The uncommon strong inhibition of α-glucosidase by multivalent glycoclusters based on cyclodextrin scaffolds

Article abstract of DOI:10.1039/c9ob01344j

The homeostasis disruption of d-glucose causes diabetes, a dramatic chronic disease worldwide. Type 1 diabetes is a successfully treatable form, where blood d-glucose is regulated by insulin treatment. In contrast type 2 diabetes, the non-insulin dependent kind, is problematic. The control of the d-glucose blood level via intestinal α-d-glucosidase inactivation can be achieved by using competitive inhibitors, such as iminosugars (e.g. acarbose) or sulfonium sugar derivatives (e.g. salacinol). Recently, an unprecedented result showed that multivalent diamond nanoparticles grafted with unmodified sugars displayed α-glucosidase inhibition at low micromolar concentrations. Herein we describe the synthesis of multivalent glycoclusters using cyclodextrins (CDs) as scaffolds and an assessment of their role as inhibitors of α-d-glucosidase. The glycoclusters were efficiently obtained from per-azido α, β and γ-CD derivatives and propargyl glycosides using click-chemistry under microwave irradiation. The methodology was successfully applied to various protected and non-protected propargylated monosaccharides, including both O- and S-glycosides, giving clear evidence of its versatility. The targeted 6-per-glycosylated CDs were isolated in moderate to excellent yields (30-90%) by silica gel chromatography. The results showed inhibition of α-glucosidase from Saccharomyces cerevisiae with IC₅₀ values in the 32-132 μM range, lower than that of acarbose (IC₅₀ = ～250 μM), a well-known competitive inhibitor used in the clinical treatment of type 2 diabetes. Preliminary experiments suggest a mixed-type non-competitive inhibition mode for these new glycoclusters.

Full text of DOI:10.1039/c9ob01344j

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O rP gl ea na si ce &d Bo i on mo to al e dc juu l sa tr mC ha er mg i ins ts ry
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ARTICLE
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Uncommon Strong Inhibition of α-Glucosidase by Multivalent
DOI: 10.1039/C9OB01344J
Glycoclusters based on Cyclodextrins Scaffolds†
a
a
a
a
b
c
Received 00th January 20xx,
Accepted 00th January 20xx
Urjwan Alali ‡, Aurélie Vallin ‡, Abed Bil , Takwa Khanchouche , David Mathiron , Cédric Przybylski ,
a
a
a
a
Rémi Beaulieu , José Kovensky , Mohammed Benazza and Véronique Bonnet *
DOI: 10.1039/x0xx00000x
The homeostasis disruption of D-glucose causes diabetes, a dramatic world wide chronic disease. The type 1 diabetes is a
succesfully treatable form, where the blood D-glucose is regulated by insulin treatement. In contrast the type 2 diabetes,
the non Insulin dependent one, is problematic. The control of the D-glucose blood level via intestinal α-D-glucosidase
inactivation can be achieved by using competitive inhibitors as iminosugars (e.g. acarbose) or sulfonium sugar derivatives
(
e.g. salacinol). Recently, an unprecedented result showed that multivalent diamond nanoparticules grafted with
unmodified sugars displayed α-glucosidase inhibition at low micromolar concentrations. We describe herein the synthesis
of multivalent glycoclusters using cyclodextrines (CDs) as scaffolds and their assessment as inhibithors of α-D-glucosidase.
The glycoclusters were efficiently obtained from per-azido α, β and γ-CDs derivatives and propargyl glycosides using click-
chemistry under microwave irradiation. The methodology was successfully applied to various protected and non-protected
propargylated monosaccharides, including both O- and S- glycosides, giving clear evidences of its versatility. The targeted 6-
per-glycosylated CDs were isolated in moderate to excellent yields (30-90 %) by silica gel chromatography. The results
showed inhibition of α-glucosidase from Saccharomyces cerevisiae with IC50 values in a 32-132 µM range, lower than that of
acarbose (IC50 ~250µM), a well knowm competitive inhibitor used in clinical treatment of type 2 diabetes. Preliminary
experiments suggest a mixed-type non-competitive inhibition mode of these new glycoclusters.
Keywords: cyclodextrin, click chemistry, microwaves, glycocluster, multivalent scaffold, glucosidase inhibitors
β-CD (cycloheptaamylose, seven Glcp units) and
γ-CD
Introduction
(cyclooctaamylose, eight Glcp units) [9]. A CD molecule is
considered as a nanoparticle (outer diameter from 1.46 nm
onwards) with a hydrophilic outer wall and a hydrophobic inner
cavity, which can include non-polar compounds [8]. Among all
CDs, β-CD is the most described in literature, due to its
availability in large amounts and its low cost.
Moreover, CDs are appealing scaffolds to design multivalent
glycoconjugates [10], as their primary hydroxyls can be
regioselectively functionalized. Some saccharide-branched CDs
synthesized chemically or chemo-enzymatically are
monosubstituted derivatives, in which mono-, di- or
oligosaccharides are grafted either directly or via a spacer arm
Diabetes is a chronic disease that affected in 2014, 8.5% of
adults aged 18 years and older according to the World Health
Organization [1]. Type 2 diabetes comprises 90% of diabetic
people around the world. It can be treated and its complications
reduced by synthetic and natural product inhibitors of
carbohydrate degrading enzymes, especially
involved in carbohydrate digestion process increasing the
glucose level in blood [2-4].
It was demonstrated that α-O-glucosides and also α-O-
mannosides, conjugated on nanodiamond particles (ND) as
multivalent scaffolds are inhibitors of glycosidases at low
micromolar concentrations [5]. Unfortunately, such type of
scaffolds does not allow to evaluate the number of saccharides
which are grafted so the efficiency of the inhibition. Multivalent
interactions involving oligosaccharides and proteins are now
well known as key factors in molecular recognition driving many
biological events [6], where the protein-saccharide binding
affinity is increased by the so-called cluster effect.
α-glucosidase
[
11]. Some CD derivatives have been previously described as
suitable glycoclusters interacting with lectins [12,13].
Moreover, -CD grafted with iminosugars were potent
β
glycosidase inhibitors [14,15]. Recently, glucosyl and mannosyl-
CD were reported as potential Trojan horses to improve effects
of antibiotics in bacteria growth, opening new potential
applications [16], a heptavalent β-CD tethered with n-heptyl α-
D-mannoside as a nanomolar adhesin FimH antagonist [17]. β-
Cyclodextrins
(CDs)
are
cyclic
oligomers
of
CDs carrying GlcNAc, Gal, Lac and LacNAc residues exhibited
enhanced inhibition of erythrocytes agglutination through
binding to wheat germ agglutinin (WGA) and Erythrina
corallodendron lectin (ECorL) [18]. Mannoside and rhamnoside
β-CD derivatives have been tested as inhibitors of binding of
ConA to yeast mannan (0.1<IC50<0.4µM) [19].
α (1→4)−glucopyranose (Glcp) units able to form well-known
host-guest complexes, driven by non-covalent interactions.
[
7,8]. Common CDs are classified according to their number of
Glcp units as follows: α-CD (cyclohexaamylose, six Glcp units),
a.
Copper-catalyzed azide alkyne cycloaddition (CuAAC) has been
extensively used for grafting sugar units onto oligosaccharides
Laboratoire de Glycochimie, des Antimicrobiens et des Agroressources, LG2A UMR
CNRS 7378, Université de Picardie Jules Verne, 33 rue Saint Leu 80039 Amiens
France. veronique.bonnet@u-picardie.fr
Plateforme Analytique, Université de Picardie Jules Verne, 33 rue Saint Leu 80039
Amiens, France.
Institut Parisien de Chimie Moléculaire, CNRS UMR 8232, Sorbonne Université, 4
place Jussieu, 75252 Paris, France.
These authors contributed equally.
[20] but, to our knowledge, only few examples were described
b.
c.
with CDs. Per-azido β-CD and alkyne functionalized mannoside
have been coupled using a CuBr and 4,4’-bypyridine (bpy)
catalyst in DMSO yielding a β-CD-(Man)
7
glycocluster [21]. β-CD
‡
†
in memory of Aloysius Siriwardena.
substituted with the iminosugar 1-deoxynojirimycin (DNJ) has
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Journal Name
been prepared by CuAAC from a hepta-propargylated β-CD 71, 88 and 79% of per-6–deoxy-6-iodo α-, β-, and γ-CDs,
View Article Online
DOI: 10.1039/C9OB01344J
derivative and N-(ω-azidoalkyl)-DNJ [22].
respectively.
Herein, we introduce a robust and versatile approach using The obtained per-6-halogenated derivatives were used for the
microwaves to synthesize a glycosyl-CD family by CuAAC from synthesis of per-6-deoxy-6-azido α-, β-, -CDs intermediates via
different propargyl glycosides and 6-deoxy-6-azido -, - or nucleophilic substitution with azide anion [27-31]. Highly pure
CDs. Reaction conditions were carefully optimized to meet as α-, β-, and -CD azido-derivatives were obtained in 78, 90 and
γ
α
β
γ-
γ
1
3
much as possible the green chemistry criteria, especially to limit 87% yields, respectively. C NMR chemical shifts of C6 within
the amount of chemical reagents used. Two compounds per-azido CDs at 51.60 ppm, 51.58 ppm and 51.79 ppm for per-
revealed to be suitable inhibitors of α-glucosidase compared to 6-deoxy-6-azido α-, β-,
γ-CDs, respectively, were in good
iminosugars as acarbose. Finally, inhibition tests showed that agreement with previous reported data [32].
α
-
glucosylated cyclodextrin acted not as a substrate but as a On the other hand, the propargylated monosaccharides
mixed-type non-competitive inhibitor showing the effect of (Scheme 2) used in this study were synthesized according to
multivalent presentation of the substrate on enzyme activity.
procedures described elsewhere [33-38].
4 2
Click reaction in aqueous medium of CuSO .5H O/sodium
ascorbate at room temperature failed to lead to expected
clicked products. Further runs were attempted by varying the
temperature (45-80 °C), the number of equivalents of the
catalyst, and the reaction time (from several days to 2 weeks).
Unfortunately, the targeted products were obtained in low
yields and/or in the form of unresolving mixtures with by-
products. To overcome such limitations, we noticed that
microwaves were previously described as an efficient tool for
click chemistry [39-41]. Herein, microwaves were combined
with CuI - N,N-diisopropylethylamine (DIPEA) instead of
Results and discussion
The synthetic route starts by the halogenation of primary
positions of unprotected cyclodextrins [23]. Thus, CDs were
2 3
separately reacted with I -PPh in DMF, leading to the
corresponding per-6-deoxy-6-iodo-CD derivatives (Scheme 1).
Characterization of the obtained products by ESI-MS revealed
the presence of pseudo-molecular ions at m/z 1655.22, 1927.25
+
and 2198.80, corresponding to [M+Na] of α-, β- and γ-CD
derivatives, respectively. 3C NMR spectra exhibited all the
1
4 2
common CuSO .5H O/sodium ascorbate couple. It is well
documented that copper(I) iodide as the catalyst in the
presence of DIPEA can significantly increase yields of CuAAC
characteristic signals of CH
downfield (Δδ = 50 ppm) with respect to the initial ones
corresponding to CH OH. Moreover, a unique peak was
observed for CH I as required by the symmetries of uniformly
2
-I at the C6 which are shifted
2
[38]. The click reactions were performed using both protected
2
and unprotected propargyl glycosides, affording a library of
glycosyl CDs (Scheme 3) in moderate to excellent yields (28-
hexa-, hepta- and octa-substituted α-, β- and γ-CD, respectively.
The main advantage of this Vilsmeier-type iodination method
9
5%).
[24, 25] is that by-products are easily removed by treatment
with sodium methanolate to cleave the formate esters
previously formed. Our procedure for iodination was modified
from previously reported ones [26-28].
First, the commercial CDs were thoroughly freeze-dried before
use. Second, the addition of sodium methoxide 3 M in methanol
to adjust the pH in the range 9-10 was performed at room
temperature without further cooling, and the solution was kept
at room temperature at least for 30 min to cleave the formate
Scheme 2: Structures of propargyl glycopyranosides.
The results obtained for the various conjugation assays
between the three CDs and the propargyl glycosides clearly
showed that the reaction is quite versatile (Scheme 3),
whatever the type of monosaccharide, the anomeric centre
(
α, β), the nature of the glycoside bond (O, S). Nevertheless, in
some case no reaction occurred, such as for propargyl 2,3,4,6-
tetra-O-acetyl- -galactopyranoside where the reaction was
Scheme 1: Synthesis pathway of per-6-azido CDs from native effective with
- and -CDN (leading to 12 and 13), but not for
CDs.
-CDN under the same conditions. Indeed, TLC, HRMS and,
α
-D
7
α
γ
3
13
β
3
C
NMR only revealed the initial propargyl glycoside and some
esters formed during the reaction. Finally, the expected iodo- partially substituted intermediates. Such results can be due to
CDs were isolated by overnight precipitation in the steric hindrance driven by acetylated groups avoiding any
dichloromethane. With these improved conditions, yields were reactivity on the
β-CD having the more rigid structure compared
to
α
- and
γ
-CD. Although the reaction with protected glycosides
2
| J. Name., 2012, 00, 1-3
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DOI: 10.1039/C9OB01344J
was efficient, the deprotection of 12 and 13 was difficult
leading to mixture of partially deprotected glycosylated
cyclodextrins.
Figure 1: HSQC NMR experiment of 12 (DMSO d6, 25°C)
the excess of monosaccharides. Then the CDs derivatives were
eluted in pure water. All the glycosyl-CDs were characterized by
NMR and by ESI-HRMS to confirm their structure. As an
example, HSQC NMR spectrum of compound 12 is shown in
Figure 1.
Product (yield)
Propargyl
The quaternary carbon atom of triazole was observed at 142
glycoside
1
13
ppm as well as the CH-triazole at H/ C 8.02/125.1 ppm, the
CH -triazole at 4.63/50.28 ppm, and the CH of the linker at
.67/60.69 ppm. In addition, the structure of this clicked
α-CD
β-CD
γ-CD
2
2
7
8
9
12 (77%)
(<5%)
(<5%)
13 (67%)
15 (95%)
4
product was confirmed by HRMS (ESI+): m/z 1743.0302,
corresponding to the doubly sodiated ion (Figure 2).
14 (95%)
17 (92%)
20 (79%)
22 (31%)
Previous studies have shown that glycosides normally accepted
as substrates by glycosyl hydrolases, can inhibit these enzymes
when presented in multivalent structures on nanodiamonds [4].
The glycoCDs prepared in this study were therefore evaluated
for α-glucosidase inhibition. As a model, we have chosen α-
16 (77%)
19 (28%)
21 (47%)
18 (93%)
1
0
1
not synthesized
23 (84%)
1
Scheme 3: Synthesis and yields of glycosyl-CDs by CuI /DIPEA glucosidase from Saccharomyces cerevisiae because
based CuAAC reaction (with R=glycoside, see Scheme 2).
mammalian glucosidases are not commercially available [4].
Some of the glycoCDs showed strong inhibition of the enzyme,
We have thus preferred to click unprotected glycosides (8-11
)
as the glucose derivatives of β-CD (17) and
γ-CD (18), and the
because of their use in aqueous inhibition tests without any mannose derivative of
γ-CD (15) with IC50 values about 2-7 fold
further modification. In most cases, yields were as high as yields lower than acarbose used as positive control inhibitor (Figure
with acetylated glycosides. Some low yields can be explained by 3). IC50 values were 31.8 µM, 67.4 µM, 132.4 µM for compounds
a limited efficiency of purification steps of glycosylated CDs by 15
, 17 and 18, respectively, while values for acarbose were
flash chromatography. Silica gel column chromatography was about 250 µM. As expected native β-CD and methyl-α-D-
performed using a acetone-methanol-water gradient to remove glucopyranoside showed no inhibition.
[
M+Na+Cu]2+
[
M+2Na+K]3+
M+2Na]2+
[
[
M+3Na]3+
Figure 2: ESI+ HRMS of compound 12
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DOI: 10.1039/C9OB01344J
1
05.00
8
6
4
2
5.00
5.00
5.00
5.00
1
5
acarbose
methyl-a-D-glucopyranoside
α
17
18
b-CD
β
5
.00
0
.75
1.25
1.75
2.25
2.75
3.25
3.75
-15.00
Log Concentration (µM)
Figure 3: Percentage of inhibition of
α-glucosidase by cyclodextrin derivatives (see experimental section for details). Acarbose is
used as positive control, -CD and methyl-
β
α
-
D
-glucopyranoside are negative controls (n=3).
As previously described, multipresentation of
mannopyranoside led to good α-glucosidases inhibitors [5,42].
In the -glucopyranoside family, the -CD derivative 17 is a
better inhibitor than the -CD 18. This could be explained by the
higher flexibility of -CD compared to -CD even if this scaffold
can graft one additional monosaccharide.
In the case of -glucopyranoside, variation of concentration
α
-D-
a)
280.00
α
-
D
β
[
[
[
[
17]=0µM
γ
2
1
1
30.00
80.00
30.00
17]=11.75µM
17]=23.5µM
17]=46.87µM
γ
β
α
-D
of inhibitor 17 from 0 to 188 µM and 15 (0 to 11.72µM) was
studied to evaluate their mode of inhibition. Kinetic study using
the double reciprocal Lineweaver-Burk plot (Figure 4) revealed
that 17 and 15 inhibited α-glucosidase thanks an unusual mixed-
type non-competitive inhibition mode compared to acarbose
well known as a competitive inhibitor.
8
0.00
0.00
3
-
4.00
-2.00
0.00
2.00
/[pNPGlcp]
4.00
6.00
-
20.00
1
b)
180.00
Experimental
130.00
Materials and methods
Wacker Chemicals, Sigma- Aldrich, Fluka, Acros and TCI
supplied reagents and solvents. DMF were distilled under argon
atmosphere (on barium oxide) and stored with molecular
[
[
[
15]=0µM
8
0.00
0.00
15]=2.93µM
15]=11,72µM
sieves. Deuterated solvents (CDCl
purchased from Eurisotop (France).
3 2 6
, D O, DMSO-d ) were
3
Microwave conditions
-5.00
-3.00
-1.00
1.00
3.00
5.00
7.00
9.00
11.00
13.00
15.00
The apparatus is a Discover from CEM (France). It was used in
dy namic mode with a pre-stirring of 2min. Pressure was
controlled at 247 psi with power 200 W, temperature at 110°C
for 1h.
-
20.00
1
/[pNPGlcp]
Figure 4 Lineweaver-Burk plots for different concentrations of
a) 17 and b) 15 (n=3)
concentration of p-nitrophenyl
substrate in mM.
-
1
Chromatography
.
V is in
Δ
α
Abs.min , [pNPGlcp] is the
-D-glucopyranoside used as
Chromatographic separations were done in glass column filled
with silica gel (GERUDAN®, MERCK). Eluent composition was
specified for each compound in experimental part. Moreover,
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flash chromatographic device (Reveleris iES Flash System) following softwares: MestRenova 6.0.2 (MestRelab) or Topspin
View Article Online
DOI: 10.1039/C9OB01344J
supplied by Grace was also used. The compound detection was 3.2 (Bruker).
achieved with an evaporative light scattering detector and a UV Enzymatic tests
detector at two wavelengths (200 nm to 400 nm).
Mass Spectrometry
We herein examine the behaviour of some our CDs multivalent
derivatives towards α-glucosidase inhibition from
Stepwise control of the reactions has been readily achieved Saccharomyces cerevisiae. The inhibitory activity of our
using electrospray ionisation mass spectrometry (ESI-MS) using compounds and Acarbose was determined
a ZQ 4000 quadrupole mass spectrometer (Waters-Micromass, spectrophotometrically (UV-Visible, Tecan infinite M1000) on
Manchester, U.K.), provided with a pneumatically assisted 96-well microplate reader by studying the reaction of hydrolysis
electrospray (Z-spray) ion source. The compounds were of p-nitrophenyl-α-D-glucopyranoside. In the sample group
dissolved in MeOH, acetonitrile or water, alone or in mixture (As), to a total of 60µL of reaction mixture contained 20µL of
and were directly introduced into the ESI source at a flow rate 100mM phosphate buffer pH 6.8, 20 µL of our compounds at
of 20 μL/min through an integrated syringe pump. Source and different concentrations, 20 µL of 2.5 mM p-NPGlcp in
desolvation temperatures were set to 80°C and 150°C, phosphate buffer then 20 µL of 0.2 U/mL α-glucosidase in
respectively. Nitrogen has been used as desolvation and phosphate buffer were added and mixed well. In the control
nebulization gas with an output of 350 and 50 L/h, respectively. group (Ac), the composition of the reaction mixture was the
The capillary voltage was around 3.5 kV and the cone voltage same as in sample group except that phosphate buffer was used
was optimized from 20 to 150 V depending on analysed instead of our compounds, acarbose and methy-α-D-
products. Spectra were recorded at 2s/scan rate. High glucopyranoside. In the sample blank group (Asb) and in control
resolution electrospray mass spectrometry (ESI-HRMS) analyses blank group (Acb), the composition was the same as in sample
were performed on a SYNAPT G2-Si-Q-TOF hybrid quadrupole and control except that phosphate buffer was used instead of
time-of-flight instrument (Waters, Manchester, UK) equipped α-glucosidase. After incubation at 37°C for 15 min, the reaction
with an electrospray ionization (ESI) source (Z-spray) and an was stopped by adding 80 µL of 0.2 M sodium carbonate
additional sprayer for the reference compound (Lock Spray). solution [43,44]. Then the absorbance was measured at 405 nm.
Automated flow injections were performed using an ACQUITY Experiments were performed in triplicates.
UPLC H-Class system (Waters, Manchester, UK) coupled to the The inhibitory activity was calculated using the following
SYNAPT G2-Si-Q-TOF. The mobile phase composition was formula:
optimized according to the solvent used to dissolve the Inhibitory activity (%): (1-((As-Asb) /(Ac-Acb)))*100
compounds and the flow rate was set to 0.4 mL/min. One Different concentrations of our products were prepared as
microliter of each sample was injected and analyzed by ESI- follows:
HRMS in the positive ionization mode over the mass range 50- - 30mM, 3mM, 1.5mM, 750µM, 375µM, 187.5µM, 93.7µM and
4
000 at 0.4s/scan during an acquisition time set to 2 min.
46.8µM for the compounds.
The electrospray ionization conditions used a capillary voltage - 6mM, 3mM, 1.5mM, 750µM, 375µM, 187.5µM, 93.7µM and
of 3 kV, a sampling cone voltage at 120 V and a source offset 46.8µM for our compounds and methy-α-
voltage at 40 V. Source and desolvation gas temperatures were negative controls.
D-glucopyranoside as
1
20 °C and 450 °C, respectively. Nitrogen (>99.5%) was - 30mM, 15mM, 7.5mM, 3.75mM, 1.87mM, 937µM, 468µM and
employed as desolvation gas and cone gas with flows set to 600 234µM for acarbose as standard reference.
L/h and 50 L/h, respectively. Mass calibration was performed
using a solution containing a mixture of sodium and cesium To create the Lineweaver-Burk plot, a kinetical study in 30 min
iodide. A Leu-enkephalin solution was used for lock mass with measurements every 5 min is performed in same
correction by using m/z 556.2771 in order to obtain accurate conditions to that used for the α-glucosidase inhibitory assay.
mass measurements. The time of flight (TOF) was operated in The substrate was added at 8 concentrations: 10, 7.5, 5, 3.75,
the resolution mode, with an average resolving power reaching 2.5, 1.25, 0.625, and 0.312 mM. The mode of inhibition was
2
5,000 (FWHM). Data acquisition was performed with defined as competitive, non-competitive or mixed-type non-
MassLynx software (V4.1, Waters).
Nuclear Magnetic Resonance (NMR)
competitive, according to the Michaelis-Menten constants and
the maximum velocity on the Lineweaver-Burk plot.
Structure elucidation of compounds has been readily Experiments were performed in triplicates.
1
13
accomplished by recording standard H, C (or DEPTQ) spectra,
1
1
1
13
and 2D experiments such as H- H COSY, and H- C HSQC. All
NMR experiments were performed at 298 K on a Bruker Avance
III 600 Mhz spectrometer or a Bruker Avance III HD 400 MHz
spectrometer (Bruker, Wissembourg, France). Samples were
Synthetic procedures
Hexakis (6-deoxy-6-iodo)-cyclomaltohexaose (1)
Under argon, PPh (3.9 g, 14.8 mmol) and I (3.75 g, 14.8 mmol)
3 2
were dissolved in anhydrous DMF (16 mL). The reaction mixture
was stirred for 10 min then lyophilized α-cyclodextrin (0.8 g,
dissolved in deuterated solvent (Methanol-d
4
, CDCl
3 6
, DMSO-d ,
D
2
O, Pyridine-d
5
). Chemical shift (δ) was expressed in ppm and
1
13
H and C spectra were calibrated using the residual solvent
signal. Spectra processing was performed thanks to the
0
.82 mmol) was added to the above mixture and stirred
overnight at room temperature. After concentration under
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
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vacuum to 5 mL, the pH was adjusted in the range 9-10 by the The same procedure for the synthesis of
Journal Name
was used. Compound
4
View Article Online
DOI: 10.1039/C9OB01344J
addition of 3 M sodium methoxide in methanol (30 mL) with
3
(1.2 g, 0.55 mmol), DMF (43 mL) and NaN
3
(0.536 g, 8.25
as a white solid (0.72 g, 87% yield).
6
H NMR (400 MHz, DMSO-d ): δ 5.86-5.92 (16H, 3s, OH), 4.94
continuous stirring at room temperature for 30 min. The mmol) gave compound
mixture was poured into ice water, and the precipitate formed
6
1
and filtrated. DCM was added to the solid and left overnight. (8H, m , H1), 3.35 (8H, m, H4), 3.74 (8H, m, H2), 3.57 (8H, m,
1
3
The precipitate was separated and extensively washed with H3), 3.40 (8H, m, H5),3.58-3.72 (16H, m, H6). C NMR in
+
DCM until the filtrate becomes transparent, and finally with agreement with reference 27. ESI-MS: m/z 1520 [M+Na] .
methanol. After drying at 60° for 24 h, a faint brown solid
1
product was obtained (0.95 g, 71% yield). H NMR (400 MHz, General procedure for the CuAAC
6
DMSO-d ): δ 5.53-5.68 (12H, m, OH), 4.92 (6H, s, H1), 3.80 (6H, In a microwave reactor (10mL) under argon, to a solution of per-
bs, H2), 3.32 (6H, bs, H4), 3.61-3.67 (12H, 2s, H6), 3.34-3.51 azido
α
-,
β
- or
γ
-CD (
4
,
5
, or
6
, respectively) (0.0445 mmol) and
12H, m, H3, H5). 1 C NMR in agreement with reference 25. ESI- the corresponding propargyl glycoside (18, 21 or 24 equivalents,
3
(
+
MS: m/z 1655.22 [M+Na] .
respectively) in 2 mL of degassed dry DMF, CuI (5.1 mg, 0.0267
mmol) was added, followed by DIPEA (34 µL, 0.2 mmol). The
Heptakis (6-deoxy-6-iodo)-cyclomaltoheptaose (2)
The same procedure for the synthesis of
1 was used. DMF (16 mixture was irradiated at 200 W, and 110°C for 1h. The reaction
mL), PPh (4.2 g, 16 mmol) and I (4.0 g, 16 mmol), lyophilized mixture was concentrated and the residue was purified by silica
3
2
β-cyclodextrin (0.8 g, 0.71 mmol) gave 1.18 g (88% yield) of
2
.
gel column chromatography.
[6-deoxy-6-(2,3,4,6-tetra-O-acetyl-α-D-
1
H NMR (400 MHz, DMSO-d
6
): δ 3.29 (7H, m, H4), 3.33 (7H, m, Hexakis
H2), 3.44 (7H, m, H6a), 3.57 (7H, m , H5), 3.65 (7H, m, H3), galactopyranosyloxyethyl)triazol-1-yl]-cyclomaltohexaose
1
3
3
.81(7H, bd, H6b), 4.99 (7H, bs, H1), 5.91-6.01 (14H, 3s, OH).
C
(12)
NMR in agreement with reference 25. ESI-MS: m/z 1927.25 The excess of propargyl glycoside was eluted in 9:1:0.2
+
[
M+Na] .
2
Acetone/Methanol/H O. The silica gel was then washed with
Octakis (6-deoxy-6-iodo)-cyclomaltooctaose (3)
The same procedure for the synthesis of
mL), PPh (4.4 g, 16.6 mmol) and I
deionized water then the filtrate was lyophilized to give a white
1
1
was used. DMF (16 solid product (0.118 g, 77% yield). H NMR (600 MHz, DMSO-d
6
)
3
2
(4.2 g, 16.6 mmol), δ 1.86-2.11 (72H, m, CH
3
CO), 3.88 (6H, m, H'3), 3.87 (6H, m,
lyophilized γ-cyclodextrin (0.9 g, 0.69 mmol) gave 1.19 g (79 % H'5), 3.26 (6H, m, H'4), 4.02 (12H, s, H6), 4.23 (6H, m, H4), 4.25
1
yield) of
3
. H NMR (400 MHz, DMSO-d
6
): δ 5.98-5.74 (16H, 2 bs, (6H, m, H'2), 4.26 (6H, m, H'2), 4.63 (12H, m, H'6) , 4.67 (12H,
-linker), 4.93 (6H, s, H3), 5.06 (6H, s, H'1 ), 5.11 (6H, m,
16 H, bs, H3, H5), 3.27 (8H, bs, H4). 13C NMR in agreement with H1), 5.11 (6H, m, H2), 5.32 (6H, s, H5), 5.66-5.72 (12H, OH), 8.08
OH), 5.02 (8H, s, H1), 3.80 (8H, bs , H2), 3.60 (16H, bs, H6) , 3.39 m, CH
2
(
+
13
references 27. ESI-MS: m/z 2198.80 [M+Na] .
(6H, s, CH-triazole). C NMR (151 MHz, DMSO-d
CH CO), 169.05-169.54 (C=O), 70.15 (C'2), 72.87 (C'5), 83.15
2
(C'4), 61.71 (C6), 66.67 (C4), 73.48 (C'3), 50.28 (C'6), 60.69 (CH -
6
) δ 20.82-20.85
(
3
Hexakis (6-azido-6-deoxy)-cyclomaltohexaose (4)
To a solution of compound
DMF (34 mL), NaN (0.567 g, 8.73 mmol) was added at room (C'1), 125.08 (CH-triazole), 142.08 (qC triazole). HRMS (ESI+): [M
temperature, the reaction mixture was left stirring under argon + 2Na] 1743.0302 (calculated [M + 2Na] 1743.0332).
atmosphere for 24 h. The suspension was evaporated from DMF Octakis [6-deoxy-6-(2,3,4,6-tetra- -acetyl-α-D-
1 (0.95g, 0.58 mmol) in anhydrous linker), 67.38 (C2), 67.87 (C3), 68.03 (C5), 94.89 (C1), 101.74
3
2+
2+
O
to 3mL under reduced pressure, then about 100 mL from fresh galactopyranosyloxyethyl)triazol-1-yl]-cyclomaltooctaose (13)
deionized water was added to the above suspension, a faint The excess of propargyl glycoside was eluted in 1:1
yellowish precipitate was formed which was filtered and Acetone/H O. The silica gel was then washed with deionized
2
washed 5 times with deionized water then dried at 60° under water then the filtrate was lyophilized to give a white solid
1
vacuum, to afford compound
yield). H NMR (400 MHz, DMSO-d
4
as a white solid (0.51 g, 78% product (0.102 g, 67% yield). H NMR (400 MHz, DMSO-d
6
) δ
CO), 4.40 (8H,
-linker), 4.25 (8H,
): δ m, H3), 4.93 (8H, m, H5), 5.14 (8H, m, H4), 5.33 (8H, m, H2), 4.02
02.22 (C1), 83.64 (C4), 72.96 (C3), 71.81 (C5), 70.65 (C2), 51.60 (8H, m, H'3), 3.70 (8H, m, H'5), 3.40 (8H, s, H'2), 3.72 (8H, m,
1
6
): δ 3.60-3.72 (12H, m, H6), 7.94 (8H, s, CH-triazole), 1.88-2.09 (96H, 4s, CH
.88 (6H, s, H1), 5.47-5.66 (12H, 3s, OH), 3.37-3.81 (18H, m, H2, m, H6a), 4.95 (8H, m, H6b), 4.04 (16H, m, CH
2
3
4
1
3
H3, H5), 3.37 (6H, m, H4). C NMR (101 MHz, DMSO-d
6
1
(
+
C6). ESI-MS: m/z 1122 [M+Na] .
Heptakis (6-azido-6-deoxy)-cyclomaltoheptaose (5)
The same procedure for the synthesis of was used. Compound 21.06 (CH
(0.33 g, 0.17 mmol), DMF (12 mL) and NaN (0.169 g, 2.60 67.18 (C5), 66.88 (C4), 67.48 (C2), 69.72 (C'3), 72.31 (C'5), 72.46
as a white solid (0.205 g, 90% yield). (C'2), 81.72 (C'4), 95.11 (C1), 102.08 (C'1), 126.53 (CH-triazole),
H'4), 5.17 (8H, m, H1), 5.15 (8H, m, H'1), 4.39 (16H, m, H'6),
1
3
5.99-6.01 (16H, OH). C NMR (101 MHz, DMSO-d
6
) δ 20.93-
4
3
CO), 61.70 (CH -linker), 60.21-60.51 (C6), 66.38 (C3),
2
2
3
mmol) gave compound
H NMR (400 MHz, DMSO-d
5
1
6
): δ 5.76-5.91 (14H, 3s, OH), 4.91 49.79 (C'6), 170.09-170.61 (C=O), 143.10 (qC triazole). HRMS
7H, bs, H1), 3.34 (7H, m, H4), 3.74 (7H, m, H2), 3.60 (7H, m, H3), (ESI+): [M + 3Na] 1552.1418 (calculated [M + 3Na]
3+
3+
(
1
3
3
.38 (7H, m, H5), 3.60-3.78 (14H, m, H6). C NMR (101 MHz, 1552.1384).
): δ 102.47 (C1), 83.62 (C4), 70.76 (C2), 73.04 (C3), Heptakis [6-deoxy-6-(α-
DMSO-d
2.49 (C5), 51.73-51.77 (C6).
6
D
-mannopyranosyloxyethyl)triazol-1-
7
yl]-cyclomaltoheptaose (14)
+
ESI-MS: m/z 1333 [M+Na] .
The excess of propargyl glycoside was eluted in 9:1:0.2
Octakis (6-azido-6-deoxy)-cyclomaltooctaose (6)
2
Acetone/Methanol/H O. The silica gel was washed with
6
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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Page 7 of 9
O rP gl ea na si ce &d Bo i on mo to al e dc juu l sa tr mC ha er mg i ins ts ry
Journal Name
ARTICLE
deionized water then the filtrate was lyophilized to give a white 1419.4931; [M+ 2Na]2+ 1441.4745 (calculated [M+2H]
2+
View Article Online
1
DOI: 10.1039/C9OB01344J
solid product (0.103 g, 95% yield). H NMR (400 MHz, D
.17-4.36 (14H, m, H'6), 4.36-4.59 (14H, m, CH -linker), 8.00 Octakis [6-deoxy-6-(α-
7H, s, CH-triazole), 3.66-3.75 (14H, m, H6), 3.53 (7H, m, H4), cyclomaltooctaose (18)
2
O) δ 1419.4932).
4
2
D
-glucopyranosyloxyethyl)triazol-1-yl]-
(
4
.19 (7H, m, H2), 3.93 (7H, m, H3), 3.32 (7H, m, H'2), 3.46 (7H, The excess of propargyl glycoside was eluted in 9:1:0.2
O. The silica gel was washed with
m, H'3), 3.57 (7H, m, H5), 3.66 (7H, m, H'5), 3.32 (7H, m, H'4), Acetone/Methanol/H
2
4
7
7
.71 (7H, m, H1), 5.15 (7H, s, H'1). 13C NMR (101 MHz, D
2
O) δ deionized water then the filtrate was lyophilized to give a white
1
2.94 (C'3), 82.61 (C'4), 71.62 (C5), 69.93 (C'5), 72.33 (C'2), solid product (0.102 g, 93% yield). H NMR (400 MHz, D
2
O) δ
-linker), 8.02
-linker), 101.77 (C'1), 126.87 (8H, s, CH-triazole), 3.72 (16H, m, H6), 3.58 (16H, m, H3, H4),
2.35 (C3), 70.05 (C2), 66.63 (C4), 60.83-60.87 (C6), 50.45-50.49 4.19-4.33 (16H,m, H'6), 4.40-4.60 (16H, m, CH
2
(
C'6), 99.34 (C1), 59.51-59.53 (CH
2
2+
(
CH-triazole), 143.73 (qC triazole). HRMS (ESI+): [M+ 2Na]
4.27 (8H, m, H2), 3.58 (16H, m, H3), 3.36 (8H, m, H'2), 3.56 (16H,
m, H2, H5), 4.21 (8H, m, H'5), 3.38 (8H, m, H'4), 4.85 (8H, m, H1),
2+
1
441.4779 (calculated [M+2Na] 1441.4755).
1
3
Octakis [6-deoxy-6-(α-
yl]-cyclomaltooctaose (15)
The excess of propargyl glycoside was eluted in 9:1:0.2 69.46 (C2, C5), 60.46 (C6), 50.24-50.25 (C'6), 97.81 (C1), 59.92-
Acetone/Methanol/H O. The silica gel was washed with 59.94 (CH -linker), 101.55 (C'1), 126.93 (CH-triazole), 143.65 (qC
deionized water then the filtrate was lyophilized to give a white triazole). HRMS (ESI+): 1643.5422 [M+2Na]
D
-mannopyranosyloxyethyl)triazol-1- 5.12 (8H, bs, H'1), 3.93 ( 8H, m, H'3). C NMR (101 MHz, D
2
O) δ
72.22 (C'3), 82.04 (C'4), 69.80 (C'5), 69.45 (C'2), 71.90 (C3, C4),
2
2
2+
(calculated
1
2+
solid product (0.123 g, 95% yield). H NMR (400 MHz, D
.20-4.32 (16H, m, H'6), 4.42-4.64 (16H, m, CH -linker), 8.05 Hexakis [6-deoxy-6-(β-
8H, s, CH-triazole), 3.78-3.87 (16H, m, H6), 3.66 (8H, m, H4), cyclomaltohexaose (19)
2
O) δ [M+2Na] 1643.5431).
4
(
2
D-glucopyranosyloxyethyl)triazol-1-yl]-
4.27 (8H, m, H2), 3.98 (8H, m, H3), 3.60 (8H, m, H'2), 3.68 (16H, The excess of propargyl glycoside was eluted in Acetone. The
m, H'3, H5), 3.66 (8H, m, H'5), 3.43 (8H, m, H'4), 4.83 (8H, m, silica gel was washed with deionized water then the filtrate was
1
3
1
H1), 5.16 (8H, bs, H'1). C NMR (101 MHz, D
2
O) δ 71.89 (C'3, lyophilized to give a white solid product (0.030 g, 28% yield). H
O/MeOD d4) δ 4.70 (12H, m, H'6), 3.74-3.92
-linker) 4.74-4.86 (12H, m, H6), 3.37 (6H, m, H'2),
-linker), 101.62 (C'1), 126.60 (CH-triazole), 4.33 (6H, m, H'5), 3.24 (6H, m, H'3), 3.46-4.04 (24H, m, H2, H3,
43.49 (qC triazole). HRMS for the purified product showed at H4, H5), 3.42 (6H, m, H'4), 4.50 (6H, m, H1), 5.22 (6H, m, H'1),
C5), 82.26 (C'4), 70.46 (C'5), 72.86 (C'2), 72.15 (C3), 69.89 (C2), NMR (400 MHz, D
6.60(C4), 60.82-60.84(C6), 50.28-50.32 (C'6), 99.26 (C1), (12H, m, CH
9.38-59.39 (CH
2
6
5
1
1
2
2
2+
2+
13
643.5400 [M+2Na] (calculated [M+2Na] 1643.5431).
-glucopyranosyloxyethyl)triazol-1-yl]- 50.46 (C'6), 60.93-60.96 (CH
(C'2), 70.26 (C'5), 73.21 (C'3), 71.45-76.07 (C2, C3, C4, C5), 82.55
8.14 (6H, s, CH-triazole). C NMR (101 MHz, D
2
O/MeOD d4) δ
-linker), 61.76-61.86 (C6), 69.80
Hexakis [6-deoxy-6-(α-
D
2
cyclomaltohexaose (16)
The excess of propargyl glycoside was eluted in 1:1 (C'4), 101.82 (C1), 101.64 (C'1), 127.07 (CH-triazole), 151.03 (qC
2
+
2+
Acetone/Methanol. The silica gel was washed with deionized triazole). HRMS (ESI+): [M+Na+H] 1227.4132; [M+2H]
2+
2+
water then the filtrate was lyophilized to give a pale yellow solid 1216.9239; [M+ 2Na]
1238.9056 (calculated [M+2H]
1
product (0.083 g, 77% yield). H NMR (400 MHz, D
2
O) δ 4.30- 1216.9240).
-linker), 3.71 (12H, m, Heptakis [6-deoxy-6-(β-
H6), 3.56-3.68 (12H, m, H2-H5), 3.35 (6H, m, H'2), 4.29 (6H, m, yl]-cyclomaltoheptaose (20)
H'5), 4.02 (6H, m, H'3), 3.46-3.47 (12H, m, H3, H4), 3.38 (6H, m, The excess of propargyl glycoside was eluted in 9:1:0.2
H'4), 4.85 (6H, s, H1), 5.15 (6H, s, H'1), 8.09 (6H, s, CH-triazol). Acetone/Methanol/H O. The silica gel was washed with
4
.63 (12H, m, H'6), 4.47-4.64 (12H, m, CH
2
D-glucopyranosyloxyethyl)triazol-1-
2
13
C NMR (101 MHz, D
2
O) δ 50.89-50.90 (C'6), 60.38-60.42 (CH
2
-
deionized water then the filtrate was lyophilized to give a pale
linker), 60.92 (C6), 69.95 (C2,C5), 69.94 (C'2), 70.58 (C'5), 73.10 yellow solid product (0.048 g, 79% yield). H NMR (400 MHz,
1
(
C'3), 71.64-73.51 (C3, C4), 82.85 (C'4), 98.25 (C1), 101.80 (C'1),
D
2
O/MeOD d4) δ 4.08–4.49 (14H, m, H'6), 3.74-3.92 (14H, m,
1
2
27.62 (CH-triazole), 154.74 (qC triazole). HRMS (ESI+): [M+ CH -linker), 3.52-3.68 (14H, m, H6), 3.69-4.32 (21H, m, H'2, H'5,
2
Na]2 1238.9033 (calculated [M+2H] 1238.9064).
+
2+
H'3), 3.54- 4.37 (28H, m, H2, H3, H4, H5), 3.04 (7H, m, H'4), 4.88
Heptakis [6-deoxy-6-(α-
yl]-cyclomaltoheptaose (17)
The excess of propargyl glycoside was eluted in 9:1:0.2 62.71 (C6), 72.10-74.95 (C'2, C’5, C’3), 70.94-77.05 (C2, C3, C4,
Acetone/Methanol/H O. The silica gel was washed with C5), 81.07 (C'4), 102.07 (C1), 102.22 (C'1), 124.04 (CH-triazole),
deionized water then the filtrate was lyophilized to give a white 143.59 (qC triazole). HRMS (ESI+): [M+ 2Na] 1441.4738
D
-glucopyranosyloxyethyl)triazol-1- (7H, m, H1), 5.05 (7H, m, H'1), 7.61 (7H, s, CH-triazole). 13C NMR
2 2
(101 MHz, D O/MeOD d4) δ 49.42 (C'6), 61.16 (CH -linker),
2
2+
1
2+
solid product (0.100 g, 92% yield). H NMR (400 MHz, D
.22-4.47 (14H, m, H'6), 3.70 (14H, m, CH -linker), 4.41-4.62 Hexakis
14H, m, H6) , 4.21 (7H, m, H'5), 3.95 (7H, m, H3), 3.42 (7H, m, galactopyranosyloxyethyl)triazol-1-yl]-cyclomaltohexaose
H'3), 3.64 (7H, m, H'2), 3.34-3.53 (21H, m, H2, H4, H5), 3.36 (7H, (21)
2
O) δ (calculated [M+2Na] 1440.9842).
4
(
2
[6-deoxy-6-(1-thio-β-D-
1
3
m, H'4), 5.03 (7H, s, H1), 5.10 (7H, s, H'1). C NMR (101 MHz, The crude coupled product was purified by crystallization using
O) δ 50.28-50.30 (C'6), 60.38 (CH -linker), 59.84-59.85 (C6), methanol as the best solvent for dissolving the impurities and
9.89 (C'5), 72.32 (C3), 72.93 (C'3), 69.34 (C'2), 69.36-69.37 (C2, the excess of propargyl thioglycoside. The product was filtrated
C4, C5), 82.28 (C'4), 97.12 (C1), 101.59 (C'1), 143.56 (qC and dried under vacuum to give a reddish brown powder (0.064
D
6
2
2
2+
2+
1
triazole). HRMS (ESI+): [M+Na+H] 1430.4838; [M+2H]
2 2
g, 47% yield). H NMR (400 MHz, D O) δ 4.07 (12H, t, CH -S-
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
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O rP gl ea na si ce &d Bo i on mo to al e dc juu l sa tr mC ha er mg i ins ts ry
Page 8 of 9
ARTICLE
Journal Name
β-CD and perglucosylated β-CD were
View Article Online
linker), 4.30-4.61(12H, m, H'6), 3.54-3.69 (12H, m, H6), 3.60 (6H, permannosylated
m, H'2), 3.97 (6H, m, H5), 4.08 (6H, m, H'3), 3.44 (6H, m, H'4), identified as the best
.64 (6H, m, H'5), 3.60-3.63 (18H, m, H2, H3, H4), 3.44 (6H, m, interacting via a mixed non-competitive, inhibition mode.
H'3), 4.38 (6H, m, H1), 5.19 (6H, m, H'1), 8.06 (6H, m, CH-
DOI: 10.1039/C9OB01344J
α
-glycosidase inhibitors are the
3
1
3
triazole). C NMR (101 MHz, D
2
O) δ 23.60 (CH
0.52 (C'6), 60.98-61.11 (C6), 69.50 (C'2), 71.30 (C'5), 73.95-
9.02 (C2, C3, C4), 82.47 (C'4), 68.75 (C5), 72.66 (C'3), 85.23
2
-S- linker), 50.50-
Acknowledgments
We thank Dr Corinne Pau-Roblot and Dr Stéphanie Guenin for
helpful discussions on enzymatic experiments.
5
7
(
C1), 101.47 (C'1), 126.45 (CH-triazole), 144.98 (qC triazole).
2+
2+
HRMS (ESI+): [M+Na+H] 1275.8466; [M+2H] 1264.8559; [M+
+
2
Na]2 1286.8374 (calculated [M+2H]2+ 1264.8555).
Heptakis
galactopyranosyloxyethyl)triazol-1-yl]-cyclomaltoheptaose
22)
[6-deoxy-6-(1-thio-β-D-
Conflicts of interest
There are no conflicts to declare.
(
The excess of propargyl glycoside was eluted using a gradient
from 1:2 to 1:4 EtOAc/MeOH and finally 9:1:0.2
Acetone/Methanol/H O. The silica gel was washed with
2
Notes and references
1
https://www.who.int/news-room/fact-
deionized water then the filtrate was lyophilized to give a white
sheets/detail/diabetes (may 2019).
1
solid product (0.042 g, 31% yield). H NMR (400 MHz, D
2
O) δ
2
D. Kalita, D.G. Holm, D.V. LaBarbera, Petrash J.M., S.S. Jayanty,
Inhibition of α-glucosidase, α-amylase, and aldose reductase
by potato polyphenolic compounds. PLoS ONE, 2018, 13
e0191025.
3
(
(
.78-3.97 (14H, m, CH
14H, m, H6), 3.61 (7H, m, H'2), 3.67 (7H, m, H'5), 3.61-3.65
21H, m, H2, H3, H4), 3.46 (7H, m, H'4), 4.40 (7H, m, H1 , 5.18
2
-S-linker), 4.25-4.47 (14H, m, H'6), 3.70
)
3
4
5
S. Ghosh, P. More, A. Derle, A.B. Patil, P. Markad, A. Asok, N.
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(
4
7H, s, H'1
)
, 7.99-8.08 (7H, 2s, CH-triazole ), 3.98 (7H, m, H5),
.04 (7H, m, H'3). 13C NMR (101 MHz, D
2
O) δ 23.17-23.32 (CH
2
-
S-linker), 50.40-50.44 (C'6), 61.07 (C6), 69.42 (C'2), 71.64 (C'5),
7
1
7
3.95-79.07 (C2, C3, C4), 82.48 (C'4), 85.12 (C1), 101.67 (C'1),
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,
Octakis
[6-deoxy-6-(1-thio-β-D-
8
galactopyranosyloxyethyl)triazol-1-yl]-cyclomaltooctaose (23)
The excess of propargyl glycoside was eluted using a gradient
from 1:2 to 1:4 EtOAc/MeOH and finally 9:1:0.2
A. Siriwardena, M. Khanal, A. Barras, O. Bande,; Mena-
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6
7
8
9
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solid product (0.113 g, 84% yield). H NMR (600 MHz, D
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3
(
3
.75-3.84 (16H, m, CH
16H, s, H6), 3.86 (8H, s, H5), 4.04 (8H, m, H'3), 3.98 (8H, m, H'5),
.59 (8H, m, H'2), 3.53-3.59 (24H, m, H2, H3, H4), 3.37 (8H, m,
H'4), 4.33 (8H, m, H1), 5.08 (8H, s, H'1), 7.91 (8H, s, CH-triazole).
2
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C NMR (151 MHz, D
2 2
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6
1
2
9.48-78.98 (C2, C3, C4), 82.08 (C'4), 85.13 (C1), 101.68 (C'1),
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017, 22, 1311.
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