2 and BaO in DMF from 0 °C to room temperature
(resulting in benzoate ester cleavage);29 and (3) NaHMDS and
propargyl bromide at ꢀ78 °C (resulting in no reaction).30 Ulti-
mately, we were able to install the propargyl group in acidic medium
using propargyl trichloroacetimidate, prepared from trichloroaceto-
nitrile and propargyl alcohol in the presence of catalytic amount of
sodium followed by distillation to obtain the fresh acetimidate.31
Treatment of compound 1a with propargyl trichloroacetimidate in
the presence of catalytic trifluoromethane sulfonic acid in dichloro-
methane at room temperature afforded the partial conversion of 1a
acetic anhydride in pyridine to afford compounds 29 (3JH4,H3
=
9.4 Hz) and 30 (3JH4,H3 = 2.7 Hz).
Scheme 3 was revised to synthesize galactoside derivative
more efficiently. Commercially available galactosamine pen-
taacetate 31 was converted into the β-benzyl galactoside 32 in
good yield and high selectivity with scandium triflate and
benzyl alcohol in dichloroethane at 90 °C (Scheme 4).24
Zemplen deacetylation reaction with sodium methoxide fol-
lowed by treatment with benzaldehyde dimethyl acetal and
p-toluenesulfonic acid in DMF at room temperature afforded
the corresponding 4,6-O-benzylidene protected benzyl-N-
acetyl galactosamine derivative 33 in quantitative yield. The
free C3-hydroxyl group was propargyluted with propargyl
bromide in the presence of BaO and Ba(OH)2 to obtain 34
in 85% yield. Cleavage of 4,6-O-benzylidene ring with p-TSA
in MeOH followed by acetylation with Ac2O in pyridine
provided the modified compound 7 in quantitative yield.
Synthesis of Heparosan Disaccharide Analogue. A 1,3-
dipolar cycloaddition reaction was carried out to couple
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Scheme 5a
a Reagent and conditions: (a) CuI, DIPEA, CH3CN, rt.
Scheme 6a
a Reagents and conditions: (a) CuI, DIPEA, CH3CN, CH2Cl2, rt.
Scheme 7a
a Reagents and conditions: (a) propargyl trichloroacetimidate, trifluoromethane sulfonic acid, CH2Cl2, mol sieves powder 4 Å, rt; (b) MOMCl, DIPEA,
DMAP, CH2Cl2, 35 °C; (c) H2,10% Pd/C, MeOH/EtOAC, 1 N HCl (cat.); (d) Ac2O, pyridine, rt; (e) SnCl4, TMSN3, CH2Cl2, 0 °C.
Scheme 8a
a Reagents and conditions: (a) CuI, DIPEA, CH3CN, rt; (b) LiOOH, THF; (c) 2 N NaOH, MeOH; (d) H2, 10% Pd/C, MeOH.
to compound 35. After chromatographic purification afforded the
desired propargylated compound 35 in 45% yield (Scheme 7).
Next, the azido group at anomeric position was installed in
disaccharide 1a. For this, at first the C4 hydroxyl was protected as
its MOM ether derivative (36) using MOMCl and H€unig’s base
with catalytic DMAP. Then, reductive removal of the benzyl
groups under hydrogenation over Pd/C at atmospheric pressure,
followed by acetylation with acetic anhydride and treatment with
trimethylsilyl azide in the presence of stannic chloride at 0 °C
furnished the β-azido compound 37 in 61% yields with in situ
removal of MOM group (Scheme 7). The compound 37 was
confirmed by mass spectrometry and the structure was analyzed
by NMR spectroscopy. The compound is confirmed with a mass
of 809.2502 (Mþ) by the high-resolution mass spectrometry.
Disaccharide analogues 35 and 37 were coupled under 1,
3-dipolar cycloaddition reaction with CuI in acetonitrile for 10 h
to form the tetrasaccharide analogue 38 in 87% yield. The
compound was confirmed with a mass of 1840.6193 (M þ
Na)þ. Finally, basic hydrolysis with LiOOH and 2 N NaOH
and hydrogenation over Pd/C16h produced a tetrasaccharide
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Scheme 9a
a Reagents and conditions: (a) propargyl trichloroacetimidate, trifluoromethane sulfonic acid, CH2Cl2, mol sieves powder 4 Å, rt; (b) MOMCl, DIPEA,
DMAP, CH2Cl2, 35 °C; (c) H2,10% Pd/C, MeOH/EtOAC, 1 N HCl (cat.); (d) Ac2O, pyridine, rt; (e) SnCl4, TMSN3, CH2Cl2, 0 °C.
Scheme 10a
a Reagents and conditions: (a) CuI, DIPEA, CH3CN, rt; (b) LiOOH, THF; (c) 2 N NaOH, MeOH; (d) H2, 10% Pd/C, MeOH.
analogue 3. Complete spectral characterization of 3, including
mass spectral analysis confirmed the synthesis of heparosan
tetrasaccharide analogue (Scheme 8).
analysis confirm the structure of chondroitin tetrasaccharide
analogue.
In a similar way, the construction of chondroitin tetrasaccha-
ride analogue was undertaken from the chondroitin disaccharide
analogue 2a, which was first transformed into two disaccharide
analogues 39 and 41 (Scheme 9). Treatment of compound 2a
with propargyl trichloroacetimidate in the presence of catalytic
trifluoromethane sulfonic acid in CH2Cl2 at room temperature
afforded compound 39. Next, for 1-azido derivative of disaccha-
ride analogue 2a, the C4 hydroxyl was protected with MOMCl
and catalytic DMAP in diisopropylethylamine as solvent to offer
the MOM-protected disaccharide 40. Reductive removal of the
anomeric benzyl groups upon hydrogenation over Pd/C, fol-
lowed by acetylation and treatment with trimethylsilyl azide in the
presence of stannic chloride at 0 °C provided β-azido derivatives 41
as confirmed by mass spectrometry and NMR spectroscopy.
Disaccharide analogues 39 and 41 were coupled under 1,3-
dipolar cycloaddition reaction with CuI in acetonitrile for 14 h at
room temperature to form the tetrasaccharide 42 in 77% yield
(Scheme 10). The compound was confirmed by high-resolution
mass spectrometry based on a peak for [M þ Na] at 1744.5462
amu. Finally, basic hydrolysis with LiOOH in THF and 2 N
NaOH followed by debenzylation under hydrogenation condition
with Pd/C in MeOH, produced tetrasaccharide analogue 4.
Complete spectral characterization of 4, including mass spectral
’ CONCLUSION
In conclusion, a highly effective approach for the preparation
of heparosan and chondroitin disaccharide analogues and tetra-
saccharide analogues bearing the triazole ring as the interglyco-
sidic linker has been described. The key reaction for the assembly
of these oligomers consists of very efficient copper catalyzed
azideꢀalkyne cycloadditions (CuAAC). The efficiency and fide-
lity of this reaction were maintained regardless of the structural
complexity of building blocks to be assembled. Very likely the
1,4-disubstituted triazole ring spacers contribute substantially to
the overall length of these non-natural glycosaminoglycans. This
tetrasaccharide building block could be extended to the higher
oligosaccharide. Our experimental observation could be valuable
for designing and synthesizing a novel drug in a rapid, cost-
efficient manner. Future studies are planned for the chemical
sulfonation of these tetrasaccharide analogues and their biologi-
cal evaluation.
’ EXPERIMENTAL SECTION
General Methods. NMR spectra were recorded on 500 MHz
spectrometer (1H NMR and 13C NMR). Optical rotation was measured
at a concentration of g/100 mL (accuracy (0.002°). Analytical thin-layer
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chromatography was performed on precoated silica gel plates. Visualiza-
tion was performed by ultraviolet light and/or by staining with anisal-
dehyde solution in ethanol. Chromatographic separations were per-
formed on a silica gel column by flash chromatography. Yields are given
after purification, unless differently stated. When reactions were per-
formed under anhydrous conditions, the mixtures were maintained
under nitrogen. Compounds were named following IUPAC rules as
applied by Beilstein-Institute AutoNom software for systematic names
in organic chemistry.
[R]25 = þ40.0 (c 0.11, CHCl3); HRMS-FAB: [M þ Na] calcd for
D
C34H30O8NaSþ 621.1559, found 621.1556.
Methyl-1-O-acetyl-2,3-di-O-benzoyl-4-O-benzyl-1-thio-β-
D-glucuronate (12). To a vigorously stirred solution of thioglycoside
11 (0.11 g, 0.183 mmol) in CH2Cl2 (10 mL) were added NIS (0.041 g,
0.183 mmol) and acetic acid (0.014 mL, 0.183 mmol) at 20 °C. After
TLC analysis showed complete consumption of starting material, the
reaction was quenched with satd aq Na2S2O3 and washed with satd aq
NaHCO3. The organic layer was dried over MgSO4 and concentrated in
vacuo. The crude reaction mixture was purified by column chromatog-
raphy to get corresponding 1-O-acetyl glycosides 12. Yield = 63%; Rf 0.6
(EtOAc/hexane, 3/7); 1H NMR (500 MHz, CDCl3) δ 7.90 (m, 4 H),
7.51 (m, 2 H), 7.38 (m, 4 H), 7.16 (m, 3 H), 7.03 (m, 2 H), 5.99 (d, 1 H, J
= 7.5 Hz), 5.74 (dd, 1 H, J = 8.8, 8.9 Hz), 5.50 (dd, 1 H, J = 8.8, 8.9 Hz),
4.50 (m, 2 H), 4.33 (d, 1 H, J = 8.9 Hz), 4.20 (dd, 1 H, J = 8.8, 8.9 Hz),
3.78 (s, 3 H), 2.05 (s, 3 H), 1.54 (br s, 1 H); 13C NMR (125 MHz,
CD3OD) δ 168.6, 165.5, 136.8ꢀ128.2, 91.2, 76.7, 74.9, 74.6, 73.5, 70.6,
52.8, 20.8; [R]25D = þ46.0 (c 0.13, CHCl3); HRMS-FAB: [M þ Na]
calcd for C30H28NaO10þ 571.1580, found 571.1581.
Phenyl 2,3-Di-O-benzoyl-4,6-O-benzylidene-1-thio-β-D-
glucopyranoside (9). To a solution of compound 8 (2.0 g, 5.54
mmol) in pyridine (20 mL), benzoyl chloride (1.54 mL, 13.31 mmol)
was added at 0 °C, and the mixture was stirred for 3 h at room
temperature. After completion of the reaction by TLC, water was added
and extracted with CH2Cl2. Organic layer was washed with NaHCO3,
water, and brine. The solution was dried over Na2SO4 and concentrated
to get dibenzoyl derivative as solid, which was recrystallized from
ethylacetate and hexane to obtain the pure dibenzoyl derivative 9. Yield
1
= 92%; Rf 0.6 (EtOAc/hexane, 1/4); H NMR (500 MHz, CDCl3) δ
7.98 (d, 2 H, J = 7.9 Hz), 7.93 (d, 2 H, J = 7.4 Hz), 7.52 (t, 1 H, J = 7.4
Hz), 7.48 (m, 3 H), 7.43 (m, 4 H), 7.38ꢀ7.30 (m, 8 H), 5.81 (t, 1 H, J =
9.5 Hz), 5.45 (m, 1 H), 5.48 (t, 1 H, J = 9.2 Hz), 5.05 (d, 1 H, J = 10.1
Hz), 4.47 (dd, 1 H, J = 5.0, 10.4 Hz), 3.98 (q, 2 H, J = 9.2 Hz), 3.76 ((dt, 1
H, J = 4.7, 9.2 Hz); 13C NMR (125 MHz, CDCl3) δ 165.6, 165.2, 136.6,
133.3, 129.9ꢀ126.1, 101.4, 87.0, 78.6, 73.2, 71.0, 70.9, 68.5; HRMS-
FAB: [M þ Na] calcd for C33H28O7NaSþ 591.1453, found 591.1446.
Phenyl 2,3-Di-O-benzoyl-4-O-benzyl-1-thio-β-D-glucopyra-
noside (10). To a solution of compound 9 (1.8 g, 3.16 mmol) in dry
CH2Cl2 (35 mL) were added PhBCl2 (0.66 mL, 5.06 mmol) and Et3SiH
(0.70 mL, 4.42 mmol) at ꢀ78 °C, and the reaction was allowed to warm to
room temperature. After 30 min, reaction mixture was quenched with Et3N
and MeOH. The reaction was diluted with CH2Cl2 and washed with aq
NaHCO3, and the organic layer was dried over anhydr Na2SO4 and con-
centrated in vacuo. The resulting residue was purified by column chroma-
tography to afford compound 10. Yield = 87%; Rf 0.5 (EtOAc/hexane, 3/7);
1H NMR (500 MHz, CDCl3) δ 7.95 (m, 2 H), 7.95 (m, 2 H), 7.50ꢀ7.43
(m, 4 H), 7.38 (m, 4 H), 7.31ꢀ7.29 (m, 3 H), 7.16 (m, 5 H), 5.79 (t, 1 H, J
= 9.4 Hz), 5.40 (t, 1 H, J= 9.2 Hz), 5.01 (d, 1 H, J=10.1Hz), 4.60(m, 2H),
3.97 (dd, 1 H, J = 2.6, 12.1 Hz), 3.93 (t, 1 H, J = 9.6 Hz), 3.81 (dd, 1 H, J =
4.1, 12.1 Hz), 3.66 (m, 1 H); 13C NMR (125 MHz, CDCl3) δ 165.7, 165.3,
137. 1, 133.3ꢀ127.9, 86.1, 79.6, 76.2, 75.4, 74.8, 70.8, 61.7; HRMS-FAB:
[M þ Na] calcd for C33H30O7NaSþ 593.1610, found 593.1604.
Phenyl 2,3-Di-O-benzoyl-1-thio-β-D-glucopyranoside (14).
To a solution of dibenzoylated compound 9 (3.0 g, 5.27 mmol) in
MeOH and CH2Cl2 (4:1) (30 mL) was added p-TSA (0.50, 2.63 mmol),
and the mixture was stirred at 35 °C for 8 h. The reaction mixture was
neutralized with NaHCO3, solvent was removed under reduced pres-
sure, and the reaction mixture was partitioned between water (50 mL)
and CH2Cl2 (80 mL). The aqueous layer was washed with CH2Cl2, and
the organic layer was dried over Na2SO4 and concentrated to get the diol
which was purified through column chromatography eluting with
EtOAc/hexane (1/1) to get pure diol 14. Yield = 83%; Rf 0.1
1
(EtOAc/hexane, 4/1); H NMR (500 MHz, CDCl3) δ 7.98 (m, 2
H), 7.95 (m, 2 H), 7.53ꢀ7.50 (m, 2 H), 7.46ꢀ7.44 (m, 2 H), 7.40ꢀ7.34
(m, 4 H), 7.31ꢀ7.29 (m, 3 H), 5.43 (m, 2 H), 4.97 (d, 1 H, J = 9.6 Hz),
4.01 (dd, 1 H, J = 3.2, 12.0 Hz), 3.94 (t, 1 H, J = 9.2 Hz), 3.90 (dd, 1 H, J =
4.8, 12.0 Hz), 3.64ꢀ3.61 (m, 1 H), 3.14ꢀ3.11 (m, 1 H), 2.33 (br s, 1 H);
13C NMR (125 MHz, CD3OD) δ 167.4, 165.2, 133.5ꢀ127.4, 87.0, 80.0,
78.1, 70.1, 69.6, 62.3; [R]25D = þ96.0 (c 1.7, CHCl3); HRMS-FAB: [M
þ Na] calcd for C26H24O7NaSþ 503.1140, found 503.1142.
Phenyl 2,3-Di-O-benzoyl-4,6-O-p-methoxy-benzylidene-
1-thio-β-D-glucopyranoside (15). To a solution of compound 14
(2.0 g, 4.16 mmol) in dry DMF (25 mL) were added anisaldehyde
dimethyl acetal (1.13 mL, 6.24 mmol) and 10-camphorsulfonic acid
(0.19 g, 0.832 mmol), and the mixture was stirred at 50 °C for 1 h. After
completion of the reaction by TLC, the mixture was neutralized with
NaHCO3, water was added, the mixture was extracted with EtOAc, the
organic layer was washed with water and brine, and the solution was
dried over anhydr Na2SO4 and concentrated to obtain the p-methox-
ybenzylated derivative 15 as solid, which was purified by column
chromatography. Yield = 89%; Rf 0.6 (EtOAc/hexane, 1/4); 1H NMR
(500 MHz, CDCl3) δ 7.98 (m, 2 H, J = 1.2 Hz), 7.94 (m, 2 H), 7.54 (m, 2
H), 7.48ꢀ7.46 (m, 3 H), 7.39 (m, 3 H), 7.33 (m, 5 H), 6.84 (m, 2 H),
5.81 (t, 1 H, J = 9.0 Hz), 5.50 (s, 1 H), 5.48 (dd, 1 H, J = 9.2, 10.1 Hz),
5.05 (d, 1 H, J = 9.6 Hz), 4.47 (dd, 1 H, J = 5.0 Hz), 3.96ꢀ3.85 (m, 3 H),
3.78 (s, 3 H; 13C NMR (125 MHz, CD3OD) δ 165.6, 165.2, 160.1,
Methyl-phenyl 2,3-Di-O-benzoyl-4-O-benzyl-1-thio-β-D-
glucuronate (11). To a vigorously stirred solution of thioglycoside
10 (1.5 g, 2.62 mmol) in CH2Cl2 (20 mL) and H2O (20 mL) were
added TEMPO (0.082 g, 0.524 mmol) and BAIB (2.11 g, 6.57 mmol).
After 1 h the reaction mixture was quenched by the addition of aqueous
Na2S2O3 solution (50 mL) and aqueous satd NaHCO3 (50 mL). The
mixture was then extracted twice with EtOAc (2 ꢁ 30 mL), and the
combined organic phase was dried (MgSO4), filtered, and concentrated.
Flash column chromatography using EtOAc/MeOH (9:1) afforded the
glycuronic acid. To a solution of acid (1.4 g, 2.39 mmol) in DMF
(20 mL) was added Cs2CO3 (3.11 g, 9.57 mmol), and the mixture was
cooled to 0 °C. Then was added CH3I (0.223 g, 3.58 mmol), and the
mixture was stirred for 45 min, quenched with water, extracted with
EtOAc, washed with brine, dried over anhydr Na2SO4, and concentrated
in vacuo. The crude reaction mixture was purified by column chroma-
tography to get 11 as thick liquid. Yield = 60% (2-steps); Rf 0.5 (EtOAc/
hexane, 1/4); 1H NMR (500 MHz, CDCl3) δ 7.95 (m, 2 H), 7.91 (m, 2
H), 7.52 (m, 2 H), 7.49 (m, 2 H), 7.46 (m, 4 H), 7.31 (m, 3 H), 7.16 (m,
3 H), 7.09 (m, 2 H), 5.74 (m, 1 H), 5.39 (t, 1 H, J = 9.3, 10.0 Hz), 4.99 (d,
1 H, J = 9.9 Hz), 4.58 (d, 1 H, J = 11.0 Hz), 4.53 (d, 1 H, J = 11.9 Hz),
4.16 (m, 2 H), 3.73 (s, 3 H); 13C NMR (125 MHz, CD3OD) δ 168.4,
165.4, 165.1, 133.3ꢀ127.9, 87.0, 77.2, 75.4, 74.7, 70.2 (ꢁ 2), 52.8;
133.3ꢀ127.4, 113.5, 101.4, 87.0, 78.6, 73.2, 71.0, 70.9, 68.4, 55.2; [R]25
aSþ 635.1716, found 635.1719.
D
= þ64.0 (c 0.8, CHCl3); HRMS-FAB: [M þ Na] calcd for C35H32O8N-
Phenyl 2,3-Di-O-benzoyl-4-O-p-methoxybenzyl-1-thio-β-
D-glucopyranoside (16). Compound 15 (1.95 g, 3.18 mmol) and
flame activated AW-300 molecular sieves were suspended in dry CH2Cl2
(30 mL) for 1 h at rt under argon and then cooled to ꢀ78 °C. PhBCl2
(0.66 mL, 5.09 mmol) and Et3SiH (0.71 mL, 4.45 mmol) were added,
and the reaction was stirred at ꢀ78 °C for 30 min. After that the reaction
mixture was quenched with Et3N and MeOH, the reaction was diluted
with CH2Cl2 and washed with NaHCO3, the organic layer was dried
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over anhydr Na2SO4, and the product was concentrated in vacuo. The
resulting residue was purified by column chromatography to give cor-
responding compound 16. Yield = 71%. Rf 0.4 (EtOAc/hexane, 3/7); 1H
NMR (500 MHz, CDCl3) δ 7.95 (m, 2 H), 7.88 (m, 2 H), 7.52ꢀ7.49
(m, 2 H), 7.45ꢀ7.43 (m, 2 H), 7.38ꢀ7.34 (m, 4 H), 7.31ꢀ7.29 (m, 3
H), 7.06 (d, 2 H, J = 8.6 Hz), 6.84 (d, 2 H, J = 8.7 Hz), 5.71 (t, 1 H, J = 9.0
Hz), 5.34 (t, 1 H, J = 9.2 Hz), 4.96 (d, 1 H, J = 9.6 Hz), 4.50 (ABq, 2 H, J
= 10.9 Hz), 3.97 (dd, 1 H, J = 2.6, 12.1 Hz), 3.88 (t, 1 H, J = 9.6 Hz), 3.77
(dd, 1 H, J = 4.1, 12.1 Hz), 3.70 (s, 3 H), 3.63 (m, 1 H); 13C NMR (125
MHz, CD3OD) δ 165.6, 165.2, 159.3, 133.3ꢀ113.7, 86.1, 79.6, 76.2,
13C NMR (125 MHz, CDCl3) δ 168.4, 166.4, 165.0, 133.5ꢀ129.4, 88.6,
76.2, 74.6, 72.4, 71.7, 70.5, 70.2, 53.1; [R]25D = þ45.0 (c 1.3, CHCl3);
þ
HRMS-FAB: [M þ H] calcd for C21H20N3O8 442.1250, found
442.1247.
Benzyl-N-acetyl D-Glucosamine (20). N-Acetyl D-glucosamine
19 (0.5 g, 2.31 mmol) was suspended in DMF (5 mL) and successively
treated with sodium hydride (0.12 g, 3.01 mmol) and benzyl bromide
(0.824 g, 6.93 mmol) at room temperature for 4 h. After standard
workup, the compound was dried and concentrated to obtain compound
20 as a mixture of R:β in a ratio of 1:4. Yield = 77%; Rf 0.6 (MeOHþ/
EtOAc 1/4); HRMS-FAB: [M þ Na] calcd for C15H21NNaO6
334.1267, found 334.1264.
74.0, 74.4, 70.8, 61.7, 55.2; [R]25 = þ62.0 (c 1.8, CHCl3); HRMS-
D
FAB: [M þ Na] calcd for C34H32O8NaSþ 623.1716, found 623.1710.
Methyl-phenyl 2,3-Di-O-benzoyl-4-O-p-methoxybenzyl-
1-thio-β-D-glucuronate (17). To a vigorously stirred solution of
thioglycoside 16 (1.32 g, 2.19 mmol) in CH2Cl2 (15 mL) and H2O
(5 mL) were added TEMPO (0.171 g, 1.09 mmol) and BAIB (1.7 g,
5.49 mmol). The reaction mixture was quenched by the addition of
Na2S2O3 solution (10% in H2O, 25 mL) and NaHCO3 (satd aq 20 mL).
The mixture was then extracted twice with EtOAc (20 mL), and the
combined organic phase was dried (MgSO4), filtered, and concentrated.
Flash column chromatography using EtOAc/petroleum ether afforded
the acids. To a solution of acid (1.18 g, 1.91 mmol) in DMF (25 mL) was
added Cs2CO3 (2.49 g, 7.67 mmol), the mixture was cooled to 0 °C,
CH3I (0.178 mL, 2.86 mmol) was added, and the mixture was stirred for
1 h, quenched with water, extracted with EtOAc, washed with brine,
dried over anhydr Na2SO4, and concentrated in vacuo. The crude
reaction mixture was purified by column chromatography to get 17 as
thick liquid. Yield = 88%; Rf 0.36 (EtOAc/hexane, 3/7); 1H NMR (500
MHz, CDCl3) δ 7.85 (d, 2 H, J = 7.2 Hz), 7.80 (d, 2 H, J = 7.2 Hz), 7.42
(m, 2 H), 7.38 (m, 2 H), 7.29 (m, 4 H), 7.21 (m, 3 H), 6.91 (m, 2 H),
6.56 (m, 2 H), 5.61 (t, 1 H, J = 9.0 Hz), 5.28 (t, 1 H, J = 9.5 Hz), 4.87 (d, 1
H, J = 9.9 Hz), 4.42 (d, 1 H, J = 10.8 Hz), 4.35 (d, 1 H, J = 10.8 Hz), 4.06
(m, 2 H), 3.73 (s, 3 H), 3.62 (s, 3 H); 13C NMR (125 MHz, CDCl3)
δ 168.2, 165.4, 165.2, 159.3, 133.4ꢀ113.7, 86.9, 78.1, 76.6, 75.4, 74.3,
Benzyl 2-Acetamido-4,6-O-benzylidene-2-deoxy-β-D-glu-
copyranoside (21β). Glucosamine pentaacetate 23 (2.46 g, 6.3
mmol) and benzyl alcohol (2.0 mL, 0.0189 mol) were coupled with
Sc(OTf)3 (0.466 g, 0.094 mmol) in dry dichloroethane (25 mL) at
90 °C for 90 min. The reaction mixture was diluted with CH2Cl2, and the
organic layer was washed with water, dried over Na2SO4, and purified by
column chromatography to give benzyl glycoside 24. Yield = 78%. This
compound (2.00 g, 4.57 mmol) was dissolved in dry CH3OH (20 mL)
under a N2-atmosphere, and 0.5 M NaOMe in MeOH (9.1 mL, 4.57
mmol) was added to it. After 3 h the reaction mixture was neutralized
with Amberlite (IR-120 Hþ) and filtered, and the resulting mixture was
concentrated under vacuum to give the crude triol. A solution of the triol
(1.00 g, 3.21 mmol), which was dissolved in dry DMF (30 mL), was
treated with p-TsOH (0.018 mg, 0.096 mmol) and benzaldehyde
dimethyl acetal (0.721 mL, 4.81 mmol). After 12 h at room temperature,
the mixture was treated with NaHCO3 (1 mL) and evaporated under
reduced pressure. The residue was purified by column chromatography
to afford 21β as a white solid. Yield = 81%; Rf 0.41 (EtOAc/hexane, 3/
2); 1H NMR (500 MHz, CDCl3) δ 7.34ꢀ7.32 (m, 2 H), 7.20ꢀ7.13 (m,
8 H), 5.41 (s, 1 H), 4.72 (d, 1 H, J = 12.0 Hz), 4.46 (d, 1 H, J = 8.2 Hz),
4.43 (d, 1 H, J = 12.0 Hz), 4.20 (dd, 1 H, J = 4.9, 10.4 Hz), 3.68ꢀ3.59 (m,
3 H), 3.40 (t, 1 H, J = 9.2 Hz), 3.29 (dt, 1 H, J = 4.9, 9.5 Hz), 1.85 (s, 3
H); 13C NMR (125 MHz, CD3OD) δ 172.4, 136.9ꢀ126.0, 101.6, 100.2,
81.3, 70.8, 70.6, 68.4, 66.1, 56.8, 23.5; HRMS-FAB: [M þ Na] calcd for
C22H25NNaO6þ 422.1580, found 422.1581.
70.4, 55.2, 52.9; [R]25 = þ51.0 (c 2.2, CHCl3); HRMS-FAB: [M þ
D
Na] calcd for C35H32O9NaSþ 651.1665, found 651.1667.
Methyl-1-O-acetyl-2,3-di-O-benzoyl-4-O-p-methoxybenzyl-
1-thio-β-D-glucuronate (18). To a vigorously stirred solution of
thioglycoside 17 (0.96 g, 1.52 mmol) in CH2Cl2 (15 mL) were added
N-iodosuccinimide (NIS) (343 mg, 1.52 mmol) and acetic acid (0.117 mL,
1.52 mmol) at 0 °C. After TLC analysis showed complete consumption of
starting material, the reaction was quenched with satd aq Na2S2O3 and
washed with satd aq NaHCO3. The organic layer was dried over anhydr
MgSO4 and concentrated in vacuo. The crude reaction mixture was treated
with pyridine and Ac2O and stirred for overnight. After removal of pyridine
the crude reaction mixture was purified by column chromatography to
obtain the β-acetyl glycoside 18. Yield = 61%; Rf 0.4 (EtOAc/hexane, 3/7);
1H NMR (500 MHz, CDCl3) δ 7.91 (m, 2 H), 7.52 (m, 2 H), 7.38 (m, 6
H), 7.02 (m, 2 H), 6.67 (m, 1 H), 6.56 (m, 1 H), 5.98 (d, 1 H, J = 7.5 Hz),
5.71 (dd, 1 H, J= 8.9, 9.0 Hz), 5.48 (dd, 1 H, J = 8.9, 9.0 Hz), 4.53 (m, 2 H),
4.44 (d, 1 H, J = 10.9 Hz) 4.32 (d, 1 H, J = 9.1 Hz), 3.73 (s, 3 H), 3.62 (s, 3
H), 2.04 (s, 3 H); 13C NMR (125 MHz, CDCl3) δ 168.8, 168.5, 165.4,
159.3, 133.4ꢀ113.7, 91.2, 76.4, 74.9, 74.2, 73.5, 70.4, 55.2, 52.8, 20.9;
Benzyl 2-Acetamido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-
β-D-glucopyranoside (22). To a mixture of BaO (1.26 g, 8.25 mmol)
and Ba(OH)2 (1.12 g, 3.57 mmol) in N,N-dimethylformamide (20 mL)
containing benzyl bromide (0.68 mL, 4.13 mmol) was added compound
21β (1.1 g, 2.75 mmol) at 0 °C. The mixture was stirred at room
temperature for 18 h, diluted with water, and neutralized with aqueous
formic acid (10%). The white precipitate obtained was filtered, washed
with water, and dried to give compound 22. Yield = 90%; Rf 0.5 (EtOAc/
hexane, 3/2); 1H NMR (500 MHz, CDCl3) δ 7.49 (m, 2 H), 7.46 (m, 2
H), 7.38ꢀ7.29 (m, 11 H), 5.61 (s, 1 H), 4.86 (dd, 2 H, J = 10.6 Hz), 4.71
(d, 1 H, J = 8.0 Hz), 4.64 (d, 1 H, J = 12.0 Hz), 4.58 (d, 1 H, J = 11.6 Hz),
3.92 (t, 1 H, J = 9.2 Hz), 3.84 (t, 2 H, J = 9.5 Hz), 3.74 (t, 2 H, J = 7.5 Hz),
3.48 (m, 2 H), 1.87 (s, 3 H); 13C NMR (125 MHz, CDCl3) δ 175.4,
142.0, 141.0 (ꢁ 3), 132.9ꢀ129.8, 105.1, 104.1, 86.0, 85.4, 78.0, 74.8,
þ
72.5, 69.8, 59.8, 26.5; HRMS-FAB: [M þ Na] calcd for C29H31NNaO6
512.2049, found 512.2046.
[R]25 = þ35.0 (c 0.9, CHCl3); HRMS-FAB: [M þ Na] calcd for
Benzyl 2-Acetamido-3,6-di-O-benzyl-2-deoxy-β-D-gluco-
pyranoside (25). To a solution of compound 22 (0.815 g, 1.66 mmol)
in dry CH2Cl2 (20 mL) at 0 °C was added Et3SiH (0.53 mL, 3.32 mmol)
followed by BF3/Et2O (0.614 mL, 4.98 mmol). After the reaction mixture
stirred at 0 °C for 1 h, it was quenched with saturated NaHCO3 solution
(20 mL) and extracted with CH2Cl2 (2 ꢁ 30 mL). The organic layer was
dried over anhydrous Na2SO4, concentrated under reduced pressure, and
purified through flash silica gel column chromatography to get compound
25 as white solid. Yield = 73%; Rf 0.4 (EtOAc/hexane, 1/1); 1H NMR (500
MHz, CDCl3) δ 7.34ꢀ7.28 (m, 15 H), 5.47 (d, 1 H, J = 5.6 Hz), 4.87 (d, 1
H, J= 6.0 Hz), 4.85 (d, 1 H, J= 9.6 Hz), 4.77 (d, 1 H, J=11.7Hz), 4.69(d, 1
D
C35H32O9NaSþ 651.1665, found 651.1664.
Methyl-2,3-di-O-benzoyl-1-azido-β-D-glucuronate (5). SnCl4
(0.14 mL, 1.24 mmol) and TMSN3 (0.17 mL, 3.11 mmol) were added to
a solution of 18 (900 mg, 1.55 mmol) in dry CH2Cl2 (6 mL), and the
mixture was stirred at room temperature for 3 h. The mixture was
washed successively with aqueous NaHCO3 and water and dried over
Na2SO4. The solvent was removed under reduced pressure to afford 5 as
colorless oil. Yield = 66%; Rf 0.4 (EtOAc/hexane, 2/3); 8.01 (m, 4 H),
7.52 (m, 2 H), 7.43 (m, 4 H), 5.65 (dd, 1 H, J = 9.1, 9.3 Hz), 5.43 (dd, 1
H, J = 9.1, 9.3 Hz), 4.95 (d, 1 H, J = 8.7 Hz), 4.21 (m, 2 H), 3.73 (s, 3 H);
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H, J = 11.7 Hz), 4.58 (m, 3 H), 3.96 (dd, 1 H, J = 8.7, 10.1 Hz), 3.72 (d, 2 H,
J= 4.9 Hz), 3.69 (t, 2 H, J= 4.9 Hz), 3.53 (penta, 1 H, J= 4.7 Hz), 3.41 (m, 1
H), 2.75 (br s, 1 H), 1.85 (s, 3 H); 13C NMR (125 MHz, CD3OD) δ170.4,
138.5ꢀ127.7, 99.2, 80.3, 74.1, 73.7, 73.2, 70.9 (ꢁ 2), 70.6, 56.8, 23.5;
4.96 (d, 1 H, J = 8.3 Hz), 4.88 (d, 1 H, J = 11.9 Hz), 4.59 (m, 2 H), 4.57
(d, 1 H, J = 11.5 Hz), 4.23 (d, 1 H, J = 2.3 Hz), 4.20 (t, 1 H, J = 3.2 Hz),
4.14 (d, 1 H, J = 2.9 Hz), 3.82 (dd, 1 H, J = 5.8, 9.9 Hz), 3.76 (dd, 1 H,
J = 5.8, 9.9 Hz), 3.70 (t, 1 H, J = 5.8 Hz), 4.53 (dd, 1 H, J = 8.3, 10.4 Hz),
2.43 (t, 1 H, J = 2.3 Hz), 1.92 (s, 3 H), 1.62 (br s, 1 H); 13C NMR (125
MHz, CD3OD) δ 171.8, 137.8ꢀ127.5, 99.7, 79.4, 77.2, 74.7, 73.6, 73.3,
70.3, 69.4, 65.2, 56.3, 51.9, 22.7. HRMS-FAB: [M þ Na] calcd for
C25H29NNaO6þ 462.1893, found 462.1884.
þ
HRMS-FAB: [M þ Na] calcd for C29H33NNaO6 514.2206, found
514.2211.
Benzyl 2-Acetamido-3,6-di-O-benzyl-4-O-propargyl-2-deoxy-
β-D-glucopyranoside (6). To a solution of compound 25 (0.5 g, 1.01
mmol) in dry DMF (20 mL) were added Ba(OH)2 (1.3 g, 1.31 mmol)
and BaO (0.046 g, 3.03 mmol), the mixture was cooled to 0 °C, and then
propargyl bromide (0.20 mL, 1.53 mmol) was added and stirred at rt for
10 h. After standard workup compound 6 was obtained as white solid.
Yield = 88%; Rf 0.4 (EtOAc/hexane, 2/3); 1H NMR (500 MHz,
CDCl3/CD3OD) δ 7.30ꢀ7.23 (m, 15 H), 4.80 (d, 1 H, J = 12.0 Hz),
4.73 (d, 1 H, J = 11.3 Hz), 4.64 (d, 1 H, J = 8.1 Hz), 4.60 (m, 4 H), 4.29
(dd, 1 H, J = 2.5, 15.2 Hz), 4.21 (dd, 1 H, J = 2.5, 15.2 Hz), 3.85 (dd, 1 H,
J = 8.4, 10.1), 3.69 (dd, 1 H, J = 1.9, 10.1 Hz), 3.56 (dd, 1 H, J = 8.2, 9.9
Hz), 3.49 (dd, 1 H, J = 8.3, 9.5 Hz), 3.45 (dd, 1 H, J = 1.8, 4.9 Hz), 2.40 (t,
1 H, J = 2.4 Hz), 1.78 (s, 3 H); 13C NMR (125 MHz, CD3OD) δ 171.2,
138.1ꢀ127.6, 99.2 (anomeric), 80.9, 79.6, 78.0, 74.6, 74.5, 74.4, 73.4,
Benzyl 2-Acetamido-3-O-propargyloxy-4-O-acetyl-6-O-ben-
zyl-2-deoxy-β-D-glucopyranoside (29). To a solution of compound
27 (0.075 g, 0.17 mmol) in pyridine (1 mL) was added acetic anhydride
(0.02 mL, 0.22 mmol) at 0 °C, and the mixture was stirred for 3 h. After
completion of the reaction by TLC, water was added and extracted with
CH2Cl2. Organic layer was washed with NaHCO3, water, and brine. The
solution was dried over Na2SO4 and concentrated to get 4-acetyl
1
derivative 29 as oil. Yield = 85%; Rf 0.5 (EtOAc/hexane, 1/1); H
NMR (500 MHz, CDCl3) δ 7.33 (m, 10 H), 5.76 (s, 1 H), 5.06 (d, 1 H,
J = 8.3 Hz), 4.93 (d, 2 H, J = 9.3 Hz), 4.90 (d, 1 H, J = 11.8 Hz), 4.59 (d, 1
H, J = 11.8 Hz), 4.54 (m, 2 H), 4.27 (m, 4 H), 3.68 (q, 1 H, J = 9.7 Hz),
3.28 (m, 1 H), 2.41 (t, 1 H, J = 2.3 Hz), 1.98 (s, 3 H); 1.94 (s, 3 H); 13C
NMR (125 MHz, CDCl3) δ 169.2 (ꢁ 2), 137.9ꢀ127.5, 99.8, 79.9, 74.4,
73.6, 73.2, 72.4, 71.8, 64.8, 60.2, 57.7, 54.3, 21.0, 20.7; HRMS-FAB: [M
þ Na] calcd for C27H31NNaO7þ 504.1998, found 504.1996.
70.5, 69.1, 59.6, 56.0, 22.9; [R]25 = þ35.0 (c 0.3, CHCl3); HRMS-
D
FAB: [M þ Na] calcd for C32H35NNaO6þ 552.2362, found 552.2356.
Benzyl 2-Acetamido-3-O-propargyl-4,6-O-benzylidene-2-
deoxy-β-D-glucopyranoside (26). Following the procedure used
for compound 6, compound 26 was obtained from compound 21β as a
white solid. Yield = 83%. Rf 0.3 (EtOAc/hexane, 3/2); 1H NMR (500
MHz, CDCl3) δ 7.25 (m, 2 H), 7.14ꢀ7.10 (m, 8 H), 5.37 (s, 1 H), 4.67
(d, 1 H, J = 12.0 Hz), 4.60 (d, 1 H, J = 8.3 Hz), 4.39 (d, 1 H, J = 12.0 Hz),
4.20ꢀ4.14 (m, 4 H), 3.78 (t, 1 H, J = 9.5 Hz), 3.67 (t, 1 H, J = 10.3 Hz),
3.45 (q, 1 H, J = 8.5 Hz), 3.30 (m, 1 H), 2.34 (m, 1 H), 1.75 (s, 3 H); 13C
NMR (125 MHz, CD3OD) δ 171.7, 136.9ꢀ125.7, 101.6, 100.0, 81.9,
79.7, 76.8, 74.2, 70.8, 68.4, 65.6, 59.2, 55.5, 22.4; [R]25D = þ40.0 (c 0.09,
CHCl3); HRMS-FAB: [M þ Na] calcd for C25H27NNaO6þ 460.1736,
found 460.1733.
Benzyl 2-Acetamido-3-O-propargyloxy-4-O-acetyl-6-O-
benzyl-2-deoxy-β-D-galactopyranoside (30). Using the same
procedure as described form the synthesis of compound 29, compound
30 was obtained from compound 28. Yield = 88%; Rf 0.55 (EtOAc/
hexane, 1/1); 1H NMR (500 MHz, CDCl3) δ 7.33 (m, 10 H), 5.60 (s, 1
H), 5.59 (d, 1 H, J = 2.7 Hz), 5.05 (d, 1 H, J = 8.2 Hz), 4.90 (d, 1 H, J =
12.0 Hz), 4.60 (dd, 2 H, J = 10.6, 11.2 Hz), 4.47 (d, 1 H, J = 11.9 Hz),
4.28 (dd, 1 H, J = 2.9, 10.9 Hz), 4.20 (dd, 1 H, J = 2.9, 10.9 Hz), 4.19 (d, 1
H, J = 1.8, 5.9 Hz), 3.83 (t, 1 H, J = 6.3 Hz), 3.58 (dd, 1 H, J = 7.1, 9.3
Hz), 3.46 (q, 1 H, J = 10.3 Hz), 3.47 (m, 1 H), 2.40 (t, 1 H, J = 2.3 Hz),
2.42 (s, 3 H), 1.93 (s, 3 H); 13C NMR (125 MHz, CDCl3) δ 170.7,
170.4, 137.6ꢀ127.9, 99.2, 79.5, 74.8, 73.6, 72.0, 71.2, 67.9, 65.4, 56.7,
Benzyl 2-Acetamido-3-O-propargyl-6-O-benzyl-2-deoxy-
β-D-glucopyranoside (27). To a solution of compound 26 (0.62 g,
1.41 mmol) in dry CH2Cl2 (20 mL) at 0 °C was added Et3SiH
(0.452 mL, 2.8 mmol) followed by BF3/Et2O (0.52 mL, 4.23 mmol).
After the reaction mixture stirred at 0 °C for 1 h, it was quenched with
saturated NaHCO3 solution (20 mL) and extracted with CH2Cl2 (2 ꢁ
30 mL). The organic layer was dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The compound was purified
through flash column chromatography to get pure compound 27. Yield
= 77%; Rf 0.26 (EtOAc/hexane, 4/6); 1H NMR (500 MHz, CDCl3) δ
7.28 (m, 5 H), 7.23 (m, 5 H), 4.80 (d, 1 H, J = 12.0 Hz), 4.62 (d, 1 H, J =
8.3 Hz), 4.54 (m, 3 H), 4.31 (m, 2 H), 3.75 (dd, 1 H, J = 2.9, 10.7 Hz),
3.66 (q, 1 H, J = 5.2 Hz), 3.67 (m, 1 H), 3.47 (m, 1 H), 3.42 (m, 1 H),
3.40 (m, 1 H), 2.34 (d, 1 H, J = 2.3 Hz), 1.87 (s, 3 H); 13C NMR (125
MHz, CD3OD) δ 171.4, 137.8ꢀ127.6, 99.3, 80.7, 80.3, 74.7, 74.3, 73.6,
71.5, 70.5, 69.6, 59.5, 55.5, 23.0; [R]25D = þ31.0 (c 0.2, CHCl3); HRMS-
FAB: [M þ Na] calcd for C25H29NNaO6þ 462.1893, found 462.1887.
Benzyl 2-Acetamido-3-O-propargyl-6-O-benzyl-2-deoxy-
β-D-galactopyranoside (28). To a stirred solution of trifluorometha-
nesulfonic anhydride (0.096 mL, 0.568 mmol) in CH2Cl2 (10 mL)
at ꢀ18 °C were added dropwise pyridine (0.09 mL) diluted with CH2Cl2
(1 mL) and then a solution of compound 27 (0.125 g, 0.284 mmol) in
CH2Cl2 (45 mL). After 30 min, the mixture was diluted with CH2Cl2,
washed with 2 M HCl, satd aq NaHCO3 and with water, dried (MgSO4),
and evaporated. The residue containing the 4-O-triflate derivative was
treated with NaNO2 (0.195 g, 2.84 mmol) in DMF (12 mL) under
stirring at room temperature for 1 h. The crude mixture was diluted with
CH2Cl2, washed with water, dried (Na2SO4), and evaporated. The
residue was purified to give 28. Yield = 40%. Rf 0.2 (EtOAc/hexane,
4/6); 1H NMR (500 MHz, CDCl3/CD3OD (2:1)) δ 7.33 (m, 10 H),
þ
54.3, 23.5, 20.7; HRMS-FAB: [M þ Na] calcd for C27H31NNaO7
504.1998, found 504.1995.
β-D-Benzyl-N-acetyl-galactosamine Triacetate (32). To a
solution of β-D-N-acetyl-galactosamine tetraacetate 31 (820 mg, 2.3
mmol) in dichloroethane (5 mL) were added benzyl alcohol (0.66 mL,
0.006 mmol) and Sc(OTf)3 (0.15 mg, 0.031 mmol), and the mixture was
heated to reflux at 90 °C for 90 min. After cooling, the reaction mixture
was washed with sat aq NaHCO3, dried with anhydr MgSO4, concen-
trated, and purified by column chromatography to obtain the benzyl
galactoside 32 as a white powder. Yield = 81%; Rf = 0.37 (EtOAc); 1H
NMR (500 MHz, CDCl3) δ 7.34ꢀ7.29 (m, 5 H), 5.36 (m, 1 H), 5.34
(dd, 1 H, J = 1.0, 3.3 Hz), 5.19 (dd, 1 H, J = 3.3, 11.2 Hz), 4.90 (dd, 1 H, J
= 12.0 Hz), 4.65 (m, 2 H), 4.21ꢀ4.15 (m, 1 H), 4.10ꢀ4.09 (m, 1 H),
3.89 (dt, 1 H, J = 1.0, 6.5 Hz), 2.04 (s, 3 H), 1.98 (s, 3 H), 1.90 (s, 3 H);
13C NMR (125 MHz, CDCl3) δ 170.4, 170.4, 170.3 (ꢁ 2),
136.9ꢀ128.0, 99.6, 70.7, 70.6, 69.9, 66.7, 61.5, 51.þ3, 23.5, 20.7, 20.6;
HRMS-FAB: [M þ Na] calcd for C21H27NNaO9 460.1584, found
460.1587.
Benzyl 2-Acetamido-4,6-O-benzylidene-2-deoxy-β-D-ga-
lactopyranoside (33). Using the same procedure as described for
the synthesis of glucosamine derivative 21β, benzylidine galactoside 33
was obtained from the β-D-benzyl-N-acetyl-galactosamine triacetate 32.
Yield = 77%; Rf 0.2 (EtOAc/hexane, 7/3); 1H NMR (500 MHz, CDCl3:
CD3OD (2:1)) δ 7.34 (m, 2 H), 7.16 (m, 8 H), 5.41 (s, 1 H), 4.74 (d, 1
H, J = 12.0 Hz), 4.44 (d, 1 H, J = 12.0 Hz), 4.40 (d, 1 H, J = 8.3 Hz), 4.15
(dd, 1 H, J = 1.9, 12.0 Hz), 3.68ꢀ3.59 (m, 3 H), 3.93 (dd, 1 H, J = 1.8,
12.4 Hz), 3.83 (dd, 1 H, J = 8.3, 10.8 Hz), 3.60 (dd, 1 H, J = 3.6, 10.8 Hz),
3.91 (m, 1 H), 1.77 (s, 3 H); 13C NMR (125 MHz, CDCl3:CD3OD
(2:1)) δ 172.2, 137.4ꢀ126.2, 101.1, 99.7, 75.3, 70.2 (ꢁ 2), 68.9, 66.4,
3190
dx.doi.org/10.1021/jo200076z |J. Org. Chem. 2011, 76, 3181–3193

The Journal of Organic Chemistry
ARTICLE
52.8, 22.5; HRMS-FAB: [M þ Na] calcd for C22H25NNaO6þ 422.1580,
found 422.1583.
2 H), 4.22 (d, 1 H, J = 6.4 Hz), 4.20 (d, 1 H, J = 6.9 Hz), 3.89 (s, 3 H),
3.69 (m, 1 H), 2.14 (s, 3 H), 2.10 (s, 3 H), 1.96 (s, 3 H); 13C NMR (125
MHz, CDCl3) δ 170.9, 170.5, 170.4, 168.1, 165.6, 165.4, 145.3,
137.2ꢀ127.7, 121.6, 99.9, 85.8, 74.4, 71.6, 70.5, 70.4, 70.2, 70.0, 64.6,
Benzyl 2-Acetamido-3-O-propargyl-4,6-O-benzylidene-2-
deoxy-β-D-galactopyranoside (34). Using the same procedure as
described form the synthesis of glucosamine derivative 26, compound
34 was obtained from compound 33. Yield = 85%. Rf 0.35 (EtOAc/
hexane, 7/3); 1H NMR (500 MHz, CDCl3:CD3OD (2:1)) δ 7.44 (m,
2 H), 7.25 (m, 8 H), 5.49 (s, 1 H), 4.96 (d, 1 H, J = 8.3 Hz), 4.83 (d, 1 H,
J = 11.9 Hz), 4.51 (d, 1 H, J = 11.8 Hz), 4.28 (dd, 1 H, J = 1.5, 3.2 Hz),
4.24 (dd, 1 H, J = 1.9, 9.3 Hz), 4.16 (t, 2 H, J = 1.8 Hz), 4.08 (dd, 1 H,
J = 1.5, 12.2 Hz), 3.53 (t, 1 H, J = 9.6 Hz), 3.44 (m, 1 H), 3.30 (m, 1 H),
2.42 (t, 1 H, J = 2.3 Hz), 1.85 (s, 3 H); 13C NMR (125 MHz, CDCl3:
CD3OD (2:1)) δ 171.7, 137.5ꢀ126.0, 101.6, 98.8, 79.7, 74.9, 74.2, 72.6,
70.7, 69.2, 66.3, 56.3, 53.1, 22.9; [R]25D = þ25.0 (c 0.1, CHCl3); HRMS-
FAB: [M þ Na] calcd for C25H27NNaO6þ 460.1736, found 460.1735.
Benzyl 2-Acetamido-3-O-propargyl-4,6-di-O-acetyl-2-deoxy-
β-D-galactopyranoside (7). To a solution of compound 34 (0.62 g,
1.41 mmol) in MeOH was added p-TSA (0.053 g, 0.028 mmol), and the
mixture was stirred for 5 h. The product was neutralized with triethylamine,
concentrated, and purified through column chromatography to obtain the
diol derivative, which was dissolved in pyridine (5 mL) and acetic anhydride
(0.531 mL, 5.64 mmol). After stirring at rt for 4 h, pyridine was removed
under reduced pressure and purified through column chromatography using
EtOAc and hexane to obtain pure compound 7. Yield = 86%; Rf 0.35
(EtOAc/hexane, 7/3); 1H NMR (500 MHz, CDCl3) δ 7.33ꢀ7.26 (m, 5
H), 5.59 (d, 1 H, J = 7.2 Hz), 5.36 (d, 1 H, J = 3.2 Hz), 511 (d, 1 H, J = 8.3
Hz), 4.90 (d, 1 H, J = 12.0 Hz), 4.60 (d, 1 H, J = 11.8 Hz), 4.34 (dd, 1 H,
J= 2.9, 10.9 Hz), 4.18- 4.15 (m, 4 H), 3.88 (t, 1 H, J=6.5Hz), 3.41(m, 1H),
2.42 (m, 1 H), 2.12 (s, 3 H), 2.08 (s, 3 H), 1.93 (s, 3 H); 13C NMR (125
MHz, CDCl3) δ 170.7, 170.5, 170.4, 136.9ꢀ127.9, 98.9, 79.3, 74.9, 73.7,
71.2, 70.6, 65.4, 62.0, 56.7, 54.3, 23.5, 20.7, 20.6; [R]25D = þ31.0 (c 0.35,
62.2, 61.7, 53.1, 51.9, 22.9, 20.9, 20.7 (ꢁ 2); [R]25 = þ30.0 (c 0.06,
D
CHCl3); HRMS-FAB: [M þ Na] calcd for C43H46N4NaO16þ 897.2807,
found 897.2805.
4-O-Propargyl-heparosan Disaccharide Analogue 35. To a
mixture of propargyl alcohol (1.00 g, 17.8 mmol) and trichloroacetoni-
trile (2 mL, 19.6 mmol) was added a catalytic amount of sodium at 0 °C.
After stirring for 3 h at rt, the reaction mixture was quenched with 0.2 mL
of acetic acid, diluted with ether, washed with brine, and dried over
anhydr MgSO4. The solvent was evaporated at reduced pressure, and the
residue was distilled (100 °C, 5 mmHg) to afford the corresponding
trichloroacetimidate (2.30 g, 65%, colorless oil). To a solution of this
trichloroacetimidate (52 mg, 0.262 mmol) and compound 1a (85 mg,
0.087 mmol) in CH2Cl2 (5 mL) were added a catalytic amount of
trifluoromethanesulfonic acid and molecular sieves powder at room
temperature. After stirring overnight, the mixture was filtered through a
pad of Celite, washed with NaHCO3, dried over anhydrous MgSO4, and
evaporated. The residue was purified by flash chromatography to afford
compound 35. Yield = 45%; Rf 0.6 (MeOH/CH2Cl2, 1/2.4); 1H NMR
(500 MHz, CDCl3) δ 7.95 (d, 1 H, J = 7.5 Hz), 7.87 (d, 1 H, J = 7.2 Hz),
7.80 (br s, 1 H), 7.60 (d, 1 H, J = 1.3 Hz), 7.74 (dd, 2 H, J = 8.4, 9.3 Hz),
7.53 (m, 2 H), 7.43 (m, 2 H), 7.38 (m, 3 H), 7.32ꢀ7.28 (m, 13 H), 6.10
(d, 1 H, J = 9.2 Hz), 5.99 (d, 2 H, J = 9.2 Hz), 5.85ꢀ5.79 (m, 3 H), 5.43
(d, 1 H, J = 7.7 Hz), 4.85- 4.79 (m, 2 H), 4.71 (m, 1 H), 4.65ꢀ4.50 (m, 4
H), 4.37ꢀ4.35 (m, 2 H), 4.25 (dd, 1 H, J = 2.5, 3.4 Hz), 3.95 (t, 1 H,
J = 8.1 Hz), 3.81 (s, 3 H), 3.74 (m, 1 H), 3.60 (m, 1 H), 3.53 (m, 2 H),
2.28 (t, 1 H, J = 1.9 Hz), 1.78 (s, 3 H); 13C NMR (125 MHz, CDCl3)
δ 171.0, 167.4, 165.7, 164.7, 145.8, 138.0ꢀ129.4, 120.9, 98.4, 85.7, 80.4,
80.1, 79.3, 78.5, 76.6, 76.7, 75.7, 74.5, 74.1, 73.4, 71.3, 70.4, 68.4, 65.8,
64.3, 59.7, 53.0, 26.4; [R]25D = þ27.0 (c 0.08, CHCl3); HRMS-FAB: [M
þ Na] calcd for C56H56N4NaO14þ 1031.3691, found 1031.3687.
4-O-Methoxymethyl Ether of Heparosan Disaccharide
Analogue 36. To a stirred solution of 1a (75 mg, 0.077 mmol) in
dry CH2Cl2 (1 mL) was added N,N-diisopropylethylamine (0.195 mL,
1.54 mmol), and the mixture was cooled to 0 °C. MOMCl (0.11 mL,
0.154 mmol) was added dropwise, and the mixture was stirred overnight
at 30 °C. Saturated aq NaHCO3, (20 mL) was added, and the two phases
were separated. The organic phase was washed with 5% hydrochloric
acid (2 ꢁ 5 mL) and brine (2 ꢁ 5 mL), dried (MgSO4), and
concentrated to obtain compound 36. Yield = 78%. Rf 0.5 (MeOH/
CH2Cl2,1/2.4);1H NMR (500 MHz, CDCl3) δ 7.95 (m, 2 H), 7.73 (br
s, 1 H), 7.72 (m, 1 H), 7.71 (d, 2 H, J = 1.9 Hz), 7.52 (m, 1 H), 7.43ꢀ7.38
(m, 6 H), 7.32ꢀ7.28 (m, 13 H), 6.10 (d, 1 H, J = 9.2 Hz), 5.84ꢀ5.81 (m,
2 H, J = 9.0 Hz), 5.62 (d, 1 H, J = 7.7 Hz), 4.87 (d, 1 H, J = 12.0 Hz), 4.85
(d, 1 H, J = 12.0 Hz), 4.80 (d, 1 H, J = 4.4 Hz), 4.78 (s, 2 H), 4.66ꢀ4.60
(m, 3 H), 4.57ꢀ4.52 (m, 3 H, J = 12.5 Hz), 4.40ꢀ4.37 (m, 2 H), 3.95
(dd, 1 H, J = 8.1, 9.4 Hz), 3.79 (s, 3 H), 3.77 (d, 1 H, J = 5.4 Hz), 3.73 (m,
1 H), 3.65 (m, 1 H), 3.53 (m, 1 H), 3.18 (s, 3 H), 1.78 (s, 3 H); 13C NMR
(125 MHz, CDCl3) δ 171.0, 167.2, 165.4, 164.6, 145.6, 138.3ꢀ127.8,
121.0, 99.3, 97.8, 85.7, 80.1, 78.5, 77.1, 76.1, 74.6, 74.3, 73.8, 73.3, 70.6,
70.5, 68.8, 65.3, 56.4, 5.2, 53.0, 23.4. [R]25D = þ33.0 (c 0.06, CHCl3);
HRMS-FAB: [M þ Na] calcd for C55H58N4NaO15þ 1037.3796, found
1037.3793.
þ
CHCl3); HRMS-FAB: [M þ Na] calcd for C22H27NNaO8 456.1634,
found 456.1632.
Heparosan Disaccharide Analogue 1a. To a solution of alkyne
derivative 6 (0.143 g, 0.271 mmol) and azide derivative 5 (0.120 g, 0.271
mmol) in acetonitrile (10 mL) were added CuI (0.027 g, 0.5 mmol) and
N,N-diisopropylethylamine (0.103 mL, 0.813 mmol) at rt, and the
mixture was stirred for 1ꢀ2 h. The reaction mixture was diluted with
EtOAc (20 mL), 10 mL of NH4Cl was added and, the aqueous layer was
extracted with EtOAc (3 ꢁ 15 mL), and the combined organic layer was
washed with brine solution, dried over anhydr Na2SO4, and concen-
trated in vacuo to obtain a crude residue that was purified by flash
chromatography to obtain the desired triazole derivative 1a as a solid.
1
Yield = 83%; Rf 0.5 (MeOH/CH2Cl2, 1/2.4); H NMR (500 MHz,
CDCl3) δ 7.84 (m, 2 H), 7.80 (br s, 1 H), 7.60 (m, 2 H), 7.41 (m, 1 H),
7.33 (m, 1 H), 7.28ꢀ7.26 (m, 3 H), 7.21 (m, 5 H), 7.18ꢀ7.15 (m, 11H),
6.10 (d, 1 H, J = 9.2 Hz), 5.71 (dd, 1 H, J = 9.1, 9.2 Hz), 5.60 (dd, 1 H,
J = 9.1, 9.2 Hz), 4.75 (m, 1 H), 4.72 (m, 1 H), 4.65 (d, 1 H, J = 11.2 Hz),
4.52ꢀ4.40 (m, 6 H), 4.29 (d, 1 H, J = 9.7 Hz), 4.16 (dd, 1 H, J = 9.2, 9.6
Hz), 3.85 (s, 3 H), 3.64 (m, 1 H), 3.60 (m, 2 H), 3.49 (m, 2 H), 3.35 (m,
1 H), 1.78 (s, 3 H); 13C NMR (125 MHz, CD3OD) δ 171.2, 167.9,
165.9, 164.7, 145.2, 138.1ꢀ127.5, 121.4, 99.4, 85.6, 81.0, 78.3, 77.5,
74.7, 74.4 (ꢁ 2), 73.4, 70.5, 70.3, 69.6, 68.6, 65.1, 55.6, 52.7, 22.6;
[R]25 = þ37.0 (c 0.09, CHCl3); HRMS-FAB: [M þ H] calcd for
D
C53H55N4O14þ 971.3715, found 971.3717.
Chondroitin Disaccharide Analogue 2a. Using the same
procedure as described for the synthesis of compound 1a, compound
2a was obtained from compound 5 and 7. Yield = 85%; Rf 0.4 (MeOH/
CH2Cl2, 1/2.4); 1H NMR (500 MHz, CDCl3) δ 7.98 (dd, 2 H, J = 1.2,
7.3 Hz), 7.90 (s, 1 H), 7.66 (dd, 2 H, J = 1.2, 7.3 Hz), 7.58 (m, 1 H), 7.51
(m, 1 H), 7.47 (m, 1 H), 7.43 (m, 2 H), 7.30 (m, 4 H), 7.23 (m, 2 H),
6.15 (d, 1 H, J = 9.3 Hz), 6.05 (d, 1 H, J = 8.7 Hz), 5.69 (dd, 1 H, J = 9.3,
Hz), 5.50 (d, 1 H, J = 9.3, Hz), 4.92 (d, 1 H, J = 12.3 Hz), 4.68 (d, 2 H,
J = 2.2 Hz), 4.62 (d, 1 H, J = 12.2 Hz), 4.56 (d, 1 H, J = 8.4 Hz), 4.39 (m,
1-Azido-heparosan Disaccharide Analogue 37. Compound
36 (80 mg, 0.081 mmol) underwent hydrogenation over 10% Pd/C (10
mg) in MeOH (5 mL) in the presence of a catalytic amount of 1 N HCl
(0.1 mL) at room temperature for 2 days to produce triol, which was
treated with acetic anhydride (0.045 g, 0.484 mmol) in pyridine (1 mL)
and stirred for overnight to afford the triacetate. After chromatographic
separation the triacetate (26 mg, 0.03 mmol) was treated with SnCl4
(0.003 mL, 0.03 mmol) and TMSN3 (0.008 mL, 0.06 mmol) were in dry
3191
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The Journal of Organic Chemistry
ARTICLE
CH2Cl2 (6 mL), and the mixture was stirred at room temperature for 3 h.
The mixture was washed successively with aq NaHCO3 and water and
dried over anhydr Na2SO4. The solvent was removed under reduced
pressure to afford 37 as colorless oil. Yield = 61%, Rf 0.45 (MeOH/
CH2Cl2, 1/2.4); 1H NMR (600 MHz, CDCl3) δ 7.98 (m, 2 H), 7.96 (br
s, 1 H), 7.75 (m, 2 H), 7.56 (m, 1 H), 7.50 (m, 1 H), 7.42 (m, 2 H), 7.30
(m, 2 H), 6.16 (d, 1 H, J = 9.3 Hz), 5.91 (dd, 1 H, J = 9.3, 9.5 Hz), 5.79
(m, 1 H), 5.61 (d, 1 H, J = 9.6 Hz), 5.07 (dd, 1 H, J = 8.9, 10.7 Hz), 4.76
(ABq, 2 H, J = 12.2 Hz), 4.50 (d, 1 H, J = 9.1 Hz), 4.45 (dd, 1 H, J = 2.2,
12.1 Hz), 4.39 (m, 2 H), 4.23 (dd, 1 H, J = 4.5, 7.6 Hz), 4.05 (dd, 1 H, J =
9.5, 19.4 Hz), 3.77 (s, 3 H), 3.71 (t, 1 H, J = 9.5 Hz), 3.65 (m, 1 H), 3.42
(br s, 1 H), 2.08 (s, 3 H), 2.00 (s, 3 H), 1.92 (s, 3 H); 13C NMR (125
MHz, CDCl3) δ 171.8, 170.7 (ꢁ 2), 168.1, 166.2, 164.7, 144.6,
133.7ꢀ127.9, 121.7, 88.4, 85.6, 76.8, 75.8, 75.0, 74.7, 74.5, 70.3, 70.2,
65.6, 62.2, 53.4, 52.8, 23.0, 20.9, 20.8; [R]25D = þ35.0 (c 0.07, CHCl3);
HRMS-FAB: [M] calcd for C36H39N7O15þ 809.2504, found 809.2502.
Triazole-Containing Heparosan Tetrasaccharide Ana-
logue 38. To a solution of alkyne 35 (10 mg, 10.0 μmol) and azide
37 (8 mg, 10.0 μmol) in CH3CN were added CuI (1 mg, 5.0 μmol) and
DIPEA (1.0 μL, 60.0 μmol), and the mixture was stirred for 10 h. After a
standard workup followed by purification desired heparosan triazole
derivative 38 was obtained as a solid. Yield = 87%, Rf 0.4 (MeOH/
CH2Cl2, 1/1.9); 1H NMR (500 MHz, CDCl3) δ 7.96 (br s, 1 H), 7.95
(m, 1 H), 7.91 (br s, 1 H), 7.90 (br s, 1 H), 7.87ꢀ7.81 (m, 3 H),
7.73ꢀ7.71 (m, 3 H), 7.68 (m, 3 H), 7.48 (m, 3 H), 7.33 (m, 5 H),
7.28ꢀ7.23 (m, 17 H), 6.16 (d, 1 H, J = 2.0, 9.2 Hz), 6.08 (m, 1 H), 6.05
(m, 1 H), 6.83 (m, 4 H), 5.45ꢀ5.68 (m, 2 H), 4.81 (dd, 2 H, J = 4.7, 12.0
Hz), 4.74ꢀ4.64 (m, 2 H), 4.62 (m, 2 H), 4.57ꢀ4.45 (m, 6 H), 4.35 (m, 4
H), 4.28 (m, 2 H), 3.78 (s, 3 H), 3.76 (m, 1 H), 3.70 (m, 4 H), 3.63 (s, 3
H), 3.58 (m, 3 H), 3.44 (m, 2 H), 2.07 (s, 3 H), 1.96 (s, 3 H), 1.77 (s, 3
H); 13C NMR (125 MHz, CDCl3) δ 171.4, 171.3, 170.7, 170.6, 167.9,
167.4, 165.8, 165.3, 164.7, 164.4, 145.3, 144.5, 144.4, 138.0ꢀ127.4,
121.9 (ꢁ 2), 99.5, 85.6, 85.3, 81.3, 78.2, 77.7, 76.2, 76.1, 76.0, 75.6, 75.3,
74.6, 74.4, 74.3, 74.2, 74.0, 73.2, 65.4, 65.2, 65.0, 64.7, 62.2, 60.0, 55.5,
53.1, 53.0, 52.8, 52.6, 22.4, 22.0, 21.9, 20.4, 20.3 (ꢁ 3); [R]25D = þ31.0
H), 3.63 (t, 1 H, J = 6.5 Hz), 3.21 (s, 3 H), 2.14 (s, 3 H), 1.95 (s, 3 H),
1.82 (s, 3 H); 13C NMR (125 MHz, CDCl3) δ 170.6, 170.5, 170.4, 167.4,
165.3, 165.2, 145.1, 137.2ꢀ127.7, 121.5, 100.0, 97.7, 85.4, 76.9, 75.8,
74.2, 73.4, 71.8, 70.5, 70.1, 64.6, 61.7, 61.2, 56.3, 53.0, 51.7, 22.9, 20.9,
20.7; [R]25D = þ36.0 (c 0.078, CHCl3); HRMS-FAB: [M þ Na] calcd
for C45H50N4NaO17þ 941.3069, found 941.3045.
1-Azido-chondroitin Disaccharide Analogue 41. Using the
same procedure as described for the synthesis of compound 37,
compound 41 was obtained from compound 40. Rf 0.3 (MeOH/
1
CH2Cl2, 1/2.4); H NMR (500 MHz, CDCl3) δ 7.93 (br s, 1 H),
7.86 (m, 2 H), 7.68 (m, 2 H), 7.44 (m, 2 H), 7.30ꢀ7.20 (m, 4 H), 6.39
(d, 1 H, J = 8.4 Hz), 6.18 (dd, 1 H, J = 9.2 Hz), 5.84 (dd, 2 H, J = 9.2, 9.4
Hz), 5.75 (dd, 1 H, J = 9.2, 9.4 Hz), 5.41 (d, 2 H, J = 2.8 Hz), 4.56 (m, 2
H), 4.43 (m, 2 H), 4.34 (t, 1 H, J = 9.4 Hz), 4.07 (dd, 1 H, J = 1.9, 6.3 Hz),
3.84 (dd, 2 H, J = 9.0, 19.4 Hz), 3.80 (s, 3 H), 3.14 (dd, 1 H, J = 3.1, 10.8
Hz), 2.09 (s, 3 H), 1.95 (s, 3 H), 1.82 (s, 3 H); 13C NMR (125 MHz,
CDCl3) δ 171.6, 170.5, 170.4, 168.0, 165.9, 165.6, 145.2, 134.1ꢀ127.7,
121.8, 89.0, 85.8, 74.4, 74.3, 72.7, 71.5, 70.5, 70.1, 64.6, 61.7, 61.2, 53.0,
51.2, 22.9, 20.9, 20.7; [R]25D = þ29.0 (c 0.25, CHCl3); HRMS-FAB: [M
þ Na] calcd for C36H39N7NaO15þ 832.2402, found 832.2391.
Triazole-Containing Chondroitin Tetrasaccharide Ana-
logue 42. Using the same procedure as described for the synthesis
of compound 38, compound 42 was obtained from compound 39 and
1
41. Rf 0.3 (MeOH/CH2Cl2, 1/1.9); H NMR (500 MHz, CDCl3) δ
8.06 (br s, 1 H), 8.04 (br s, 1 H), 8.01 (br s, 1 H), 7.85ꢀ7.74 (m, 8 H),
7.55 (m, 2 H), 7.44 (m, 5 H), 7.31 (m, 5 H), 7.20 (m, 5 H), 7.12 (m, 1
H), 7.04 (m, 1 H), 6.15ꢀ6.11 (m, 2 H), 5.80 (m, 1 H), 5.73 (m, 2 H),
5.63 (m, 1 H), 5.58 (m, 1 H), 5.54 (m, 1 H), 5.53 (m, 1 H), 5.37 (m, 2
H), 4.77 (t, 1 H, J = 12.3 Hz), 4.71 (m, 2 H), 4.64ꢀ4.55 (m, 4 H), 4.49
(m, 2 H), 4.44 (dd, 1 H, J = 8.3, 8.6 Hz), 4.37 (dd, 1 H, J = 5.7, 5.8 Hz),
4.32 (m, 2 H), 4.21 (m, 2 H), 4.08 (m, 2 H), 3.99 (m, 2 H), 3.91 (m, 1
H), 3.84 (m, 1 H), 3.75 (s, 3 H), 3.63 (s, 3 H), 3.41 (m, 1 H), 2.06 (s, 3
H), 1.99 (s, 3 H), 1.98 (s, 3 H), 1.97 (s, 3 H), 1.95 (s, 3 H), 1.89 (s, 3 H);
13C NMR (125 MHz, CDCl3 and CD3OD) δ 171.7, 170.8, 170.7, 170.6,
170.4, 170.0, 167.9, 167.7, 165.8, 165.6, 165.2, 165.0, 145.0, 144.2, 143.7,
136.9ꢀ127.4, 122.6, 122.5, 121.8, 99.9, 87.8, 85.7, 85.2, 77.6, 76.6, 74.8,
74.4, 74.2, 74.0, 73.7, 73.5, 71.6, 71.4, 70.4, 70.2, 69.5, 65.0, 64.5, 64.2,
61.8, 60.9, 52.8, 52.7, 51.7, 51.5, 49.5, 49.3, 22.5 (ꢁ 2), 22.3, 20.4ꢀ20.3;
þ
(c 0.27, CHCl3); HRMS-FAB:[M þ Na] calcd for C92H95N11NaO29
1840.6195, found 1840.6193.
4-O-Propargyl-chondroitin Disaccharide Analogue 39.
Using the same procedure as described for the synthesis of compound
35, compound 39 was obtained from compound 2a. Yield = 49%; Rf 0.65
(MeOH/CH2Cl2,1/2.4). Yield = 49%; Rf 0.65 (MeOH/CH2Cl2, 1/
2.4); 1H NMR (500 MHz, CDCl3) δ 7.94 (br s, 1 H), 7.93 (m, 2 H), 7.57
(m, 2 H), 7.41 (m, 3 H), 7.26ꢀ7.24 (m, 6 H), 7.14 (m, 2 H), 6.16 (d, 1
H, J = 9.2 Hz), 5.82 (t, 2 H, J = 8.7 Hz), 5.66 (t, 1 H, J = 9.0 Hz), 5.45 (br
d, 1 H, J = 2.0 Hz), 4.87 (d, 2 H, J = 12.3 Hz), 4.63 (m, 2 H), 4.57 (d, 1 H,
J = 12.2 Hz), 4.44 (d, 1 H, J = 8.4 Hz), 4.39 (d, 1 H, J = 8.2 Hz), 4.30 (t, 1
H, J = 2.3 Hz), 4.29 (dd, 2 H, J = 2.3, 5.4 Hz), 4.15 (m, 1 H), 3.80 (s, 3
H), 3.62 (m, 1 H), 3.54 (dd, 1H, J = 3.0, 10.3 Hz), 2.31 (t, 1 H, J = 2.5
Hz), 2.14 (s, 3 H), 2.05 (s, 3 H), 1.92 (s, 3 H); 13C NMR (125 MHz,
CDCl3) δ 170.6, 170.5, 170.4, 167.4, 165.3, 165.2, 145.6, 137.2ꢀ127.7,
121.5, 100.8, 85.4, 78.2, 76.9, 76.3, 75.8, 74.3, 73.5, 71.7, 70.5, 70.1, 64.6,
[R]25 = þ36.0 (c 0.08, CHCl3); HRMS-FAB: [M þ Na] calcd for
D
C82H87N11NaO31þ 1744.5467, found 1744.5462.
General Procedure for Saponification of Methyl Esters,
De-O-benzoylation, De-O-acetylation, and Debenzylation.
LiOOH (prepared from 30% solution of H2O2 in water (100 equiv per
CO2Me) and 1 M LiOH (50 equiv per CO2Me)) were added to a
solution of the starting material in THF (0.02 M). The reaction mixture
was stirred at room temperature for 16 h, and then a 2 N solution of
NaOH (1.0 mL) was added until pH 14. The reaction mixture was
stirred for 18 h at room temperature and then neutralized with Amberlite
IR-120 (Hþ) resin, and the solvent was concentrated in vacuo. Next,
10% Pd/C (1 equiv) was added to a solution of the starting material in
MeOH (3 mL for 5 mg) and 1 N HCl (0.1 mL for 5 mg). The mixture
was placed under an atmosphere of hydrogen, and the progress of the
reaction was monitored by TLC (silica gel, CHCl3/MeOH/H2O (20/
5/1). The mixture was filtered, and the residue was washed with H2O
(5 mL). The filtrate was freeze-dried, and the residue was passed through
a short reversed phase chromatography using H2O as the eluent and
freeze-dried to provide the final product.
Unnatural Heparosan Tetrasaccharide Analogue 3. Using
the above-mentioned procedure compound 3 was obtained from
compound 38. Yield = 55%. 1H NMR (500 MHz, D2O) δ 8.21 (br s,
1 H), 8.12 (br s, 2 H), 5.73ꢀ5.68 (m, 4 H), 4.91 (m, 1 H), 4.80 (m, 3 H),
4.70 (m, 2 H), 4.52 (m, 1 H), 4.23 (m, 1 H), 4.20ꢀ4.11 (m, 2 H), 3.97
(t, 2 H, J = 8.5 Hz), 3.70 (m, 2 H), 3.65ꢀ3.59 (m, 7 H), 3.54 (m, 3 H),
3.38 (m, 2 H), 1.89 (s, 3 H), 1.68 (s, 3 H); 13C NMR (125 MHz, D2O)
61.7, 61.3, 59.3, 53.1, 51.8, 23.2, 20.7 (ꢁ 2); [R]25 = þ37.0 (c 0.09,
D
þ
CHCl3); HRMS-FAB: [M þ Na] calcd for C46H48N4NaO16
935.2963, found 935.2954.
4-O-Methoxymethyl Ether of Chondroitin Disaccharide
Analogue 40. Using the same procedure as described for the synthesis
of compound 36, compound 40 was obtained from compound 2a. Yield
= 81%; Rf 0.5 (MeOH/CH2Cl2, 1/2.4); 1H NMR (500 MHz, CDCl3) δ
7.97 (br s, 1 H), 7.95 (m, 2 H), 7.59 (m, 2 H), 7.48 (m. One H), 7.41 (m,
1 H), 7.27 (m, 8 H), 7.14 (m, 1 H, J = 7.2 Hz), 6.16 (d, 1 H, J = 9.2 Hz),
5.84 (dd, 2 H, J = 8.0, 9.0 Hz), 5.75 (t, 1 H, J = 9.3 Hz), 5.48 (d, 2 H,
J = 2.8 Hz), 4.88 (dd, 2 H, J = 12.3 Hz), 4.68 (d, 2 H, J = 11.8 Hz), 4.65
(s, 2 H), 4.58 (d, 1 H, J = 12.2 Hz), 4.48 (t, 3 H, J = 12.4 Hz), 4.39 (t, 1 H,
J = 8.0 Hz), 4.18 (d, 1 H, J = 6.3 Hz), 4.14 (d, 2 H, J = 7.0 Hz), 3.80 (s, 3
3192
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ARTICLE
δ 174.7, 174.5, 174.1, 171.3, 144.0, 143.7, 125.6, 124.6, 123.9, 94.9, 90.6,
86.8, 86.1, 77.9, 77.8, 76.9, 75.3, 74.8, 73.7, 73.5, 73.2, 71.6, 70.6, 70.2,
69.1, 64.4, 64.3, 60.3, 60.1, 59.9, 58.8, 56.6, 55.2, 54.0, 53.1, 21.8, 21.5;
W. FASEB J. 1993, 7, 1233–1242. (e) Fraser, J. R.; Laurent, T. C.;
Laurent, U. B. J. Intern. Med. 1997, 242, 27–33.
(13) (a) Huisgen, R. In 1,3-Dipolar Cycloaddition Chemistry; Padwa,
A., Ed.; Wiley: New York, 1984; Vol. 1, pp 1ꢀ176. (b) Tornoe, C. W.;
Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057–3064. (c)
Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew.
Chemie. Int. Ed. 2002, 41, 2596–2599.
[R]25 = þ23.0 (c 0.5, water); HRMS-FAB: [M þ Na] calcd for
D
C37H53N11NaO23þ 1042.3213, found 1042.3197.
Unnatural Chondroitin Tetrasaccharide Analogue 4. Using
the above-mentioned procedure compound 4 was obtained from
compound 42. Yield = 52%. 1H NMR (800 MHz, D2O) δ 8.21 (br s,
1 H), 8.18 (br s, 2 H), 5.77 (m, 2 H), 5.68 (m, 2 H), 4.80 (m, 1 H), 4.79
(m, 1 H), 4.65ꢀ4.60 (m, 3 H), 4.57 (m, 1 H), 4.33 (t, 1 H, J = 10.2 Hz),
4.26 (m, 3 H), 4.06 (m, 3 H), 3.89 (m, 2 H), 3.79 (t, 1 H, J = 8.5 Hz), 3.73
(m, 2 H), 3.69 (m, 7 H), 3.57 (m, 1 H), 1.89 (s, 3 H), 1.68 (s, 3 H); 13C
NMR (200 MHz, D2O) δ 173.9, 171.3, 170.1, 169.3, 144.5, 144.5, 143.9,
124.7, 124.1, 123.7, 94.9, 90.9, 86.8, 86.8, 78.1, 76.7, 76.6, 75.3, 75.2,
74.9, 70.7, 65.1, 64.4, 64.3, 61.3, 61.2, 61.0, 60.8, 58.8, 56.9, 54.4, 53.2,
52.3, 50.6, 48.9, 21.8, 21.5; [R]25D = þ38.0 (c 0.62, water); HRMS-FAB:
(14) (a) Calvo-Flores, F. G.; Isac-García, J.; Hernꢀandez-Mateo, F.;
Pꢀerez-Balderas, F.; Calvo-Asín, J. A.; Sanchꢀez-Vaquero, E.; Santoyo-
Gonzꢀalez, F. Org. Lett. 2000, 2, 2499–2502. (b) Appukkuttan, P.; Dehaen,
W.; Fokin, V. V.; Van der Eycken, E. Org. Lett. 2004, 6, 4223–4225.
(15) Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.; Sharpless,
K. B.; Finn, M. G. J. Am. Chem. Soc. 2003, 125, 3192–3193.
::
(16) (a) Sinay, P. Biochimie 1988, 70, 1455–1458. (b) Reference 11b.
(c) Karst, N. A.; Linhardt, R. J. Curr. Med. Chem. 2003, 10, 1993–2031.
(d) Lopin, C.; Jacquinet, J. C. Angew. Chem., Int. Ed. 2006, 10, 2574–2578.
(e) Weiwer, M.; Linhardt, R. J. In Comprehensive Glycosciences, Boons, G. J.,
Lee, Y. C., Suzuki, A., Taniguchi, N., Voragen, A. G. J., Kamerling, J. P., Eds.;
Elsevier BV: Amsterdam, 2007; pp 713ꢀ745. (f) Polat, T.; Wong, C.-H.
J. Am. Chem. Soc. 2007, 129, 12795–12800. (g) Rawat, M.; Gama, C. I.;
Matson, J. B.; Hsieh-Wilson, L. C. J. Am. Chem. Soc. 2008, 130, 2959–2961.
(h) Arungundram, S.; Al-Mafraji, K.; Asong, J.; Leach, F. E., III; Amster, I. J.;
Venot, A.; Turnbull, J. E.; G.-J. J. Am. Chem. Soc. 2009, 131, 17394–17405.
(17) Leendert, J.; van den Bos; Jeroen, D. C.; Codꢀee, J. C; van der
Toorn; Boltje, T. J.; van Boom, J. H.; Overkleeft, H. S.; van der Marel,
G. A. Org. Lett. 2004, 6, 2165–2168.
þ
[M þ Na þ H] calcd for C37H53N11NaO23 1043.3292, found
1043.3244.
’ ASSOCIATED CONTENT
S
Supporting Information. NMR spectra for all com-
b
pounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
(18) Anjos, J. V. Dos; Sinou, D.; Melo, S. J. De; Srivastava, R. M.
Carbohydr. Res. 2007, 342, 2440–2449.
’ AUTHOR INFORMATION
(19) Veeneman, G. H.; van Leeuwen, S. H.; Van Boom, J. H.
Tetrahedron Lett. 1990, 31, 1331–1334.
Corresponding Author
*E-mail: linhar@rpi.edu.
(20) Huang, L.; Huang, X. Chem.—Eur. J. 2007, 13, 529–540.
(21) (a) Schmidt, R. R. Angew. Chem., Int. Ed. Engl. 1986, 25, 212–235.
(b) Schmidt, R. R. Pure Appl. Chem. 1989, 61, 1257–1270. (c) Schmidt,
R. R. In Modern Methods in Carbohydrate Synthesis; Khan, S. H., O’Neil,
R. A., Eds.; Harwood: The Netherlands, 1996; pp 20ꢀ54. (d) Klotz, W.;
Schmidt, R. R. Liebigs Ann. Chem. 1993, 683–690.
’ ACKNOWLEDGMENT
The authors are grateful for funding from the National
Institutes of Health in the form of grants AI065786, HL62244,
and HL094463.
(22) Vauzeilles, B.; Dausse, B.; Palmier, S.; Beau, J.-M. Tetrahedron
Lett. 2001, 42, 7567–7570.
(23) Sharma, M.; Bernacki, R. J.; Hillman, M. J.; Korytnyk, W.
Carbohydr. Res. 1990, 198, 205–221.
’ REFERENCES
(24) Christensen, H.; Christiansen, M. S.; Petersen, J.; Jensen., H. H.
Org. Biomol. Chem. 2008, 6, 3276–3283.
(1) McLean, J. Am. J. Physiol. 1916, 41, 250–257.
(2) (a) Capila, I.; Linhardt, R. J. Angew. Chem., Int. Ed. 2002,
41, 391–412. (b) Rabenstein, D. L. Nat. Prod. Rep. 2002, 19, 312–331.
(3) Damus, P. S.; Hicks, M.; Rosenberg, R. D. Nature 1973, 246,
355–357.
(25) Sheryl, D. D.; Eric, J. T. Tetrahedron: Asymmetry 2000,
11, 385–387.
(26) Fernandez-Bolanos, J. G.; Garcia, S.; Fernandez-Bolanos, J.;
Dianez, M. J.; Estrada, M. D.; Lopez-Castro, A.; Perez-Garrido, S.
Tetrahedron: Asymmetry 2003, 14, 3761–3768.
(27) Bera, S.; Zhanel, G. G.; Schweizer, F. Bioorg. Med. Chem. Lett.
2010, 20, 3031–3035.
(28) Ikeda, K.; Sato, K.; Kitani, S.; Suzuki, T.; Maki, N.; Suzukib, Y.;
Sato, M. Bioorg. Med. Chem. 2006, 14, 7893–7897.
(29) Kim, Jin-H.; Huang, F.; Ly, M.; Robert, L. J. J. Org. Chem. 2008,
(4) Rosenberg, R. D.; Damus, P. S. J. Biol. Chem. 1973, 248,
6490–6505.
::
(5) Sinay, P.; Jacquinet, J. C.; Petitou, M.; Duchaussoy, P.; Lederman,
I.; Choay, J.; Torri, G. Carbohydr. Res. 1984, 132, C5–C9.
(6) Petitou, M.; Herault, L. P.; Bernat, A.; Driguez, P. A.; Duchaussoy,
P.; Lormeau, J. C.; Herbert, J. M. Nature 1999, 398, 417–422.
(7) Hardingham, T.; Semin, M. B. Arthritis Rheu. 1990, 20, 12–33.
(8) Fawthrop, F.; Yaqub, R.; Belcher, C.; Bayliss, M.; Ledingham, J.;
Doherty, M. Ann. Rheum. Dis. 1997, 56, 119–122.
73, 9497–9500.
(30) Hudlicky, T.; Moser, M.; Banfield, S. C.; Rinner, U.; Jean-
Charles, C.; George, R. P. Can. J. Chem. 2006, 84, 1313–1337.
(31) Adrio, J.; Carretero, J. C. J. Am. Chem. Soc. 2007, 129, 778–779.
(9) Conte, A.; Volpi, N.; Palmieri, L.; Bahous, I.; Ronca, G. Arzneim.
Forsch. 1995, 45, 918–925.
(10) Gressner, A. M.; Koster-Eiserfunke, W.; Van de Leur, E.;
Greiling, H. J. Clin. Chem. Clin. Biochem. 1980, 18, 279–285.
(11) (a) Reference 2a. (b) Das, S. K.; Mallet, J. M.; Esnault, J.;
Driguez, P. A.; Duchaussoy, P.; Sizun, P.; Herault, J. P.; Herbert, J. M.;
::
Petitou, M.; Sinay, P. Angew. Chem., Int. Ed. 2001, 40, 1670–1673. (c)
Raman, R.; Venkataraman, G.; Ernst, S.; Sasisekharan, V.; Sasisekharan,
R. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 2357–2362. (d) Faham, S.;
Linhardt, R. J.; Rees, D. C. Curr. Opin. Struct. Biol. 1998, 8, 578–586.
(12) (a) Vann, W. F.; Schmidt, M. A.; Jann, B.; Jann, K. Eur. J.
Biochem. 1981, 116, 359–364. (b) Yamagata, T.; Saito, H.; Habuchi, O;
Suzuki, S. J. Biol. Chem. 1968, 243, 1523–1 535. (c) 1Meyer, K.; Palmer,
J. W. J. Biol. Chem. 1934, 107, 629–634. (d) Knudson, C. B.; Knudson,
3193
dx.doi.org/10.1021/jo200076z |J. Org. Chem. 2011, 76, 3181–3193