Synthesis of chondroitin/dermatan sulfate-like oligosaccharides and evaluation of their protein affinity by fluorescence polarization

Article abstract of DOI:10.1039/c3ob40306h

Here, we present a novel approach for the chemical synthesis of chondroitin and dermatan sulfate oligosaccharides. A key point of this strategy is the preparation and use of an N-trifluoroacetyl galactosamine building block containing a 4,6-O-di-tert-butylsilylene group. Glycosylation reactions proceeded in good yields (74-91%) with our protecting group distribution. Using this approach, we have synthesized, for the first time, a chondroitin/dermatan sulfate-like tetrasaccharide that contains both types of uronic acids, d-glucuronic and l-iduronic acid. Moreover, we have employed a fluorescence polarization competition assay to evaluate the interactions between the synthesized oligosaccharides and FGF-2 (basic fibroblast growth factor). Our results show that this method, using standard instrumentation and minimal sample consumption, is a powerful tool for the rapid analysis of the glycosaminoglycan affinity for proteins in solution. The Royal Society of Chemistry.

Full text of DOI:10.1039/c3ob40306h

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Synthesis of chondroitin/dermatan sulfate-like
oligosaccharides and evaluation of their protein
Cite this: Org. Biomol. Chem., 2013, 42,
3510
affinity by fluorescence polarization†
Susana Maza, M. Mar Kayser, Giuseppe Macchione, Javier López-Prados,
Jesús Angulo, José L. de Paz* and Pedro M. Nieto*
Here, we present a novel approach for the chemical synthesis of chondroitin and dermatan sulfate oligo-
saccharides. A key point of this strategy is the preparation and use of an N-trifluoroacetyl galactosamine
building block containing a 4,6-O-di-tert-butylsilylene group. Glycosylation reactions proceeded in good
yields (74–91%) with our protecting group distribution. Using this approach, we have synthesized, for the
first time, a chondroitin/dermatan sulfate-like tetrasaccharide that contains both types of uronic acids,
D-glucuronic and L-iduronic acid. Moreover, we have employed a fluorescence polarization competition
assay to evaluate the interactions between the synthesized oligosaccharides and FGF-2 (basic fibroblast
growth factor). Our results show that this method, using standard instrumentation and minimal sample con-
sumption, is a powerful tool for the rapid analysis of the glycosaminoglycan affinity for proteins in solution.
Received 11th February 2013,
Accepted 21st March 2013
DOI: 10.1039/c3ob40306h
www.rsc.org/obc
Introduction
Chondroitin sulfate (CS) and dermatan sulfate (DS) are highly
heterogeneous and sulfated, linear polysaccharides that
belong to the glycosaminoglycan (GAG) family.1,2 CS is formed
by the repetition of disaccharide units of D-glucuronic acid
(GlcA) and N-acetyl-D-galactosamine (GalNAc), following the
sequence GlcA-β(1→3)-GalNAc-β(1→4). The disaccharide
repeating unit of the structurally related DS mainly contains
L-iduronic acid (IdoA) in place of GlcA. Both polysaccharides
may contain sulfate groups at different positions of the chain
(Fig. 1). These sulfate groups are introduced during the biosyn-
thesis of these polymers, through the action of specific sulfo-
transferases, giving rise to GAG chains with a high level of
structural diversity. The microheterogeneity of CS and DS can
be considered as a capacity to encode information and control
a wide variety of biological processes by specific interactions
with certain proteins.3 As in the case of other members of the
GAG family, such as heparin and heparan sulfate,4–8 it is pro-
posed that defined CS and DS oligosaccharide sequences are
responsible for specific protein recognition and subsequent
activity.3 However, little is known about the exact structural
Fig. 1 Disaccharide repeating units of chondroitin (left) and dermatan (right)
sulfate with potential sites of sulfation indicated.
requirements for these interactions. In this context, synthetic
CS and DS oligosaccharides9–13 are useful tools for the estab-
lishment of structure–activity relationships and the prepa-
ration of mimetics that potentially modulate the biological
functions of the natural products.14–16
Interestingly, CS and DS are often found as co-polymeric
structures.17 These hybrid CS/DS chains, containing both
types of uronic acids, GlcA and IdoA, are involved in growth
factor signalling and neuronal growth and development.17
The interaction between CS/DS and several chemokines and
growth factors, including FGF-2 (basic fibroblast growth
factor), is mediated by oversulfated oligosaccharide sequences
containing GlcA/IdoA–GalNAc (4,6-OSO3).18–21 In order to
study these interactions at the molecular level, it would be
very useful to have access to well-defined synthetic CS/DS
oligosaccharides. However, to the best of our knowledge, the
synthesis of a CS/DS oligomer, containing both GlcA and IdoA,
has not yet been reported.
Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), Centro de
Investigaciones Científicas Isla de La Cartuja, CSIC and Universidad de Sevilla,
Americo Vespucio, 49, 41092 Sevilla, Spain. E-mail: jlpaz@iiq.csic.es;
Fax: +34 954 460565
†Electronic supplementary information (ESI)
available. See DOI:
10.1039/c3ob40306h
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Scheme 2 (a) TEMPO, Bu4NBr, KBr, Ca(ClO)2, NaHCO3, CH2Cl2/H2O, 0 °C; BnBr,
DMF, Bu4NI, 60 °C, 56%; (b) LevOH, DCC, DMAP, CH2Cl2, 81%; (c) CAN, toluene/
CH3CN/H2O, 60%; (d) Cl3CCN, K2CO3, CH2Cl2, 96%; (e) Lev2O, Py/CH2Cl2, DMAP,
98%; (f) (HF)n·Py, THF, 0 °C, 77%; (g) Cl3CCN, K2CO3, CH2Cl2, 95%.
Scheme 1 Building blocks required for the synthesis of tetrasaccharide 1.
Despite significant advances in the field,22–33 the synthesis
of GAG oligosaccharides, including DS and CS, is still challen-
ging, mainly due to the low reactivity of the building blocks
required.34–37 There is still a great demand for efficient syn-
thetic strategies involving a robust and reliable set of carbo-
hydrate building blocks. This is an important point for the
successful automation of oligosaccharide synthesis.33,38–40
Here, we present a novel approach for the synthesis of CS
and DS oligomers that is based on the use of an N-trifluoro-
acetyl-protected galactosamine building block. Glycosylation
reactions proceeded in high yields using our design of protect-
ing groups. The efficiency of this strategy is illustrated with the
total synthesis of the oversulfated tetrasaccharide 1 (Scheme 1)
that contains both IdoA and GlcA and bears sulfate groups at
positions 4 and 6 of the GalNAc units, position 2 of the uronic
acid moieties, and position 4 of the non-reducing terminus.
All these positions may be sulfated in the natural products
(Fig. 1), except position 4 of the non-reducing end. Interest-
ingly, the introduction of a “non-natural” sulfate group at the
non-reducing end did not significantly affect the FGF-2 affinity
of a synthetic heparin hexasaccharide41 and an “artificial”
IdoA monosaccharide, sulfated at positions 2 and 4, showed
considerable binding to several proteins.42,43 Moreover, we
have developed a fluorescence polarization assay to analyse the
binding of tetrasaccharide 1 and its di-O-benzylated precursor
to a model heparin-binding protein, FGF-2. Fluorescence
polarization measurements allowed the study of GAG oligo-
saccharide–protein interactions in solution, with minimal
time and sample consumption, and using a standard fluore-
scence reader. Therefore, our results indicate that this method
can be considered as a powerful tool to evaluate the binding
affinities of synthetic oligosaccharides for receptors of
biological relevance, helping to establish structure–activity
relationships.
2–4 (Scheme 1). Glucuronic acid trichloroacetimidate 2 was
prepared from known diol 5,44 as shown in Scheme 2. Selective
oxidation at position 6 was performed by treatment of the diol
5 with calcium hypochlorite and catalytic TEMPO, under
phase-transfer conditions.44 Strict control of the reaction time
and temperature and quenching with Na2SO3 were required to
avoid the chlorination of the electron-rich 4-methoxyphenyl
ring.45 The carboxylate intermediate was then esterified with
BnBr and Bu4NI in DMF at 60 °C to give the benzyl uronate 6.
Levulinoylation at position 4, followed by oxidative removal of
the 4-methoxyphenyl group with CAN, and trichloroacetimi-
date formation, afforded the glycosyl donor 2. Starting from
diol 9,46,47 iduronic acid trichloroacetimidate 3 was obtained
by levulinoylation, selective desilylation and anomeric acti-
vation with Cl3CCN and K2CO3.
Regarding the galactosamine unit, two types of building
blocks, possessing different protections on the amino group,
have been employed, to date, for the synthesis of CS and DS
oligosaccharides.10 2-Azido-2-deoxy-galactose derivatives11,12,48–51
present some limitations for their general use due to the non-
participating character of the azido moiety. In contrast,
2-deoxy-2-trichloroacetamido-galactose building blocks lead to
the stereoselective formation of the required 1,2-trans glycosi-
dic bond. Impressive synthesis of CS oligomers has been
reported using N-trichloroacetyl(TCA)-protected units.3,32,52–55
However, this amino protecting group is associated with some
problems. For example, we56 and others57 have detected the
formation of stable trichlorooxazoline side products during
glycosidation of 2-deoxy-2-trichloroacetamido donors. More-
over, several difficulties are occasionally encountered in the
final transformation to the desired 2-acetamido group. Thus,
the deprotection of multiple N-TCA groups by basic hydrolysis,
followed by selective N-acetylation, requires very long reaction
times.57 Alternatively, radical reduction using tributylstannane
affords, in some cases, significant amounts of mono- and
dichloroacetamide intermediates.58,59 For these reasons, we
considered the use of an alternative amine-protecting group
for the synthesis of CS/DS oligosaccharides. We chose an N-tri-
fluoroacetyl (TFA) group because it can be easily removed
under mild conditions while ensuring high β selectivities in
glycosylation reactions.60
Results and discussion
Synthesis of chondroitin/dermatan sulfate-like
oligosaccharides
For the synthesis of the CS/DS-related tetrasaccharide 1, we
first prepared the required monosaccharide building blocks
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Scheme 3 (a) 4-Methoxyphenol, TMSOTf, CH2Cl2, 0 °C, 42% + 40% starting
material; (b) NaOMe, MeOH, quantitative; (c) PhCH(OMe)2, p-TsOH, CH3CN,
87%; (d) tBu2Si(OTf)2, Py, 89%; (e) BnNH2, THF; TDSCl, imidazole, CH2Cl2, 51%;
(f) NaOMe, MeOH; PhCH(OMe)2, p-TsOH, CH3CN/DMF; (g) NaOMe, MeOH;
(ClAc)2O, collidine, CH2Cl2, −60 °C, 77%.
Scheme 4 (a) TMSOTf, CH2Cl2, 0 °C, 91%; (b) NH2NH2·H2O, Py–AcOH, CH2Cl2,
84%; (c) TMSOTf, CH2Cl2, 0 °C, 79%.
Thus, we first planned the preparation of an N-TFA-pro-
tected galactosamine unit that should act as an efficient glyco-
syl acceptor in coupling reactions with uronic acid donors. The
synthesis of such a compound was challenging (Scheme 3).
Known tetraacetate 1261,62 was synthesized from galactosamine
hydrochloride in 66% yield by treatment with NaOMe and
then with trifluoroacetic anhydride and Et3N in MeOH, fol-
lowed by extensive acetylation (Ac2O, Py, DMAP). Compound
12 was transformed into 4-methoxyphenyl glycoside 15 by gly-
cosylation with 4-methoxyphenol, followed by de-O-acetylation
and benzylidenation. This compound was an ideal candidate
for our synthetic approach because the 4,6-O-benzylidene
acetal would allow the selective sulfation of these positions at
the end of the synthesis to generate, among others, biologi-
2 gave the desired β (1→3) disaccharide 19 in excellent yield
(Scheme 4). These results highlight the profound effect that
protecting group distribution of the building blocks has on
glycosylation reactions.65 We also performed a glycosylation
reaction between 4 and iduronic acid donor 3. The target α
(1→3) disaccharide 20 was also obtained in high yield. The
small-to-zero coupling constants for IdoA protons indicated
that this residue mainly exists in 1C4 conformation, and the
1
value of the JC,H (172 Hz) confirmed the α configuration of
the new glycosidic linkage.66
Next, we studied the 2 + 2 assembly of the disaccharide
units 19 and 20 to generate a tetrasaccharide sequence con-
taining both GlcA and IdoA. The 4,6-di-tert-butylsilylene
group67 of galactose and galactosamine donors leads to the
selective formation of α glycosides despite the presence of par-
ticipating groups at position 2.68 In some cases, even 4,6-O-
benzylidene derivatives of galactosamine donors may lead to
loss of stereocontrol in coupling reactions with glucuronic
acid-derived acceptors.69 For these reasons, we decided to
transform disaccharide 20 in a suitable protected donor con-
taining less sterically hindered acetyl groups at positions 4 and
6 to obtain the desired 1,2-trans glycoside with excellent stereo-
selectivity. Cleavage of the silylene group gave diol 21
(Scheme 5). The isolation of this compound was tricky because
21 formed gels in several solvents, such as toluene, CH2Cl2
and EtOAc. Therefore, diol 21 was directly acetylated, without
further purification, to yield compound 22. Removal of the
4-methoxyphenyl group followed by treatment with Cl3CCN
and catalytic DBU gave donor 24. On the other hand, 19 was
transformed into acceptor 25 by treatment with hydrazine
monohydrate in a pyridine/acetic acid solution (Scheme 4).
Coupling of disaccharides 24 and 25 gave the target β tetra-
saccharide 26 in high yield. This result demonstrates the
utility of our synthetic route for the assembly of CS and DS
oligosaccharides, paving the way for other sequences with
different sulfation patterns.
cally relevant type
E
sulfation sequences.3 However, 15
suffered from poor solubility in organic solvents such as
dichloromethane, toluene and acetonitrile, and glycosylation
attempts with the uronic acid donor 263 in THF or THF–
CH2Cl2 mixtures did not give any desired disaccharide. We
then decided to prepare compound 17 with a silyl ether group
at the anomeric position. Selective cleavage of the anomeric
acetyl group of 12 was followed by treatment with TDSCl to
afford derivative 16. Acetate hydrolysis and benzylidenation
gave compound 17. Surprisingly, this derivative proved to be
unstable during silica gel chromatography. Therefore, 17 was
not considered anymore as a building block for our synthetic
scheme. We then directed our attention to the 6-O-chloroacetyl-
ated building block 18, efficiently synthesized from 16 in two
steps. It was anticipated that diol 18 would be selectively glyco-
sylated at position 3. Unfortunately, glycosylation reactions
between diol 18 and uronic acid donor 263 proceeded in
low yield (<15%) and regioselectivity. Finally, we decided to
prepare monosaccharide 4 containing a di-tert-butylsilylene
group. It has been reported that this protecting group, com-
pared to benzylidene acetals, confers desirable properties on
glycosyl acceptors, such as better solubility in most organic
solvents and higher stability under acidic glycosylation con-
ditions.58,64 Thus, we treated monosaccharide 14 with di-tert-
butylsilyl bistriflate in pyridine to obtain 4 in high yield. Grati-
fyingly, coupling of 4 with glucuronic acid trichloroacetimidate
Tetrasaccharide 26 was submitted to deprotection/sulfation
steps to obtain the final compound 1 (Scheme 6). Cleavage of
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the silylene group was performed by treatment with (HF)n·Py showed considerable signal overlap. NMR spectra showed the
complex. Hydrolysis of the acyl groups, benzyl and methyl characteristic downfield shifts of the proton and carbon
esters, and trifluoroacetamides was carried out by treatment signals at positions bearing a sulfate group (see Tables 1 and
with lithium hydroperoxide and then NaOH to give compound 2). COSY, HSQC and TOCSY NMR experiments were employed
28. The amine groups were selectively acetylated with Ac2O in for the structural assignment. Additionally, mass spectroscopic
MeOH to provide intermediate 29, which was purified by gel analysis confirmed the structure of 30. Finally, hydrogenolysis
permeation chromatography. Then, extensive O-sulfation using of 30 gave the fully deprotected tetrasaccharide 1 in good
SO3·Me3N in DMF at 100 °C under microwave heating41,70 gave yield. The structure of 1 was confirmed by NMR and mass
1
cleanly the corresponding hepta-O-sulfated tetrasaccharide 30, spectroscopic analysis. The values of H and 13C NMR chemi-
which proved to be soluble in water. This compound was con- cal shifts for sulfated positions are in good agreement with
verted into the corresponding calcium salt for NMR character- those reported in the literature for similar GAG sulfated
ization because the 1H-NMR spectrum of the sodium salt sequences.41,53
Fluorescence polarization measurements
Fluorescent polarization is a powerful tool for the study of bio-
molecular interactions in solution.71 It is based on the ob-
servation that when a fluorescent molecule is excited with
plane-polarized light, the remaining polarization of the
emitted light depends on the rotational rate of the fluorescent
molecule in solution that is inversely related to its molecular
weight. Thus, the light emitted by a small fluorescent com-
pound, which rotates quickly in solution, is highly depolarized
and, therefore, the polarization value is low. If the fluorescent
probe binds to a high molecular weight molecule, for example,
a protein, the large complex rotates slower in solution, the
emitted light remains polarized, and the polarization value is
Scheme 5 (a) (HF)n·Py, THF, 0 °C, 97%; (b) Ac2O, Py, DMAP, 89%; (c) CAN,
higher. Importantly, fluorescence polarization measurements
do not require immobilization of protein or ligand to a surface
for the analysis of the interaction. Thus, ligand’s bound/free
toluene/CH3CN/H2O, 0 °C, 76%; (d) Cl3CCN, DBU, CH2Cl2, 84%; (e) 25, TMSOTf,
CH2Cl2, 0 °C, 74%.
ratio can be directly measured in solution, avoiding potential
misleading effects derived from the attachment of the bio-
molecules to solid supports. Moreover, this technique is
adequate for high throughput screening, requires very little
amount of samples, and is well suited for the binding analysis
Table 2 13C-NMR chemical shifts for sulfated positions of compounds 30 and
1 and the corresponding non-sulfated positions of 29
Compound C-4A C-6A
C-2B C-4C C-6C
C-2D C-4D
70.6 71.2
71.2 72.6
72.6 74.8
29a
30b
1c
68.8 62.7 or
62.3
75.7 67.8 or
67.4
76.4 68.5 or
67.9
74.0 69.4 62.7 or
62.3
79.1 75.3 67.8 or
67.4
80.1 76.1 68.5 or
67.9
Scheme 6 (a) (HF)n·Py, THF, 0 °C; (b) LiOH, H2O2, THF; NaOH, MeOH; (c) Ac2O,
MeOH, Et3N; (d) SO3·Me3N, DMF, 100 °C, MW, 34% from 26, 4 steps, 76%
average yield per step; (e) H2, Pd(OH)2, H2O/MeOH, 82%.
a Sodium salt, in MeOD. b Calcium salt, in D2O. c Sodium salt, in D2O.
Table 1 1H-NMR chemical shifts for sulfated positions of compounds 30 and 1 and the corresponding non-sulfated positions of 29
Compound
H-4A
H-6A
H-2B
H-4C
H-6C
H-2D
H-4D
29a
30b
1c
4.13
5.11
4.95
3.84–3.58
4.47–4.19
4.35–4.20
3.47–3.44
4.52
4.22
4.05
5.02
4.76
3.84–3.58
4.47–4.19
4.35–4.20
3.65–3.58
4.43
4.20
3.99–3.95
4.89
4.66
a Sodium salt, in MeOD. b Calcium salt, in D2O. c Sodium salt, in D2O.
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Scheme 7 (a) Fluorescein hydrazide, DMSO/phosphate buffer pH 5.5 (1 : 1), 30 °C, 91% (36), 91% (37), 94% (38), 92% (39), 95% (40).
of small ligands, such as oligosaccharides, to a protein recep-
tor. Despite these advantages, fluorescence polarization has
had a limited use in the study of carbohydrate–protein inter-
actions,72,73 in part due to the lack of sensitive and appropriate
instrumentation until the late 1990s. Interestingly, compe-
tition assays can be easily designed to study the binding
affinities of nonfluorescent ligands.72 In fact, we employed a
competition experiment to evaluate the affinity of synthetic
tetrasaccharides 1 and 30 to FGF-2, as described below.
First, we prepared five different fluorescein-conjugated
Fig. 2 Fluorescence polarization values (right, in grey) from wells containing
glycosaminoglycan oligosaccharides (36–40) to select an optimal
probe for binding studies with FGF-2 (Scheme 7 and ESI†).
Commercially available oligosaccharides 31–35, derived from
natural heparin or hyaluronic acid by enzymatic depolymeriza-
tion, were functionalized by reaction of the aldehyde group of
the reducing end of the chain with a hydrazide-containing
fluorescein molecule.74 The corresponding glycosyl hydrazides
were obtained in good yield, after purification by reverse phase
C-18 chromatography. Then, fluorescent labelled sugars 36–40
were mixed with a fixed concentration of FGF-2, and polariz-
ation was measured in 384-well microplates using a standard
fluorescence microplate reader (Fig. 2). Control wells contain-
ing only the fluorescent probe, without any protein, were
included in the experiment. As expected,75,76 heparin hexasac-
charide 38 and tetrasaccharide 37 bound to FGF-2 since a sig-
nificant increased polarization value was observed in FGF-2
containing wells. No interaction was detected for heparin dis-
accharide 36 and hyaluronic acid oligosaccharides 39 and 40,
ruling out any binding of FGF-2 to the fluorescein tag. Hexa
38, which gave the best binding, was chosen as an optimal
probe for inhibition experiments (see below). Importantly, the
use of 384-well plates allowed the minimization of the sample
quantities required for these experiments: the standard assay
was performed with a 10 nM fluorescent probe and an approxi-
mately 100 nM protein in a final volume of 40 μL per well.
Next, the binding of 38 to FGF-2 was measured with increas-
ing concentrations of protein (see ESI, Fig. S1†), giving the cor-
responding binding curve that was analyzed as a Langmuir
isotherm to determine the dissociation constant (KD). The
fluorescent GAG oligosaccharides 36–40 (10 nM) and FGF-2 (97 nM) are com-
pared with the values obtained in the absence of the protein (left, in white). For
each oligosaccharide, polarization values are the average of three replicate wells
and the error bars show the standard deviations for these measurements.
obtained value (117 10 nM) was consistent with a previous
measurement, in solution, of the binding affinity between a
similar heparin hexasaccharide and FGF-2.75
A competition binding assay was then optimized to analyze
the binding affinities of non-fluorescent ligands, such as tetra-
saccharides 1 and 30, to FGF-2. Thus, the polarization
of samples containing fixed concentrations of protein and a
fluorescent probe were recorded in the presence of a certain
concentration of potential competitors (Fig. 3). In these experi-
ments, we chose an FGF-2 concentration close to the KD of the
interaction with 38 in order to obtain a high enough polariz-
ation value while still using the minimal amount of inhibitors.
Besides 1 and 30, synthetic oligosaccharides 41–4641,77 were
included in the screening (Fig. 4). The displacement of fluore-
scent 38 by an active competitor resulted in a decrease of the
polarization value (Fig. 3 and 4). In this way, the inhibitory
capacity of non-labelled compounds could be easily and
quickly screened. As shown in Fig. 4, at 25 μM concentration,
monosaccharide 41 and disaccharides 42 and 46 did not sig-
nificantly affect the interaction between fluorescent 38 and
FGF-2, while hexasaccharides 44 and 45 strongly inhibited the
binding. Interestingly, the presence of 25 μM of 1 and 30 gave
63–67% inhibition.
A similar effect was observed with
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Fig. 3 Schematic representation of the competition assay. The displacement of a fluorescent sugar from a protein receptor by an active competitor results in a
decrease of the polarization value. Thus, the binding affinities of non-fluorescent compounds can be estimated.
Fig. 5 Inhibition curve showing the ability of tetrasaccharide 1, at different
concentrations (from 0.025 μM to 100 μM), to inhibit the interaction between
FGF-2 (103 nM) and probe 38 (10 nM). The concentration required to inhibit
50% binding (IC50 value) was calculated from data analysis (see the main text).
Control wells, with no inhibitor and no protein, were included in the fitting. All
the polarization values are the average of six replicate wells.
the ligand or the protein on a solid support and are based on
the inhibition of the interaction that occurs at the surface,
because this surface interaction can be affected by multiva-
Fig. 4 Competition assay to analyse the inhibitory potency of a collection of
lency, involves unknown amounts of one of the biomolecules
synthetic oligosaccharides (1, 30, 41–46). The graph presents the polarization
and requires additional washing and incubation steps.
values obtained from wells containing 25 μM inhibitor, 103 nM FGF-2, and 10
The binding of FGF-2 to cell surface heparan sulfate GAGs
is essential for tumor angiogenesis and growth. Inhibition of
angiogenesis is a well established and important anti-cancer
strategy and, therefore, there is great interest in compounds
that potentially inhibit the FGF-2–GAG interaction.78 Our data
indicate that CS/DS tetrasaccharides 1 and 30 display consider-
able inhibitory activity and can be considered as the starting
point for the design and synthesis of more active compounds.
Moreover, recent studies78,79 indicate that the introduction of
lipophilic groups on the structure of synthetic antiangiogenic
molecules improved the properties of these anticancer agents,
and, in this context, the activity showed by the di-O-benzylated
30 is particularly remarkable.
nM fluorescent 38. Control wells (in white) correspond to samples with probe
only (left) and no inhibitor (right) and indicate the expected values for 100%
and 0% inhibition, respectively. All the measurements are the averages of three
replicate wells and the error bars show the standard deviations for these
measurements.
tetrasaccharide 43. These results indicated that CS/DS-related
tetrasaccharides 1 and 30 are able to interact with FGF-2 and
that their relative inhibitory potencies are similar to the one
displayed by a heparin tetrasaccharide. After demonstrating
that this fluorescence polarization assay can be used for
the rapid screening of a library of compounds, we studied the
inhibitory potency of oversulfated 1 in more detail. We
measured the polarization of samples containing FGF-2, fluore-
scent 38 and increasing concentrations of tetrasaccharide 1
(Fig. 5). The obtained curve was fitted to the equation for a
simple one-site competitive interaction. An IC50 value of 15 μM
was estimated for compound 1. In terms of screening for
inhibitors, our fluorescence polarization competition assay is
advantageous over methods that require the immobilization of
Conclusions
We have prepared a N-TFA-protected galactosamine unit as a
key building block for the synthesis of CS and DS oligosacchar-
ides. While the participating N-TFA group ensures the desired
1,2-trans stereochemistry of the glycosidic bond, the temporary
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introduction of a di-tert-butylsilylene group at positions 4 and Velos spectrometer at CCiT, Universitat de Barcelona. Micro-
6 transforms this moiety into an excellent glycosyl acceptor for wave-based sulfation reactions were performed using a Biotage
coupling reactions with uronic acid donors. Our results Initiator Eight synthesizer in sealed reaction vessels.
provide an additional illustration of the profound impact that
Benzyl (4-methoxyphenyl 2-O-benzoyl-3-O-benzyl-β-D-gluco-
protecting groups have on the success of a glycosylation reac- pyranoside) uronate (6). TEMPO (2 mL of a 0.016 M solution
tion. Moreover, our protecting group design is compatible with in CH2Cl2), Bu4NBr (2 mL of a 0.08 M solution in CH2Cl2) and
the deprotection/sulfation steps required for the preparation KBr (0.65 mL of a 0.5 M solution in H2O) were added dropwise
of final CS and DS sequences. Following this strategy, over- at 0 °C to a solution of diol 5 (1.6 g, 3.33 mmol) in CH2Cl2
sulfated CS/DS-like tetrasaccharide 1 was successfully syn- (33 mL). A solution of Ca(ClO)2 (1.2 g, 8.3 mmol) and NaHCO3
thesized. Importantly, our approach can be easily applied to (1.2 g, 14.3 mmol) in H2O (31 mL) was then added dropwise at
the synthesis of other CS and DS oligosaccharides, bearing 0 °C. After stirring for 1 h at 0 °C, the reaction was quenched
different sulfate patterns.
by adding Na2SO3 (25 mL of a 0.8 M solution in H2O). After
On the other hand, we have developed a fluorescence polar- stirring for 15 min at 0 °C, the reaction mixture was diluted
ization assay for the rapid screening of the interactions with additional CH2Cl2 and H2O, and the organic layer was
between the synthesized oligosaccharides and proteins. The then separated, washed with a solution of Na2SO3 (0.8 M) and
binding is analysed in solution, avoiding the potential arte- brine, dried (MgSO4), filtered, and concentrated. The residue
facts and the additional washing steps that are typically associ- was dissolved in DMF (45 mL) and benzyl bromide (0.8 mL,
ated with assays where the receptor or the ligand is 6.7 mmol) and Bu4NI (0.6 g, 1.7 mmol) were added. The
immobilized on a solid surface. The only requirement to mixture was stirred for 3 h at 60 °C, diluted with EtOAc,
perform these experiments is the preparation of an adequate washed with H2O, dried (MgSO4), filtered, and concentrated.
fluorescent probe. With this probe at hand, we could evaluate Flash chromatography (toluene–EtOAc, 13 : 1) gave 6 (1.1 g,
the relative binding affinities of a small library of non-fluo- 56%). TLC (6 : 1 toluene–EtOAc) Rf 0.41; [α]2D0 −3.4° (c 1.0,
1
rescent synthetic GAG oligosaccharides, including synthesized CH2Cl2); H-NMR (300 MHz, CDCl3): δ 7.94 (m, 2H, Ar), 7.50
CS/DS tetrasaccharides 1 and 30, to a model heparin-binding (m, 1H, Ar), 7.36 (m, 2H, Ar), 7.28 (m, 5H, Ar), 7.09 (m, 5H,
protein (FGF-2), by using a competition experiment. Our Ar), 6.83 (m, 2H, Ar), 6.62 (m, 2H, Ar), 5.42 (dd, 1H, H-2), 5.17
results show that this method is an excellent platform for the (2d, 2H, CH2(Bn)), 4.93 (d, 1H, J1,2 = 7.6 Hz, H-1), 4.70 (2d, 2H,
fast screening of GAG–protein interactions. It requires a very CH2(Bn)), 4.12 (dd, 1H, J3,4 = J4,5 = 9.5 Hz, H-4), 3.96 (d, 1H,
little amount of samples (nmol/pmol per well) and can be H-5), 3.71 (dd, 1H, J2,3 = 9.2 Hz, H-3), 3.63 (s, 3H, Me(OMP));
useful for the determination of structure–activity relationships 13C-NMR (75 MHz, CDCl3): δ 168.9, 165.5 (CO(COOBn, Bz)),
of synthetic GAG sequences, contributing to the understand- 155.8, 151.3, 138.1, 135.3 (Ar-C), 133.5 (Ar-CH), 130.0, 129.8,
ing of the role of these polysaccharides in various biological 128.8, 128.7, 128.6, 128.4, 128.2, 127.8, (Ar-C, Ar-CH), 119.3,
processes.
114.6 (Ar-CH), 101.5 (C-1), 81.0 (C-3), 74.8, 74.7 (CH2(Bn), C-5),
73.1 (C-2), 72.0 (C-4), 67.5 (CH2(Bn)), 55.6 (Me(OMP)); HR
MS: m/z: calcd for C34H32O9Na: 607.1944; found: 607.1946
[M + Na]+.
Benzyl (4-methoxyphenyl 2-O-benzoyl-3-O-benzyl-4-O-levuli-
noyl-β-D-glucopyranoside) uronate (7). Compound 6 (2.1 g,
Experimental
General procedures
Thin layer chromatography (TLC) analyses were performed on 3.6 mmol), 1,3-dicyclohexylcarbodiimide (1.11 g, 5.39 mmol),
silica gel 60 F254 precoated on aluminium plates (Merck) and DMAP (44 mg, 0.36 mmol) and levulinic acid (1.83 mL,
the compounds were detected by staining with sulfuric acid– 18.0 mmol) were dissolved in CH2Cl2 (25 mL). After stirring for
ethanol (1 : 9), with a cerium(IV) sulfate (10 g), phosphomolyb- 3 h, the mixture was diluted with CH2Cl2 and washed with
dic acid (13 g), and sulfuric acid (60 mL) solution in water saturated aqueous NaHCO3. The organic phase was dried with
(1 L) or with an anisaldehyde solution (anisaldehyde (25 mL) MgSO4, filtered, and concentrated in vacuo. The residue was
with sulfuric acid (25 mL), ethanol (450 mL) and acetic acid purified by flash chromatography on silica gel (2 : 1 hexane–
(1 mL)) followed by heating at over 200 °C. Column chromato- EtOAc) to give 7 as a white solid (1.98 g, 81%). TLC
graphy was carried out on silica gel 60 (0.2–0.5 mm, (2 : 1 hexane–EtOAc) Rf 0.28; [α]2D0 +8.4° (c 1.0, CH2Cl2);
0.2–0.063 mm or 0.040–0.015 mm; Merck). Optical rotations 1H-NMR (300 MHz, CDCl3): δ 7.94 (m, 2H, Ar), 7.52 (m, 1H,
were determined with a Perkin-Elmer 341 polarimeter. 1H- and Ar), 7.38 (m, 2H, Ar), 7.28 (m, 5H, Ar), 7.09 (m, 5H, Ar), 6.83
13C-NMR spectra were acquired on Bruker DPX-300, Avance (m, 2H, Ar), 6.65 (m, 2H, Ar), 5.49 (dd, 1H, J1,2 = 7.2 Hz, J2,3
=
III-400 and DRX-500 spectrometers. Unit A refers to the redu- 8.9 Hz, H-2), 5.40 (dd, 1H, J3,4 = 9.0 Hz, J4,5 = 9.6 Hz, H-4), 5.08
cing end monosaccharide in the NMR data. Electrospray mass (s, 2H, CH2(Bn)), 4.99 (d, 1H, H-1), 4.58 (2d, 2H, CH2(Bn)),
spectra (ESI MS) were carried out with an Esquire 6000 ESI-Ion 4.08 (d, 1H, H-5), 3.88 (dd, 1H, J2,3 = J3,4 = 8.9 Hz, H-3), 3.66 (s,
Trap from Bruker Daltonics. High resolution mass spectra (HR 3H, Me(OMP)), 2.60–2.17 (m, 4H, CH2(Lev)), 2.05 (s, 3H,
MS) were carried out by the Mass Spectrometry Service, CH3(Lev)); 13C-NMR (75 MHz, CDCl3): δ 205.9 (CO(Lev)), 171.2,
CITIUS, Universidad de Sevilla. HR MS (electrospray) of com- 166.9, 165.0 (CO (COOBn, Bz, Lev)), 155.9, 151.1, 137.4, 135.1
pounds 30 and 1 were obtained with a Thermo LTQ Orbitrap (Ar-C), 133.4 (Ar-CH), 129.8, 129.5, 128.6, 128.5, 128.3, 128.0,
3516 | Org. Biomol. Chem., 2013, 42, 3510–3525
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127.7, (Ar-C, Ar-CH), 119.3, 114.5 (Ar-CH), 101.0 (C-1), 78.9 (4.7 mL, 45.4 mmol) was added at 0 °C to a solution of 1,3-
(C-3), 73.9 (CH2(Bn)), 72.9 (C-5), 72.8 (C-2), 71.0 (C-4), 67.7 dicyclohexylcarbodiimide (4.68 g, 22.7 mmol) in CH2Cl2
(CH2(Bn)), 55.6 (Me(OMP)), 37.6 (CH2(Lev)), 29.7 (CH3(Lev)), (38 mL). After stirring for 5 min at room temperature, the
27.7 (CH2(Lev)); HR MS: m/z: calcd for C39H38O11Na: 705.2312; mixture was cooled and filtered to give a solution of Lev2O in
found: 705.2316 [M + Na]+.
Benzyl 2-O-benzoyl-3-O-benzyl-4-O-levulinoyl-α,β-D-glucopyr-
CH2Cl2.
Lev2O (15.0 mL of a 0.76 M solution in CH2Cl2) was added
anosuronate (8). CAN (8.1 g, 14 mmol) was added to a solu- at room temperature to a mixture of 9 (1.0 g, 2.3 mmol) and
tion of the glycoside 7 (1.9 g, 2.8 mmol) in toluene– DMAP (41 mg, 0.34 mmol) in dry Py (40 mL). The mixture was
acetonitrile–water (1 : 6 : 1, 75 mL) and the mixture was stirred stirred for 22 h, diluted with CH2Cl2, and washed with 1 M
for 1.5 h at r.t. The mixture was then diluted with EtOAc, HCl, saturated aqueous NaHCO3, and H2O. The organic phase
washed with H2O and NaHCO3, dried (MgSO4), filtered and was dried (MgSO4), filtered and concentrated to dryness. The
concentrated under reduced pressure. Flash chromatography residue was purified by column chromatography (hexane–
on silica gel (toluene–EtOAc 1 : 0 → 3 : 1) afforded the corre- EtOAc 2 : 1) to afford 10 (1.55 g, 98%). TLC (hexane–EtOAc
sponding hemiacetal 8 (960 mg, 60%) as a mixture of α–β 1 : 1) Rf 0.26; [α]2D0 +19° (c 1.0, CHCl3); 1H-NMR (300 MHz,
anomers (9 : 1). TLC (2 : 1 toluene–EtOAc) Rf 0.47; 1H-NMR CDCl3): δ 7.34 (m, 5H, Ar), 5.16 (m, 1H, H-4), 5.08 (d, 1H, J1,2
=
(300 MHz, CDCl3) (for α anomer): δ 8.03 (m, 2H, Ar), 7.58 (m, 1.6 Hz, H-1), 4.93 (m, 1H, H-2), 4.76, 4.69 (2d, 2H, CH2(Bn)),
1H, Ar), 7.43 (m, 2H, Ar), 7.34 (m, 5H, Ar), 7.21 (m, 5H, Ar), 4.59 (d, 1H, J4,5 = 2.1 Hz, H-5), 3.86 (t, 1H, J2,3 = J3,4 = 2.7 Hz,
5.68 (d, 1H, J1,2 = 3.4 Hz, H-1), 5.29 (dd, 1H, J3,4 = J4,5 = 8.9 Hz, H-3), 3.78 (s, 3H, COOMe), 2.92–2.50 (m, 8H, CH2(Lev)), 2.18
H-4), 5.16 (dd, 1H, H-2), 5.06 (2d, 2H, CH2(Bn)), 4.70 (2d, 2H, (s, 6H , CH3(Lev)), 1.61 (hp, 1H, CH(CH3)2), 0.87–0.83 (12H,
CH2(Bn)), 4.61 (d, 1H, J4,5 = 8.9 Hz, H-5), 4.23 (dd, 1H, J2,3 = J3,4 CH(CH3)2, C(CH3)2), 0.23, 0.14 (2s, 6H, Si(CH3)2); 13C-NMR
= 8.9 Hz, H-3), 2.58–2.21 (m, 4H, CH2(Lev)), 2.09 (s, 3H, (75 MHz, CDCl3): δ 206.5, 206.3 (CO(Lev)), 172.3, 172.1 (CO-
CH3(Lev)); 13C-NMR (75 MHz, CDCl3) (for α anomer): δ 206.4 (Lev)), 167.9 (COOMe), 137.3 (Ar-C), 128.6, 128.2, 127.8
(CO(Lev)), 171.4, 168.5, 165.7 (CO (COOBn, Bz, Lev)), (Ar-CH), 93.1 (C-1), 74.4 (C-3), 73.1 (CH2(Bn)), 72.7 (C-5), 68.0
137.9–127.7 (Ar), 90.3 (C-1), 76.1(C-3), 74.8 (CH2(Bn)), 72.9 (C-2), 67.2 (C-4), 52.4 (COOMe), 38.0, 37.8 (CH2(Lev)), 34.1 (CH
(C-2), 71.0 (C-4), 68.9 (C-5), 68.0 (CH2(Bn)), 37.6 (CH2(Lev)), (CH3)2), 30.0, 29.9 (CH3(Lev)), 28.1 (CH2(Lev)), 25.0 (C(CH3)2),
29.7 (CH3(Lev)), 27.7 (CH2(Lev)); 1H-NMR (300 MHz, CDCl3) 20.3, 20.0, 18.7, 18.5 (CH(CH3)2, C(CH3)2), −1.8, −3.5
(selected data for β anomer): δ 7.99 (m, 2H, Ar), 7.58 (m, 1H, (Si(CH3)2); HR MS: m/z: calcd for C32H48O11SiNa: 659.2864;
Ar), 7.43 (m, 2H, Ar), 7.26 (m, 5H, Ar), 7.18 (m, 5H, Ar), 5.37 found: 659.2847 [M + Na]+.
(dd, 1H, J3,4 = J4,5 = 9.0 Hz, H-4), 5.20 (dd, 1H, H-2), 4.86 (d,
Methyl 3-O-benzyl-2,4-di-O-levulinoyl-α,β-L-idopyranosuro-
1H, J1,2 = 6.9 Hz, H-1), 4.15 (d, 1H, J4,5 = 9.2 Hz, H-5), 3.95 (dd, nate (11). An excess of (HF)n·Py (7.2 mL) was added at −10 °C
1H, J2,3 = J3,4 = 8.3 Hz, H-3), 2.58–2.21 (m, 4H, CH2(Lev)), 2.09 under an argon atmosphere to a solution of 10 (1.63 g,
(s, 3H, CH3(Lev)); 13C-NMR (75 MHz, CDCl3) (selected data for 2.56 mmol) in dry THF (37 mL). After 19 h at 0 °C the mixture
β anomer from HMQC experiment): δ 96.3 (C-1), 77.6 (C-3), was diluted with CH2Cl2 and washed with H2O and saturated
72.7 (C-5), 70.8 (C-4); HR MS: m/z: calcd for C32H32O10Na: NaHCO3 solution until neutral pH. The organic layers were
599.1893; found: 599.1911 [M + Na]+.
dried (MgSO4), filtered and concentrated in vacuo. The residue
O-(Benzyl 2-O-benzoyl-3-O-benzyl-4-O-levulinoyl-α,β-D-gluco- was purified by column chromatography (toluene–EtOAc 1 : 3)
pyranosyluronate) trichloroacetimidate (2). Trichloroaceto- to afford 11 as a mixture of α–β anomers (1 : 1) (970 mg, 77%).
nitrile (0.83 mL, 8.3 mmol) and K2CO3 (126 mg, 0.91 mmol) TLC (hexane–EtOAc 1 : 2) Rf 0.13; 1H-NMR (300 MHz, CDCl3): δ
were added to 8 (480 mg, 0.83 mmol) in dry CH2Cl2 (4.8 mL). 7.34 (m, 10H, Ar, Ar′), 5.30 (m, 1H, H-1), 5.26 (m, 1H, H-4),
After stirring at room temperature for 4 h, the mixture was fil- 5.16 (m, 1H, H-4′), 5.14 (bs, 1H, H-1′), 4.98 (d, 1H, J4,5 = 2.3 Hz,
tered off and concentrated in vacuo to obtain 2 (574 mg, 96%). H-5), 4.95 (m, 1H, H-2′), 4.85 (m, 1H, H-2), 4.78, 4.74 (2m, 4H,
1
TLC (2 : 1 toluene–EtOAc) Rf 0.55; H-NMR (300 MHz, CDCl3): CH2(Bn), CH2(Bn)′), 4.69 (d, 1H, J4,5 = 2.0 Hz, H-5′), 4.18 (bd,
(for α anomer): δ 8.59 (s, 1H , NH), 7.95 (m, 2H, Ar), 7.58 (m, 1H, OH), 3.96 (bt, 2H, H-3, H-3′), 3.81, 3.80 (2s, 6H, COOMe,
1H, Ar), 7.43 (m, 2H, Ar), 7.37 (m, 5H, Ar), 7.20 (m, 5H, Ar), COOMe′), 2.91–2.45 (m, 16H, CH2(Lev), CH2(Lev)′), 2.19–2–17
6.74 (d, 1H, J1,2 = 3.3 Hz, H-1), 5.44 (dd, 1H, H-2), 5.38 (dd, 1H, (3s, 12H, CH3(Lev), CH3(Lev)′); 13C-NMR (75 MHz, CDCl3): δ
H-4), 5.15 (2d, 2H, CH2(Bn)), 4.72 (2d, 2H, CH2(Bn)), 4.52 (d, 207.3, 206.5, 206.3 (CO(Lev), CO(Lev)′), 172.4, 171.9, 171.8 (CO
1H, J4,5 = 10.1 Hz, H-5), 4.29 (dd, 1H, J2,3 = J3,4 = 9.7 Hz, H-3), (Lev), CO(Lev)′), 168.8, 168.1 (COOMe, COOMe′), 137.0, 136.6
2.66–2.20 (m, 4H, CH2(Lev)), 2.12 (s, 3H, CH3(Lev)); 13C-NMR (Ar-C), 129.1, 128.7, 128.6, 128.4, 128.3, 128.2, 128.0, 127.8
(75 MHz, CDCl3) (for α anomer): δ 206.4 (CO(Lev)), 171.4, (Ar-C, Ar-CH), 93.0 (C-1), 92.1 (C-1′), 73.5, 73.4 (CH2(Bn),
168.5, 165.7 (CO(COOBn, Bz, Lev)), 160.0 (CvNH), 137.4–127.7 CH2(Bn)′), 73.1, 72.7, 72.3 (C-3, C-3′, C-5′), 68.0 (C-2′), 67.1
(Ar), 93.1 (C-1), 90.8 (CCl3), 75.7 (C-3), 74.9 (CH2(Bn)), 71.3, (C-4, C-4′), 66.7 (C-2), 65.6 (C-5), 52.6 (COOMe, COOMe′), 38.1,
71.2, 70.9 (C-2, C-5, C-4), 67.9 (CH2(Bn)), 37.6 (CH2(Lev)), 29.7 37.8, 37.7 (CH2(Lev), CH2(Lev)′), 29.8 (CH3(Lev), CH3(Lev)′),
(CH3(Lev)), 27.7 (CH2(Lev)); HR MS: m/z: calcd for 28.0, 27.9, 27.8 (CH2(Lev), CH2(Lev)′); HR MS: m/z: calcd for
C34H32Cl3NO10Na: 742.0989; found: 742.0984 [M + Na]+.
Methyl (dimethylthexylsilyl 3-O-benzyl-2,4-di-O-levulinoyl-
C24H30O11Na: 517.1686; found: 517.1666 [M + Na]+.
O-(Methyl 3-O-benzyl-2,4-di-O-levulinoyl-α,β-L-idopyranosyl-
β-L-idopyranoside) uronate (10). Lev2O preparation: LevOH uronate) trichloroacetimidate (3). K2CO3 (207 mg, 1.67 mmol)
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and trichloroacetonitrile (3.0 mL, 30 mmol) were added at
4-Methoxyphenyl
3,4,6-tri-O-acetyl-2-deoxy-2-trifluoroacet-
room temperature to a solution of 11 (750 mg, 1.52 mmol) in amido-β-D-galactopyranoside (13). TMSOTf (320 μL, 1.7 mmol)
dry CH2Cl2 (15 mL). After stirring for 11 h at room tempera- was added to a cooled (0 °C) solution of 12 (5.1 g, 11.5 mmol)
ture, the reaction mixture was filtered through a pad of celite and 4-methoxyphenol (2.6 g, 20.7 mmol) in dry CH2Cl2
and concentrated to dryness to obtain 3 as a mixture of α–β (51 mL). The mixture was stirred for 1 h at 0 °C and TEA
anomers (920 mg, 95%). TLC (toluene–EtOAc 1 : 4) Rf 0.44, (1 mL) was then added. The mixture was diluted with EtOAc
1
0.58 (α and β anomers); H-NMR (300 MHz, CDCl3) (data for and washed with H2O, saturated aqueous NaHCO3 and H2O.
major anomer): δ 8.68 (s, 1H, NH), 7.34 (m, 5H, Ar), 6.23 (d, The organic phase was dried (MgSO4), filtered, and concen-
1H, J1,2 = 1.8 Hz, H-1), 5.27 (m, 1H, H-2), 5.22 (m, 1H, H-4), trated. The residue was purified by silica gel chromatography
4.80 (d, 1H, J4,5 = 2.0 Hz, H-5), 4.80–4.68 (m, 2H, CH2(Bn)), (toluene–EtOAc 4 : 1 → 1 : 1) to give 13 (2.4 g, 42%) and starting
3.99 (t, 1H, J2,3 = J3,4 = 3.0 Hz, H-3), 3.78 (s, 3H, COOMe), material (α anomer, 2.4 g, 40%). TLC (4 : 1 toluene–EtOAc)
1
2.90–2.49 (m, 8H, CH2(Lev)), 2.16 (s, 6H , CH3(Lev)); 13C-NMR Rf 0.17; [α]2D0 −3.5° (c 1.0, CHCl3); H-NMR (300 MHz, CDCl3):
(75 MHz, CDCl3) (data for major anomer): δ 206.4, 206.3 (CO δ 6.98 (d, 1H, J2,NH = 9.1 Hz, NH), 6.92 (m, 2H, Ar), 6.78
(Lev)), 172.1, 172.0 (CO(Lev)), 167.2 (COOMe), 160.4 (CvNH), (m, 2H, Ar), 5.41 (d, 1H, J3,4 = 2.9 Hz, H-4), 5.31 (dd, 1H, J2,3
=
136.9 (Ar-C), 128.6, 128.3, 127.9 (Ar-CH), 94.4 (C-1), 90.8 (CCl3), 11.3 Hz, H-3), 5.04 (d, 1H, J1,2 = 8.3 Hz, H-1), 4.40 (m, 1H,
73.6 (C-3), 73.4 (CH2(Bn)), 73.2 (C-5), 67.3 (C-4), 65.8 (C-2), 52.7 H-2), 4.26–4.12 (2dd, 2H, J6a,6b = 11.5 Hz, H-6a, H-6b), 4.03 (br
(COOMe), 37.8, 37.7 (CH2(Lev)), 29.9, 29.8 (CH3(Lev)), 28.0, dd, 1H, J5,6a = J5,6b = 6.5 Hz, H-5), 3.76 (s, 3H, Me(OMP));
27.9 (CH2(Lev)); 1H-NMR (300 MHz, CDCl3) (data for minor 2.18–2.00 (3s, 9H, CH3(Ac)); 13C-NMR (75 MHz, CDCl3):
2
anomer): δ 8.69 (s, 1H, NH), 7.34 (m, 5H, Ar), 6.40 (bs, 1H, δ 170.7, 170.6, 170.3 (COCH3), 158.0 (q, JC,F = 34.1 Hz,
H-1), 5.27 (m, 1H, H-4), 5.11 (m, 1H, H-2), 5.04 (d, 1H, J4,5
=
COCF3), 155.9, 151.0 (Ar-C), 118.8, 114.7 (Ar-CH), 115.6 (q, 1JC,F
1.8 Hz, H-5), 4.80–4.68 (m, 2H, CH2(Bn)), 3.87 (m, 1H, H-3), = 287.0 Hz, COCF3), 100.4 (C-1), 71.1 (C-5), 69.5 (C-3), 66.4
3.78 (s, 3H, COOMe), 2.90–2.49 (m, 8H, CH2(Lev)), 2.17 (s, 6H, (C-4), 61.6 (C-6), 55.7 (Me(OMP)), 51.8 (C-2), 20.5 (CH3(Ac)); HR
CH3(Lev)); 13C-NMR (75 MHz, CDCl3) (data for minor anomer): MS: m/z: calcd for C21H24NO10F3Na: 530.1250; found: 530.1268
δ
206.4, 206.3 (CO(Lev)), 171.8, 171.7 (CO(Lev)), 168.1 [M + Na]+.
(COOMe), 160.1 (CvNH), 137.2 (Ar-C), 128.4, 127.9, 127.7 (Ar- 4-Methoxyphenyl
3,4,6-trihydroxy-2-deoxy-2-(trifluoroacet-
CH), 95.0 (C-1), 90.5 (CCl3), 72.6 (CH2(Bn)), 71.5 (C-3), 67.8 amido)-β-D-galactopyranoside (14). Compound 13 (2.8 g,
(C-5), 67.3 (C-4), 65.2 (C-2), 52.7 (COOMe), 37.9, 37.8 5.5 mmol) was dissolved in MeOH (39 mL) and NaOMe
(CH2(Lev)), 29.9, 29.8 (CH3(Lev)), 28.0, 27.9 (CH2(Lev)); HR MS: (365 μL, 2.17 M solution in MeOH) was added. After 50 min,
m/z: calcd for C26H30Cl3NO11Na: 660.0782; found: 660.0782 Amberlite acidic resin was added until pH 7. The Amberlite
[M + Na]+.
resin was filtered off, and the solvent was removed in vacuo to
1,3,4,6-Tetra-O-acetyl-2-deoxy-2-trifluoroacetamido-α,β-D- give 14 (2.1 g, quantitative). TLC (16 : 1 CH2Cl2–MeOH) Rf 0.21;
galactopyranose (12).61,62 Galactosamine hydrochloride (21 g, 1H NMR (300 MHz, MeOD): δ 6.97 (m, 2H, Ar), 6.82 (m, 2H,
93.4 mmol) was suspended in MeOH (250 mL). NaOMe Ar), 4.91 (d, 1H, J1,2 = 8.5 Hz, H-1), 4.25 (dd, 1H, J2,3 = 10.7 Hz,
(105 mL, 1.3 M solution in MeOH) was added at room H-2), 3.91 (d, 1H, J3,4 = 3.1 Hz, H-4), 3.87–3.71 (m, 6H, H-6a,
temperature. After stirring for 30 min, TFA anhydride H-6b, H-3, CH3(OMP)), 3.64 (dd, 1H, H-5); HR MS: m/z: calcd
(14.2 mL, 98.1 mmol) was added at 0 °C. After stirring for for C15H18NO7F3Na: 404.0933; found: 404.0923 [M + Na]+.
10 min, Et3N (13.6 mL, 94.0 mmol) was added. After stirring
4-Methoxyphenyl 4,6-O-benzylidene-2-deoxy-2-trifluoroacet-
for 28 h at room temperature, the reaction mixture was con- amido-β-D-galactopyranoside (15). Benzaldehyde dimethyl
centrated in vacuo. The residue was dissolved in Py (420 mL) acetal (1.0 mL, 6.7 mmol) and p-toluenesulfonic acid (0.08 g,
and DMAP (6.4 g, 46.7 mmol) and acetic anhydride (105.8 mL, 0.45 mmol) were added to a solution of 14 (1.7 g, 4.5 mmol) in
1.1 mol) were added at 0 °C. The reaction was stirred for MeCN (31 mL). After stirring at room temperature for 4 h,
72 h at room temperature, diluted with EtOAc, washed EtOAc was added and the mixture was washed with saturated
with H2O, 1 M HCl and NaHCO3, dried (MgSO4), filtered, and aqueous NaHCO3. The organic phase was dried with MgSO4,
concentrated. Flash chromatography (hexane–EtOAc, 3 : 1) filtered and concentrated in vacuo. Flash chromatography
gave 12 (28.4 g, 66%) as a mixture of α–β anomers (1 : 0.8). (toluene–acetone 5 : 1 → 1 : 1) gave 15 (1.8 g, 87%). TLC (2 : 1
1
TLC (2 : 1 hexane–EtOAc) Rf 0.27; 1H-NMR (300 MHz, CDCl3) toluene–acetone) Rf 0.43; [α]2D0 −8.4° (c 1.0, acetone); H-NMR
(data for α anomer): δ 6.55 (bs, 1H, NH), 6.28 (d, 1H, J1,2
=
(400 MHz, (CD3)2CO): δ 8.56 (d, 1H, J2,NH = 9.4 Hz, NH), 7.59
3.6 Hz, H-1), 5.46 (dd, 1H, H-4), 5.29 (dd, 1H, J2,3 = 11.4 Hz, (m, 2H, Ar), 7.40 (m, 3H, Ar), 7.02 (m, 2H, Ar), 6.86 (m, 2H,
J3,4 = 3.5 Hz, H-3), 4.68 (m, 1H, H-2), 4.28–4.03 (m, 3H, Ar), 5.72 (s, 1H, PhCHO), 5.19 (d, 1H, J1,2 = 8.3 Hz, H-1),
H-5, H-6a, H-6b), 2.18–2.00 (m, 12H, CH3); 1H-NMR 4.48–4.35 (m, 3H, H-2, H-4, OH), 4.32–4.18 (m, 2H, H-6a,
(300 MHz, CDCl3) (data for β anomer): δ 7.02 (bs, 1H, H-6b), 4.16–4.06 (m, 1H, H-3), 3.85 (s, 1H, H-5), 3.76 (s, 3H,
2
NH), 5.77 (d, 1H, J1,2 = 8.7 Hz, H-1), 5.40 (dd, 1H, H-4), 5.18 Me (OMP)), 13C-NMR (100 MHz, (CD3)2CO): δ 157.3 (q, JC,F
=
1
(dd, 1H, J2,3 = 11.3 Hz, J3,4 = 3.3 Hz, H-3), 4.49 (m, 1H, H-2), 36.0 Hz, COCF3), 155.5–115.5 (Ar), 116.3 (q, JC,F = 289.0 Hz,
4.28–4.03 (m, 3H, H-5, H-6a, H-6b), 2.18–2.00 (m, 12H, CH3); COCF3), 100.7 (PhCHO), 100.3 (C-1), 75.4 (C-4), 69.8 (C-3), 68.8
ESI MS: m/z: calcd for C16H20F3NO10Na: 466.1; found: 466.1 (C-6), 66.9 (C-5), 55.0 (Me(OMP)), 53.8 (C-2); HR MS: m/z: calcd
[M + Na]+.
for C22H22NO7F3Na: 492.1246; found: 492.1234 [M + Na]+.
3518 | Org. Biomol. Chem., 2013, 42, 3510–3525
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Paper
Dimethylthexylsilyl 3,4,6-tri-O-acetyl-2-deoxy-2-trifluoroacet- (0
°C).
Di-tert-butylsilyl
bis(trifluoromethanesulfonate)
amido-β-D-galactopyranoside (16). Benzylamine (3.0 mL, (0.63 mL, 1.9 mmol) was added and the mixture was stirred at
28 mmol) was added to a solution of 12 (4.0 g, 9.0 mmol) in room temperature for 12 min. The reaction was quenched with
THF (40 mL). After stirring for 3 h at room temperature, the MeOH (3 mL), diluted with EtOAc (120 mL), and washed with
mixture was diluted with CH2Cl2, and washed with 1 M HCl 1 M HCl, saturated aqueous NaHCO3, and H2O. The organic
and H2O. The organic phase was dried over Mg2SO4, filtered phase was dried (MgSO4), filtered and concentrated to dryness.
and the solvent was removed under reduced pressure. The The residue was purified by column chromatography (toluene–
residue [TLC (3 : 2 hexane–EtOAc) Rf 0.36] (3.9 g) was dissolved EtOAc 5 : 1) to afford 4 (0.8 g, 89%). TLC (toluene–EtOAc 3 : 1)
1
in CH2Cl2 (19.5 mL). Imidazole (1.78 g, 26.2 mmol) and thexyl- Rf 0.30; [α]2D0 −17° (c 1.0, CHCl3); H-NMR (300 MHz, CDCl3):
dimethylsilyl chloride (2.30 mL, 11.7 mmol) were added. After δ 6.92 (m, 2H, Ar), 6.87 (d, 1H, J2,NH = 9.1 Hz, NH), 6.78 (m,
24 h, the mixture was diluted with CH2Cl2 and washed with 2H, Ar), 4.97 (d, 1H, J1,2 = 8.4 Hz, H-1), 4.39 (d, 1H, J3,4
=
H2O. The organic layer was dried over MgSO4, filtered, and the 2.7 Hz, H-4), 4.24 (m, 2H, H-6a, H-6b), 4.09 (bq, 1H, H-2), 3.89
solvent was removed in vacuo. Flash chromatography on silica (dd, 1H, J2,3 = 10.5 Hz, H-3), 3.75 (s, 3H, Me(OMP)), 3.46 (bs,
gel (3 : 1 hexane–EtOAc) afforded 16 (2.43 g, 51%). TLC 1H, H-5), 2.68 (bs, 1H, OH), 1.09, 1.06 (2s, 18H, C(CH3)3);
2
(3 : 1 hexane–EtOAc) Rf 0.33; [α]2D0 −8.7° (c 1.0, CHCl3); 1H-NMR 13C-NMR (75 MHz, CDCl3): δ 158.2 (q, JC,F = 37.2 Hz, COCF3),
1
(400 MHz, CDCl3): δ 6.71 (d, 1H, J2,NH = 9.3 Hz, NH), 5.38 (dd, 155.9, 151.2 (Ar-C), 119.7 (Ar-CH), 115.9 (q, JC,F = 288.0 Hz,
1H, J3,4 = 3.8 Hz, H-4), 5.22 (dd, 1H, J3,4 = 3.5 Hz, J2,3 = 11.3 Hz, COCF3), 114.6 (Ar-CH), 100.2 (C-1), 72.0 (C-4), 71.5 (C-5), 70.7
H-3), 4.84 (d, 1H, J1,2 = 8.0 Hz, H-1), 4.29–4.08 (m, 3H, H-2, (C-3), 66.9 (C-6), 55.7 (Me(OMP)), 55.1 (C-2), 27.5 (C(CH3)3),
H-6a, H-6b), 3.95 (m, 1H, H-5), 2.28–1.94 (3s, 9H, COCH3), 1.62 23.4, 20.9 (C(CH3)3); HR MS: m/z: calcd for C23H34F3NO7SiNa:
(m, 1H, CH(CH3)2), 0.97–0.76 (m, 12H, C(CH3)2 and CH(CH3)2), 544.1954; found: 544.1968 [M + Na]+.
0.29–0.07 (2s, 6H, Si(CH3)2); 13C-NMR (100 MHz, CDCl3):
4-Methoxyphenyl 3-O-(benzyl 2-O-benzoyl-3-O-benzyl-4-O-
2
δ 170.9, 170.6, 170.4 (COCH3), 157.3 (q, JC,F = 36.9 Hz, levulinoyl-β-D-glucopyranosyluronate)-4,6-O-di-tert-butylsily-
1
COCF3), 115.8 (q, JC,F = 290.0 Hz, COCF3), 96.0 (C-1), 71.0 lene-2-deoxy-2-trifluoroacetamido-β-D-galactopyranoside (19).
(C-5), 69.8 (C-3), 66.8 (C-4), 61.9 (C-6), 53.6 (C-2), 33.9, 24.8, Acceptor 4 (0.18 g, 0.3 mmol) and glucuronic acid trichloroace-
20.7, 20.5, 19.8, 18.4 (TDS, OAc), −2.0, −3.0 (TDS); HR MS: m/z: timidate 2 (0.38 g, 0.5 mmol) were combined in a flask, co-
calcd for C22H36NO9F3NaSi: 566.2009; found: 566.1991 evaporated with toluene and dried under vacuum. The starting
[M + Na]+.
materials were dissolved in dry CH2Cl2 (5 mL) and further
Dimethylthexylsilyl 6-O-chloroacetyl-2-deoxy-2-trifluoroaceta- dried by stirring over freshly activated 4 Å molecular sieves for
mido-β-D-galactopyranoside (18). Compound 16 (2.2 g, 15 min. TMSOTf (10 μL, 0.05 mmol) was added at 0 °C. After
4.1 mmol) was dissolved in MeOH (31 mL) and NaOMe 10 min, the reaction was quenched with Et3N (2.5 mL) and
(273 μL, 2.17 M solution in MeOH) was added. After 50 min, filtered, and the solvent was removed under reduced pressure.
Amberlite acidic resin was added until pH 7. The Amberlite Purification by flash chromatography (8 : 1 toluene–EtOAc)
resin was filtered off, and the solvent was removed in vacuo to yielded 19 (345 mg, 91%). TLC (5 : 1 toluene–EtOAc) Rf 0.57;
1
give the desired triol (1.7 g, quantitative). An aliquot of this [α]2D0 +16.5° (c 1.0, CH2Cl2); H-NMR (500 MHz, CDCl3) δ 8.01
triol (0.5 g, 1.2 mmol) was dissolved in anhydrous CH2Cl2 (m, 2H, Ar), 7.62 (m, 1H, Ar), 7.47 (m, 2H , Ar), 7.42–7.31 (m,
(45 mL) and collidine (4.5 mL), and chloroacetic anhydride 5H, Ar), 7.18–7.07 (m, 5H, Ar), 6.94 (m, 2H, Ar), 6.81 (m, 2H,
(112 μL, 1.4 mmol) was then added dropwise at −60 °C. After Ar), 5.41–5.30 (m, 4H, H-2B, H-1B, H-4B, H-1A), 5.18 (2d, 2H,
stirring for 1 h at −60 °C, the mixture was diluted with EtOAc CH2(Bn)), 4.68–4.53 (m, 3H, H-4A, CH2(Bn)), 4.43 (dd, 1H, J2,3
and washed with 1 M HCl, NaHCO3 and H2O. The organic = 11.3 Hz, J3,4 = 2.4 Hz, H-3A), 4.21–4.03 (m, 4H, H-6aA, H-6bA,
phase was dried (MgSO4), filtered, and concentrated. Flash H-5B, H-2A), 3.86 (dd, 1H, J2,3 = J3,4 = 8.7 Hz, H-3B), 3.78 (s,
silica chromatography (3 : 2 hexane–EtOAc) afforded 18 3H, Me(OMP)), 3.42 (s, 1H, H-5A), 2.62–2.22 (m, 4H, CH2(Lev)),
(454 mg, 77%). TLC (3 : 2 hexane–EtOAc) Rf 0.14; [α]D20 +17° 2.13 (s, 3H, CH3(Lev)), 1.09, 0.98 (2s, 18H, C(CH3)3); 13C-NMR
(c 1.0, MeOH); 1H-NMR (300 MHz, CDCl3): δ 7.31 (d, 1H, (100 MHz, CDCl3): δ 206.0 (CO(Lev)), 171.3, 167.0, 164.9
2
J2,NH = 7.9 Hz, NH), 4.70 (d, 1H, J1,2 = 7.5 Hz, H-1), 4.56–4.30 (CO(COOBn, Bz, Lev)), 157.6 (q, JC,F = 38.6 Hz, COCF3), 155.8,
(m, 2H, H-6a, H-6b), 4.10 (s, 2H, COCH2Cl), 4.02–3.80 (m, 3H, 151.1, 137.2, 134.6 (Ar-C), 133.5 (Ar-CH), 129.8–127.8 (Ar),
1
H-3, H-4, H-2), 3.75 (m, 1H, H-5), 1.58 (m, 1H, CH(CH3)2), 120.0 (Ar-CH), 115.5 (q, JC,F = 288.5 Hz, COCF3), 114.5 (Ar-
0.98–0.70 (m, 12H, C(CH3)2 and CH(CH3)2), 0.23–0.03 (2s, 6H, CH), 99.5, 99.4 (C-1B, C-1A), 79.6 (C-3B), 74.5 (C-3A), 74.2
Si(CH3)2); 13C-NMR (75 MHz, CDCl3): δ 167.7 (COCH2Cl), 159.0 (CH2(Bn)), 73.2 (C-4A), 72.5 (C-4B or C-2B), 72.2 (C-5B), 71.3
2
1
(q, JC,F = 36.8 Hz, COCF3), 115.8 (q, JC,F = 285.0 Hz, COCF3), (C-5A), 70.7 (C-2B or C-4B), 68.1 (CH2(Bn)), 67.0 (C-6A), 55.6
95.8 (C-1), 72.5 (C-5), 70.4, 68.3 (C-3, C-4), 65.1 (C-6), 55.9 (Me(OMP)), 53.7 (C-2A), 37.6 (CH2(Lev)), 29.8 (CH3(Lev)), 27.7,
(C-2), 40.5 (CH2Cl), 33.9, 24.8, 19.8, 18.5 (Ac, TDS), −2.0, −4.0 27.6, 27.4 (CH2(Lev), C(CH3)3), 23.2, 20.8 (C(CH3)3); HR MS:
(TDS); ESI MS: m/z: calcd for C18H31ClF3NO7SiNa: 516.2; m/z: calcd for C55H64NO16F3NaSi: 1102.3844; found: 1102.3820
found: 516.2 [M + Na]+.
[M + Na]+.
4-Methoxyphenyl
4,6-O-di-tert-butylsilylene-2-deoxy-2-tri-
4-Methoxyphenyl 3-O-(methyl 3-O-benzyl-2,4-di-O-levulinoyl-
fluoroacetamido-β-D-galactopyranoside (4). Compound 14 α-L-idopyranosyluronate)-4,6-O-di-tert-butylsilylene-2-deoxy-2-
(0.66 g, 1.73 mmol) was dissolved in dry Py (30 mL) and cooled trifluoroacetamido-β-D-galactopyranoside (20). Donor
3
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Paper
Organic & Biomolecular Chemistry
(0.92 g, 1.44 mmol) and acceptor 4 (0.5 g, 0.96 mmol) were dis- 48 h at room temperature, the reaction mixture was diluted
solved in dry CH2Cl2 (12 mL) in the presence of freshly acti- with CH2Cl2 and washed with a 1 M HCl aqueous solution, a
vated 4 Å molecular sieves. After stirring for 30 min at 0 °C, saturated NaHCO3 aqueous solution and brine. The organic
TMSOTf (348 μL of a 0.41 M solution in dry CH2Cl2) was added layers were dried (MgSO4), filtered and concentrated in vacuo.
under an argon atmosphere. After stirring for 15 min at 0 °C, The residue was purified by column chromatography (toluene–
the reaction mixture was neutralized with Et3N and concen- acetone 3 : 1) to afford 22 (149 mg, 89%). TLC (toluene–
trated to dryness. The residue was purified by column chrom- acetone 3 : 2) Rf 0.49; [α]2D0 −17° (c 1.0, CHCl3); 1H-NMR
atography (toluene–EtOAc 1 : 1) to afford 20 (756 mg, 79%). (300 MHz, CDCl3): δ 7.77 (d, 1H, J2,NH = 7.4 Hz, NH), 7.36–7.27
TLC (toluene–EtOAc 1 : 1) Rf 0.19; [α]2D0 −18° (c 1.0, CHCl3); (m, 5H, Ar), 6.95 (m, 2H , Ar), 6.79 (m, 2H, Ar), 5.44 (d, 1H, J1,2
1H-NMR (300 MHz, CDCl3): δ 7.34–7.27 (m, 5H, Ar), 7.00 (d, = 8.6 Hz, H-1A), 5.41(d, 1H, J3,4 = 3.1 Hz, H-4A), 5.28 (bt, 1H,
1H, J2,NH = 7.2 Hz, NH), 6.94 (m, 2H, Ar), 6.79 (m, 2H, Ar), 5.34 H-4B), 5.01 (d, 1H, J1,2 = 3.9 Hz, H-1B), 4.92 (bt, 1H, J2,3
=
(d, 1H, J1,2 = 8.3 Hz, H-1A), 5.28 (bt, 1H, H-4B), 5.15 (bs, 1H, 5.5 Hz, H-2B), 4.88 (d, 1H, J4,5 = 3.3 Hz, H-5B), 4.63 (q, 2H,
H-1B), 5.07 (d, 1H, J4,5 = 2.6 Hz, H-5B), 4.88 (bt, 1H, H-2B), CH2(Bn)), 4.58 (dd, 1H, J2,3 = 10.4 Hz, J3,4 = 3.9 Hz, H-3A),
4.72 (m, 2H, CH2(Bn)), 4.54 (bd, 1H, H-4A), 4.35 (dd, 1H, J2,3
=
4.18–4.06 (m, 2H, H-6aA, H-6bA), 3.99 (bt, 1H, H-5A), 3.86 (m,
11.0 Hz, J3,4 = 2.3 Hz, H-3A), 4.20 (m, 2H, H-6aA, H-6bA), 3.99 1H, H-2A), 3.80–3.75 (m, 7H, H-3B, Me(OMP), COOMe),
(m, 1H, H-2A), 3.80–3.75 (m, 7H, H-3B, Me(OMP), COOMe), 2.92–2.33 (m, 8H, CH2(Lev)), 2.19, 2.17 (2s, 6H, CH3(Lev)),
3.48 (bs, 1H, H-5A), 2.83–2.44 (m, 8H, CH2(Lev)), 2.16 (s, 6H , 2.03, 1.92 (2s, 6H, CH3(Ac)); 13C-NMR (75 MHz, CDCl3):
CH3(Lev)), 1.06, 0.97 (2s, 18H, C(CH3)3); 13C-NMR (75 MHz, δ 207.8, 206.3 (CO(Lev)), 171.5, 171.4, 170.5, 170.4 (CO(Lev,
2
CDCl3): δ 206.8, 206.3 (CO(Lev)), 171.7, 171.6 (CO(Lev)), 168.9 Ac)), 168.7 (COOMe), 157.9 (q, JC,F = 37.5 Hz, COCF3), 155.9,
2
(COOMe), 157.8 (q, JC,F = 36.8 Hz, COCF3), 155.9, 151.2, 138.0 151.1, 137.6 (Ar-C), 128.5, 127.9, 119.1 (Ar-CH), 115.6 (q, 1JC,F
=
1
(Ar-C), 128.4, 127.7, 127.5, 120.0 (Ar-CH), 115.6 (q, JC,F
=
288.1 Hz, COCF3), 114.6 (Ar-CH), 100.3 (C-1B), 99.0 (C-1A), 75.0
287.8 Hz, COCF3), 114.6 (Ar-CH), 100.3 (C-1B), 99.1 (C-1A), 78.1 (C-3B), 73.7 (C-3A), 73.2 (CH2(Bn)), 71.3 (C-5A), 69.9 (C-4B),
(C-3A), 73.3 (C-3B), 72.7, 72.6 (C-4A, CH2(Bn)), 71.3 (C-5A), 68.6 69.4 (C-2B), 68.8 (C-4A), 68.6 (C-5B), 61.7 (C-6A), 55.7 (COOMe
(C-4B), 67.8 (C-2B), 67.4 (C-5B), 67.0 (C-6A), 55.7 (COOMe or or Me(OMP)), 55.2 (C-2A), 52.5 (COOMe or Me(OMP)), 37.9,
Me(OMP)), 54.0 (C-2A), 52.6 (COOMe or Me(OMP)), 37.9, 37.8 37.7 (CH2(Lev)), 29.8 (CH3(Lev)), 27.9, 27.6 (CH2(Lev), 20.7,
(CH2(Lev)), 29.9 (CH3(Lev)), 28.0, 27.7, 27.4 (CH2(Lev), 20.4 (CH3(Ac)); HR MS: m/z: calcd for C43H50F3NO19Na:
C(CH3)3), 23.3, 20.9 (C(CH3)3); HR MS: m/z: calcd for 964.2827; found: 964.2841 [M + Na]+.
C47H62F3NO17SiNa: 1020.3637; found: 1020.3667 [M + Na]+.
3-O-(Methyl
3-O-benzyl-2,4-di-O-levulinoyl-α-L-idopyrano-
4-Methoxyphenyl 3-O-(methyl 3-O-benzyl-2,4-di-O-levulinoyl- syluronate)-4,6-di-O-acetyl-2-deoxy-2-trifluoroacetamido-α,β-D-
α-L-idopyranosyluronate)-4,6-di-O-acetyl-2-deoxy-2-trifluoroacet- galactopyranose (23). CAN (0.75 mL of a 0.63 M solution in
amido-β-D-galactopyranoside (22). An excess of (HF)n·Py H2O) was added to a solution of 22 (149 mg, 0.158 mmol) in
(1.26 mL, 48.3 mmol) was added at 0 °C under an argon toluene–MeCN (1 : 6; 5.25 mL), and the mixture was vigorously
atmosphere to a solution of 20 (250 mg, 0.25 mmol) in dry stirred for 1 h 20 min at 0 °C. It was then diluted with EtOAc,
THF (5 mL). After 23 h at 0 °C the mixture was diluted with and washed with H2O, saturated aqueous NaHCO3, and H2O.
CH2Cl2 and washed with H2O and saturated NaHCO3 solution The organic phase was dried (MgSO4), filtered and concen-
until neutral pH. The organic layers were dried (MgSO4), fil- trated to dryness. The residue was purified by column chrom-
tered and concentrated in vacuo to give 21 (209 mg, 97%). TLC atography (toluene–acetone 5 : 2) to afford 23 (100 mg, 76%) as
(toluene–acetone 3 : 2) Rf 0.21; 1H-NMR (300 MHz, CDCl3): a mixture of α–β anomers. TLC (toluene–acetone 3 : 2) Rf 0.34,
δ 7.87 (d, 1H, J2,NH = 7.1 Hz, NH), 7.36–7.24 (m, 5H, Ar), 6.96 0.31; 1H-NMR (300 MHz, CDCl3) (data for
α anomer):
(m, 2H, Ar), 6.80 (m, 2H, Ar), 5.47 (d, 1H , J1,2 = 8.5 Hz, H-1A), δ 7.39–7.26 (m, 5H, Ar), 6.73 (d, 1H, J2,NH = 9.3 Hz, NH), 5.40
5.23 (m, 2H, H-4B, H-1B), 4.98 (t, 1H, J1,2 = J2,3 = 5.5 Hz, H-2B), (d, 1H, J3,4 = 3.0 Hz, H-4A), 5.37 (d, 1H, J1,2 = 3.1 Hz, H-1A),
4.88 (d, 1H, J4,5 = 4.7 Hz, H-5B), 4.68 (s, 2H, CH2(Bn)), 4.48 5.20 (bt, 1H, H-4B), 5.06 (bs, 1H, H-1B), 4.92 (d, 1H, J4,5
=
(dd, 1H, J2,3 = 10.8 Hz, J3,4 = 3.1 Hz, H-3A), 4.20 (d, 1H, H-4A), 2.3 Hz, H-5B), 4.80 (m, 1H, H-2B), 4.63 (q, 2H, CH2(Bn)), 4.58
3.99 (dd, 1H, J5,6a = 6.6 Hz, J6a,6b = 11.8 Hz, H-6aA), 3.95–3.82 (m, 1H, H-2A), 4.36 (bt, 1H, H-5A), 4.13 (dd, 1H, J2,3 = 10.8 Hz,
(m, 3H, H-2A, H-6bA, H-3B), 3.80, 3.76 (2s, 6H, Me(OMP), H-3A), 4.07 (dd, 1H, J5,6a = 5.3 Hz, J6a,6b = 11.5 Hz, H-6aA), 3.97
COOMe), 3.71 (bt, 1H, H-5A), 2.94–2.28 (m, 8H, CH2(Lev)), 2.17 (dd, 1H, J5,6b = 7.0 Hz, H-6bA), 3.81 (s, 3H, COOMe), 3.75 (bt,
(s, 6H, CH3(Lev)); 13C-NMR (75 MHz, CDCl3) (selected data 1H, H-3B), 3.27 (bs, 1H, OH), 2.84–2.48 (m, 8H, CH2(Lev)),
from HSQC experiment): δ 129–127 (Ar-CH), 118.8, 114.2 2.18 (s, 6H
, CH3(Lev)), 2.03, 1.68 (2s, 6H, CH3(Ac));
(Ar-CH), 99.0 (C-1B), 98.3 (C-1A), 75.9 (C-3A), 74.9 (C-3B), 74.0 13C-NMR (75 MHz, CDCl3) (data for α anomer): δ 207.5, 206.4
(C-5A), 73.0 (CH2(Bn)), 70.5 (C-2B), 69.7 (C-5B), 69.3 (C-4B), (CO(Lev)), 171.7, 171.5, 170.7 (CO(Lev, Ac)), 169.4 (COOMe),
2
68.4 (C-4A), 62.3 (C-6A), 55.3 (COOMe or Me(OMP)), 53.9 157.8 (q, JC,F = 37.3 Hz, COCF3), 137.4 (Ar-C), 128.5, 128.2,
1
(C-2A), 52.3 (COOMe or Me(OMP)), 37.4 (CH2(Lev)), 29.4 128.0 (Ar-CH), 115.9 (q, JC,F = 288.6 Hz, COCF3), 100.4 (C-1B),
(CH3(Lev)), 27.3 (CH2(Lev); HR MS: m/z: calcd for 91.8 (C-1A), 75.0 (C-3A), 72.8 (CH2(Bn)), 72.5 (C-3B), 69.0
C39H46F3NO17Na: 880.2616; found: 880.2617 [M + Na]+.
(C-4A), 68.4 (C-4B), 67.7 (C-5A), 67.2 (C-2B), 67.1 (C-5B), 62.3
21 (152 mg, 0.177 mmol) was dissolved in dry Py (7 mL), (C-6A), 52.7 (COOMe), 49.9 (C-2A), 37.9 (CH2(Lev)), 30.0
cooled (0 °C) and Ac2O (0.5 mL) was added. After stirring for (CH3(Lev)), 27.9 (CH2(Lev), 20.9, 20.3 (CH3(Ac)); HR MS: m/z:
3520 | Org. Biomol. Chem., 2013, 42, 3510–3525
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calcd for C36H44F3NO18Na: 858.2408; found: 858.2403 (Ar-C), 133.5 (Ar-CH), 129.9–127.8 (Ar), 120.2 (Ar-CH), 115.4
1
[M + Na]+.
(q, JC,F = 286.4 Hz, COCF3), 114.5 (Ar-CH), 100.3 (C-1B),
O-[3-O-(Methyl 3-O-benzyl-2,4-di-O-levulinoyl-α-L-idopyrano- 99.0 (C-1A), 81.0 (C-3B), 74.9 (C-3A), 74.7 (CH2(Bn)), 73.8
syluronate)-4,6-di-O-acetyl-2-deoxy-2-trifluoroacetamido-α,β-D- (C-5B), 73.4 (C-4A), 72.5 (C-2B), 72.2 (C-4B), 71.3 (C-5A),
galactopyranosyl] trichloroacetimidate (24). Trichloroaceto- 68.0 (CH2(Bn)), 66.9 (C-6A), 55.6 (Me(OMP)), 54.2 (C-2A),
nitrile (180 μL, 1.8 mmol) and catalytic DBU (107 μL of a 27.6, 27.4 (C(CH3)3), 23.2, 20.8 (C(CH3)3); HR MS: m/z:
0.084 M solution in dry CH2Cl2) were added to a solution of 23 calcd for C50H58NO14F3NaSi: 1004.3476; found: 1004.3511
(100 mg, 0.12 mmol) in dry CH2Cl2 (2 mL). After stirring for [M + Na]+.
13 h at room temperature, the reaction mixture was concen-
trated to dryness. The residue was purified by flash chromato- α-L-idopyranosyluronate)-(1→3)-O-(4,6-di-O-acetyl-2-deoxy-2-tri-
graphy (toluene–acetone 5 : 2 + 1% Et3N) to afford 24 (99 mg, fluoroacetamido-β-D-galactopyranosyl)-(1→4)-O-(benzyl 2-O-
4-Methoxyphenyl O-(methyl 3-O-benzyl-2,4-di-O-levulinoyl-
84%) as a mixture of α–β anomers. TLC (toluene–acetone 5 : 2) benzoyl-3-O-benzyl-β-D-glucopyranosyluronate)-(1→3)-4,6-O-di-
1
Rf 0.42 (α); H-NMR (400 MHz, CDCl3) (data for α anomer): δ tert-butylsilylene-2-deoxy-2-trifluoroacetamido-β-D-galactopyr-
8.82 (s, 1H, NH(TCA)), 7.36–7.26 (m, 5H, Ar), 6.88 (d, 1H, J2,NH anoside (26). Donor 24 (52 mg, 53 μmol) and acceptor 25
= 9.2 Hz, NH(TFA)), 6.39 (d, 1H, J1,2 = 3.7 Hz, H-1A), 5.47 (bd, (35 mg, 35 μmol) were dissolved in dry CH2Cl2 (1.0 mL) in the
1H, J3,4 = 2.9 Hz, H-4A), 5.18 (bt, 1H, H-4B), 5.13 (bd, 1H, presence of freshly activated 4 Å molecular sieves. After stirring
H-1B), 4.87 (d, 1H, J4,5 = 2.7 Hz, H-5B), 4.78 (m, 1H, H-2B), for 30 min, TMSOTf (115 μL of a 0.092 M solution in dry
4.74 (m, 1H, H-2A), 4.61 (q, 2H, CH2(Bn)), 4.28 (bt, 1H, H-5A), CH2Cl2) was added under an argon atmosphere at 0 °C. After
4.20 (dd, 1H, J2,3 = 10.9 Hz, H-3A), 4.08 (dd, 1H, J5,6a = 5.9 Hz, stirring for 15 min at 0 °C, the reaction mixture was neutral-
J6a,6b = 11.6 Hz, H-6aA), 3.94 (dd, 1H, J5,6b = 7.1 Hz, H-6bA), ized with Et3N and concentrated to dryness. The residue was
3.78 (s, 3H, COOMe), 3.74 (bt, 1H, H-3B), 2.80–2.46 (m, 8H, purified by column chromatography (toluene–acetone 3 : 1) to
CH2(Lev)), 2.15, 2.13 (2s, 6H, CH3(Lev)), 1.96, 1.72 (2s, 6H, afford 26 (47 mg, 74%). TLC (toluene–acetone 3 : 1) Rf 0.32;
1
CH3(Ac)); 13C-NMR (100 MHz, CDCl3) (data for α anomer): δ [α]D20 −5° (c 1.0, CHCl3); H-NMR (400 MHz, CDCl3): δ 7.93 (d,
206.8, 206.2 (CO(Lev)), 171.6, 170.4, 170.3 (CO(Lev, Ac)), 168.9 2H, Ar), 7.57 (t, 1H, Ar), 7.47–7.22 (m, 12H, Ar), 7.06 (m, 5H,
2
(COOMe), 160.3 (CvNH), 157.7 (q, JC,F = 38.0 Hz, COCF3), Ar), 6.96 (d, 1H, J2,NH = 8.7 Hz, NH), 6.90 (m, 2H, Ar), 6.83 (d,
1
137.3 (Ar-C), 128.5, 128.1, 128.0 (Ar-CH), 115.7 (q, JC,F = 289.0 1H, J2,NH = 7.0 Hz, NH), 6.78 (m, 2H, Ar), 5.34 (d, 1H,
Hz, COCF3), 100.6 (C-1B), 94.9 (C-1A), 90.7 (CCl3), 75.1 (C-3A), CH2(Bn)), 5.33 (d, 1H, J1,2 = 8.6 Hz, H-1A), 5.25 (m, 3H, H-1B,
72.7 (CH2(Bn)), 72.5 (C-3B), 69.9 (C-5A), 68.2 (C-4A), 68.1 H-2B, H-4D), 5.19 (d, 1H, CH2(Bn)), 5.13 (bd, 1H, H-4C), 4.98
(C-4B), 67.6 (C-2B), 67.2 (C-5B), 61.7 (C-6A), 52.7 (COOMe), (bs, 1H, H-1D), 4.96 (d, 1H, J4,5 = 2.4 Hz, H-5D), 4.80 (m, 2H,
49.5 (C-2A), 37.8, 37.6 (CH2(Lev)), 29.8 (CH3(Lev)), 27.9, 27.7 H-2D, CH2(Bn)), 4.64 (m, 2H, CH2(Bn)), 4.57 (d, 1H, J3,4
(CH2(Lev), 20.6, 20.1 (CH3(Ac)); ESI MS: m/z: calcd for 2.5 Hz, H-4A), 4.51 (d, 1H, CH2(Bn)), 4.37 (dd, 1H, J2,3
=
=
C38H44Cl3F3N2O18Na: 1001.2; found: 1001.1 [M + Na]+.
11.3 Hz, H-3A), 4.18 (d, 1H, J1,2 = 8.5 Hz, H-1C), 4.12 (bd, 1H,
4-Methoxyphenyl 3-O-(benzyl 2-O-benzoyl-3-O-benzyl-β-D- J6a,6b = 12.3 Hz, H-6aA), 4.04 (m, 3H, H-6bA, H-4B, H-2C), 3.96
glucopyranosyluronate)-4,6-O-di-tert-butylsilylene-2-deoxy-2-tri- (d, 1H, J4,5 = 9.3 Hz, H-5B), 3.93 (m, 1H, H-2A), 3.81 (m, 4H,
fluoroacetamido-β-D-galactopyranoside (25). Compound 19 H-6aC, Me(OMP) or COOMe), 3.75 (s, 3H, Me(OMP) or
(0.25 g, 0.23 mmol) was dissolved in CH2Cl2 (4 mL), and hydra- COOMe), 3.74–3.66 (m, 3H, H-3D, H-6bC, H-3B), 3.61 (dd, 1H,
zine monohydrate (2 mL of a 0.25 M solution in Py–AcOH, J2,3 = 10.6 Hz, J3,4 = 3.4 Hz, H-3C), 3.33 (m, 2H, H-5A, H-5C),
3 : 2) was added. After stirring at room temperature for 2 h, the 2.83–2.50 (m, 8H, CH2(Lev)), 2.19, 2.18 (2s, 6H, CH3(Lev)),
reaction mixture was quenched with acetone (1 mL), diluted 1.97, 1.61 (2s, 6H, CH3(Ac)), 1.06, 1.02 (2s, 18H, C(CH3)3);
with CH2Cl2, washed with HCl (1 M), saturated aqueous 13C-NMR (100 MHz, CDCl3): δ 206.9, 206.3 (CO(Lev)), 171.7,
NaHCO3 and H2O, dried with MgSO4, filtered and concen- 171.5, 170.4, 170.3, 168.9, 168.8, 165.0 (CO(Lev), CO(Ac),
trated in vacuo. The residue was purified by flash chromato- CO(Bz), COOBn, COOMe), 158.1 (q, 2JC,F = 36.4 Hz, COCF3), 157.7
2
graphy on silica gel (9 : 1 toluene–EtOAc) to give 25 (0.19 g, (q, JC,F = 36.4 Hz, COCF3), 156.0, 151.1, 137.8, 137.6, 134.7
84%). TLC (5 : 1 toluene–EtOAc) Rf 0.43; [α]2D0 +6.1° (c 1.0, (Ar-C), 133.4, 129.9, 129.7, 129.6, 129.4, 129.1, 128.7, 128.5,
1
CH2Cl2); 1H-NMR (500 MHz, CDCl3) δ 8.00 (m, 2H, Ar), 7.61 128.1, 127.9, 127.6, 120.3 (Ar-C, Ar-CH), 116.0 (q, JC,F = 286.3
1
(m, 1H, Ar), 7.46 (m, 2H, Ar), 7.43–7.31 (m, 5H, Ar), 7.23–7.12 Hz, COCF3), 115.5 (q, JC,F = 287.9 Hz, COCF3), 114.6 (Ar-CH),
(m, 5H, Ar), 6.98 (d, 1H, J2,NH = 6.5 Hz, NH), 6.92 (m, 2H, Ar), 100.4 (C-1B), 100.3 (C-1D), 99.7 (C-1C), 99.3 (C-1A), 80.1 (C-3B),
6.81 (m, 2H, Ar), 5.44 (d, 1H, J1,2 = 8.2 Hz, H-1A), 5.39 (d, 1H, 78.0 (C-4B), 76.4 (C-3C), 75.5 (C-3A), 75.1 (CH2(Bn)), 74.3
CH2(Bn)), 5.34 (d, 1H, J1,2 = 7.9 Hz, H-1B), 5.29 (dd, 1H, H-2B), (C-5B), 73.3 (C-4A), 73.0 (C-3D), 72.6 (CH2(Bn)), 72.4 (C-2B),
5.20 (d, 1H, CH2(Bn)), 4.77 (2d, 2H, CH2(Bn)), 4.62 (d, 1H, 71.4, 71.1 (C-5A, C-5C), 68.5, 68.4 (CH2(Bn), C-4D), 67.8 (C-4C),
H-4A), 4.44 (dd, 1H, J2,3 = 11.3 Hz, J3,4 = 2.4 Hz, H-3A), 67.5 (C-2D), 67.0 (C-5D, C-6A), 61.0 (C-6C), 55.7 (COOMe or
4.19–4.05 (m, 2H, H-6aA, H-4B), 4.03–4.91 (m, 3H, H-6bA, Me(OMP)), 54.2 (C-2A), 53.5 (C-2C), 52.6 (COOMe or
H-5B, H-2A), 3.78 (s, 3H, Me(OMP)), 3.69 (dd, 1H, J2,3 = J3,4
=
Me(OMP)), 37.9, 37.7 (CH2(Lev)), 29.9 (CH3(Lev)), 28.0, 27.7,
9.2 Hz, H-3B), 3.35 (s, 1H, H-5A), 1.08, 1.00 (2s, 18H, C(CH3)3); 27.6 (CH2(Lev), C(CH3)3), 23.4 (C(CH3)3), 20.8, 20.7, 20.1
13C-NMR (100 MHz, CDCl3): δ 169.0, 165.0 (CO(COOBn, Bz)), (C(CH3)3, CH3(Ac)); HR MS: m/z: calcd for C86H100F6N2O31SiNa:
2
157.7 (q, JC,F = 37.1 Hz, COCF3), 155.9, 151.1, 137.6, 134.4 1821.5881; found: 1821.5903 [M + Na]+.
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4-Methoxyphenyl O-(3-O-benzyl-2,4-di-O-sulfo-α-L-idopyrano- chromatography (CH2Cl2–MeOH 1 : 1) to give 29 as triethyl-
syluronic acid)-(1→3)-O-(2-acetamido-2-deoxy-4,6-di-O-sulfo-β-D- ammonium salt. The sodium salt of 29 was obtained by treat-
galactopyranosyl)-(1→4)-O-(3-O-benzyl-2-O-sulfo-β-D-glucopyr- ment with Amberlite IR-120 H+ resin in MeOH (pH ∼ 3),
anosyluronic acid)-(1→3)-2-acetamido-2-deoxy-4,6-di-O-sulfo- followed by filtration, treatment with 0.1 M NaOH (pH ∼ 7)
β-D-galactopyranoside (30). An excess of (HF)n·Py (56 μL, and concentration.
2.1 mmol) was added at 0 °C under an argon atmosphere to a
solution of 26 (20 mg, 0.011 mmol) in dry THF (1.0 mL). After 2H, Ar), 7.45 (d, 2H, Ar), 7.34–7.21 (m, 6H, Ar), 6.98 (m, 2H ,
24 h at 0 °C, the mixture was diluted with CH2Cl2 and washed Ar), 6.82 (m, 2H, Ar), 5.06 (d, 1H, CH2(Bn)), 4.93 (d, 1H, J1,2
1H-NMR (500 MHz, MeOD, data for sodium salt): δ 7.58 (d,
=
with H2O and saturated NaHCO3 solution until neutral pH. 8.4 Hz, H-1A), 4.89 (d, 1H, J1,2 = 4.4 Hz, H-1D), 4.77 (q, 2H,
The organic layer was dried (MgSO4), filtered and concentrated CH2(Bn)), 4.73 (d, 1H, CH2(Bn)), 4.62 (d, 1H, J1,2 = 8.5 Hz,
in vacuo to give 27. TLC (toluene–EtOAc 1 : 2) Rf 0.14; [α]2D0 −4° H-1C), 4.43 (m, 1H, H-1B), 4.41 (d, 1H, J4,5 = 3.7 Hz, H-5D),
1
(c 0.9, CHCl3); H-NMR (400 MHz, CDCl3): δ 7.85 (d, 2H, Ar), 4.27 (dd, 1H, J2,3 = 10.6 Hz, H-2A), 4.23 (dd, 1H, J2,3 = 10.4 Hz,
7.56 (t, 1H, Ar), 7.48–7.23 (m, 12H, Ar), 7.13 (d, 1H, J2,NH = 8.2 H-2C), 4.13 (d, 1H, J3,4 = 2.7 Hz, H-4A), 4.05 (d, 1H, J3,4
=
Hz, NH), 7.06 (m, 5H, Ar), 7.00 (d, 1H, J2,NH = 6.4 Hz, NH), 6.86 2.7 Hz, H-4C), 3.99–3.95 (m, 2H, H-4B, H-4D), 3.84–3.69 (m,
(m, 2H, Ar), 6.74 (m, 2H, Ar), 5.36 (d, 1H, J1,2 = 8.4 Hz, H-1A), 9H, H-6aA, H-6aC, H-6bA or H-6bC, H-3A (3.80), Me(OMP)
5.25 (q, 2H, CH2(Bn)), 5.23 (m, 2H, H-2B, H-4D), 5.16 (bs, 1H, (3.74), H-5B (3.72), H-3C (3.71)), 3.65–3.58 (m, 3H, H-6bA or
H-4C), 4.98 (bs, 1H, H-1D), 4.93 (bs, 1H, H-5D), 4.80 (m, 3H, H-6bC, H-2D, H-5A or H-5C), 3.55 (t, 1H, J2,3 = J3,4 = 5.9 Hz,
H-1B, H-2D, CH2(Bn)), 4.63 (q, 2H, CH2(Bn)), 4.54 (m, 2H, H-3D), 3.52 (m, 1H, H-5A or H-5C), 3.47–3.44 (m, 2H, H-2B,
H-1C, CH2(Bn)), 4.46 (bd, 1H, J2,3 = 10.6 Hz, H-3A), 4.20 (t, 1H, H-3B), 2.05, 1.99 (2s, 6H, NHAc); 13C-NMR (125 MHz, MeOD)
J3,4 = J4,5 = 8.2 Hz, H-4B), 4.13 (bs, 1H, H-4A), 4.08 (bd, 1H, J4,5 (selected data from HSQC experiment): δ 129.9–128.4, 118.9,
= 8.8 Hz, H-5B), 4.02 (m, 1H, H-2C), 3.89 (m, 1H, H-6aC), 115.1 (Ar-CH), 105.6 (C-1B), 103.9 (C-1D), 102.1 (C-1A), 101.3
3.83–3.71 (m, 12H, H-2A, H-6aA, H-6bA, H-3B, H-3C, H-3D, Me (C-1C), 83.7 (C-3B), 81.8 (C-3C), 81.7 (C-3A), 80.3 (C-3D), 78.9
(OMP), COOMe), 3.63 (m, 2H, H-6bC, H-5C), 3.54 (bt, 1H, (C-4B), 77.7 (C-5B), 77.2, 76.5 (C-5A, C-5C), 76.4 (CH2(Bn)), 74.0
H-5A), 2.82–2.49 (m, 8H, CH2(Lev)), 2.18 (s, 6H, CH3(Lev)), (C-2B), 73.6 (CH2(Bn)), 71.7 (C-5D), 71.2 (C-4D), 70.6 (C-2D),
1.90, 1.64 (2s, 6H, CH3(Ac)); 13C-NMR (100 MHz, CDCl3): δ 69.4 (C-4C), 68.8 (C-4A), 62.7, 62.3 (C-6A, C-6C), 55.7
207.1, 206.4 (CO(Lev)), 171.7, 171.5, 170.5, 170.3, 168.9, 168.8, (Me(OMP)), 52.7 (C-2C), 52.6 (C-2A), 23.1, 22.8 (NAc); ESI MS:
165.2 (CO(Lev), CO(Ac), CO(Bz), COOBn, COOMe), 158.0 (bq, m/z: calcd for C49H61N2O24: 1061.4; found: 1061.2 [M + H]−.
2JC,F = 37.4 Hz, 2 × COCF3), 155.8, 151.1, 137.8, 137.6, 134.8
(Ar-C), 133.6, 129.9, 129.5, 129.3, 129.2, 128.9, 128.5, 128.2, trimethylamine complex (54 mg, 0.39 mmol) were dissolved in
Compound 29 (11.1 μmol) and the sulfur trioxide–
1
128.1, 127.9, 127.8, 127.7, 119.0 (Ar-C, Ar-CH), 116.0 (q, JC,F
=
dry DMF (1.5 mL) and heated at 100 °C for 2 h using micro-
287.6 Hz, COCF3), 115.2 (q, 1JC,F = 287.6 Hz, COCF3), 114.7 (Ar- wave radiation (20 W average power). The reaction vessel was
CH), 101.6 (C-1B), 100.5 (C-1D), 100.1 (C-1C), 98.7 (C-1A), 80.0 cooled and Et3N (150 μL) and MeOH (1 mL) were added. The
(C-3B), 78.5 (C-3A), 77.8 (C-4B), 76.5 (C-3C), 75.1 (CH2(Bn)), solution was layered on the top of a Sephadex LH-20 chromato-
74.7 (C-5C), 74.3 (C-5B), 73.3 (C-3D), 72.9 (CH2(Bn)), 72.6 graphy column which was eluted with MeOH to obtain 30 as
(C-2B), 71.3 (C-5A), 69.4 (C-4D), 68.7 (CH2(Bn)), 68.6 (C-4A), triethylammonium salt. The residue was converted into the
68.1 (C-4C), 68.0 (C-2D), 67.3 (C-5D), 62.8 (C-6C), 61.4 (C-6A), sodium salt by elution from a column of Dowex 50WX4-Na+
55.8 (COOMe or Me(OMP)), 54.7 (C-2A), 53.9 (C-2C), 52.8 with MeOH–H2O 9 : 1 (6.9 mg, 34% from 26; 4 steps, 76%
(COOMe or Me(OMP)), 37.9, 37.8 (CH2(Lev)), 29.9, 29.8 average yield per step). Due to extensive overlap of 1H-NMR
(CH3(Lev)), 28.0, 27.7 (CH2(Lev)), 20.6, 20.2 (CH3(Ac)); HR MS: signals of the sodium salt at 25 °C, 30 was characterized as a
m/z: calcd for C78H84F6N2O31Na: 1681.4860; found: 1681.4868 calcium salt at 40 °C. The calcium salt of 30 was obtained by
1
[M + Na]+.
adding a 0.9 M solution of CaCl2 in D2O. H-NMR (500 MHz,
H2O2 (30%, 0.44 mL) and an aqueous solution of LiOH D2O, 40 °C, data for calcium salt): δ 7.73 (d, 2H, Ar), 7.63–7.46
(0.7 m, 0.27 mL) were added at −5 °C to a solution of 27 (m, 8H, Ar), 7.23 (m, 2H, Ar), 7.11 (m, 2H, Ar), 5.44 (bs, 1H,
(11.1 μmol) in THF (1.5 mL). After stirring for 20 h at room H-1D), 5.37 (d, 1H, J1,2 = 8.6 Hz, H-1A), 5.30 (bs, 1H, H-5D),
temperature, MeOH (1.5 mL) and an aqueous solution of 5.11 (bd, 1H, H-4A), 5.02 (bs, 1H, H-4C), 4.98 (q, 2H, CH2(Bn)),
NaOH (4 m, 0.28 mL) were added. After stirring for 6 d at room 4.91–4.81 (m, 5H, H-1B (4.90), H-4D (4.89), CH2(Bn), H-1C
temperature, the reaction mixture was neutralized with (4.84)), 4.52 (t, 1H, J1,2 = J2,3 = 7.5 Hz, H-2B), 4.47 (dd, 1H, J5,6a
Amberlite IR-120 (H+) resin, filtered, and concentrated to = 2.6 Hz, J6a,6b = 11.3 Hz, H-6aA or H-6aC), 4.43–4.19 (m, 10H,
give 28. ESI MS: m/z: calcd for C45H57N2O22: 977.3; found: H-2D (4.43), H-6aA or H-6aC, H-3A (4.39), H-3D (4.36), H-6bA,
977.2 [M + H]−.
H-6bC, H-5A or H-5C (4.33), H-4B (4.29), H-2C (4.24), H-2A
Triethylamine (0.4 mL of a 0.36 M solution in dry MeOH) (4.22)), 4.14 (m, 1H, H-3C), 4.10 (bt, 1H, H-5A or H-5C),
and acetic anhydride (21 μL, 0.22 mmol) were added to a 3.99–3.94 (m, 5H, H-3B (3.98), H-5B (3.97), Me(OMP) (3.94)),
cooled (0 °C) solution of 28 (11.1 μmol) in dry MeOH (2.5 mL). 2.21 (s, 6H, NHAc); 13C-NMR (125 MHz, D2O, 40 °C) (selected
After stirring for 2 h at room temperature, triethylamine data from HSQC experiment): δ 129.6–128.5, 118.6, 115.2
(0.3 mL) was added and the mixture was concentrated to (Ar-CH), 102.2 (C-1B), 101.5 (C-1D), 100.6 (C-1A), 100.4 (C-1C),
dryness. The residue was purified by Sephadex LH-20 80.2 (C-3B), 79.6 (C-3C), 79.1 (C-2B), 77.5 (C-4B), 77.4 (C-5B),
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76.7 (C-3A), 75.7 (C-4A), 75.3 (C-4C), 74.2 (CH2(Bn)), 72.8 (C-5A with 10 μL of an inhibitor solution (100 μM). The total sample
or C-5C), 72.6 (C-4D), 72.4 (C-5A or C-5C), 71.9 (C-3D), 71.4 volume in each well was again 40 μL. After stirring for 5 min in
(CH2(Bn)), 71.2 (C-2D), 68.2 (C-5D), 67.8, 67.4 (C-6A, C-6C), the dark, fluorescence polarization was recorded. Two control
56.0 (Me(OMP)), 52.4 (C-2A), 52.2 (C-2C), 22.8 (NAc); ESI MS: wells containing no inhibitor and containing probe only
m/z: calcd for C49H55N2O45S7Na5: 865.0; found: 865.0 [M + 5Na (no FGF-2) were included in the study. For the determination
+ 2H]2−; HR MS: m/z: calcd for C49H60N2O45S7: 810.0262; of the IC50 value, wells containing probe and FGF-2 at fixed
found: 810.0253 [M + 7H]2−
4-Methoxyphenyl O-(2,4-di-O-sulfo-α-L-idopyranosyluronic 6 different concentrations of inhibitor, ranging from 0.025 μM
.
concentration, as described above, were incubated with
acid)-(1→3)-O-(2-acetamido-2-deoxy-4,6-di-O-sulfo-β-D-galacto- to 100 μM. The average polarization values of six replicate
pyranosyl)-(1→4)-O-(2-O-sulfo-β-D-glucopyranosyluronic acid)- wells were plotted against the logarithm of inhibitor con-
(1→3)-2-acetamido-2-deoxy-4,6-di-O-sulfo-β-D-galactopyranoside centration, and the curve was fitted to the simplified formula
(1). A solution of 30 (4.9 mg, 2.7 μmol, sodium salt) in H2O/ corresponding to a one-site competitive interaction. All the
MeOH (4.5 mL/0.5 mL) was hydrogenated (1.5 atm) in the pres- experiments were repeated at least twice.
ence of Pd(OH)2. After 22 h, the suspension was filtered over
Occasionally, we found loss of FGF-2 activity when working
celite and concentrated. The residue was purified by Sephadex with low concentrated aliquots in PBS buffer. For this reason,
G-25 chromatography (H2O–MeOH 9 : 1) to give 1 as sodium all measurements were alternatively done in PBS + 0.5% BSA
salt after lyophilisation (3.6 mg, 82%; 28% from 26, 5 steps, (data not shown), getting similar results to those obtained
78% average yield per step). 1H-NMR (500 MHz, D2O): δ 7.10 with PBS.
(d, 2H, Ar), 6.98 (d, 2H, Ar), 5.29 (bs, 1H, H-1D), 5.22 (d, 1H,
J1,2 = 8.4 Hz, H-1A), 4.98 (bs, 1H, H-5D), 4.95 (bs, 1H, H-4A),
4.76 (m, 1H, H-4C), 4.72 (m, 1H, H-1C), 4.70 (d, 1H, J1,2
=
Acknowledgements
7.7 Hz, H-1B), 4.66 (bs, 1H, H-4D), 4.43 (m, 1H, H-3D), 4.35
(bd, 1H, H-6aA or H-6aC), 4.31–4.20 (m, 7H, H-6aA or H-6aC,
H-3A (4.27), H-6bA, H-6bC, H-5A or H-5C (4.23), H-2B (4.22),
H-2D (4.20)), 4.14–4.07 (m, 4H, H-2A, H-2C, H-5A or H-5C,
H-3C), 3.93 (t, 1H, J3,4 = J4,5 = 9.1 Hz, H-4B), 3.82–3.81 (m, 4H,
Me(OMP), H-3B), 3.73 (d, 1H, J4,5 = 9.4 Hz, H-5B), 2.11, 2.05
(2s, 6H, NHAc); 13C-NMR (125 MHz, D2O) (selected data from
HSQC experiment): δ 118.8, 115.5 (Ar-CH), 103.1 (C-1B), 101.3
(C-1C), 101.0 (C-1A), 100.7 (C-1D), 81.0 (C-4B), 80.1 (C-2B), 77.0
(C-5B, C-3A), 76.6 (C-3C), 76.4 (C-4A), 76.1 (C-4C), 74.8 (C-4D),
73.9 (C-3B), 73.2, 72.7 (C-5A, C-5C), 72.6 (C-2D), 68.5, 67.9
(C-6A, C-6C), 67.0 (C-5D), 66.5 (C-3D), 56.2 (Me(OMP)), 52.8,
52.4 (C-2A, C-2C), 23.2, 22.8 (NAc); ESI MS: m/z: calcd for
C35H43N2O45S7Na5: 774.9; found: 774.8 [M + 5Na + 2H]2−; HR
MS: m/z: calcd for C35H48N2O45S7: 719.9792; found: 719.9786
We thank the Spanish Ministry of Economy and Competitive-
ness (Grants CTQ2009-07168 and CTQ2012-32605), the
Regional Government of Andalusia (Grant P07-FQM-02969),
the Spanish National Research Council (CSIC, Grant
201180E021) and the European Union (FEDER support) for
financial support. J. A. acknowledges financial support from
the Spanish Ministry of Economy and Competitiveness
through the Ramón y Cajal program.
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Fluorescence polarization assays
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