Multivalent iminosugars to modulate affinity and selectivity for glycosidases

Article abstract of DOI:10.1039/b815408b

A series of mono-, di- and tri-valent iminosugars based on oligoethylene scaffolds and N-substituted deoxynojirymicin epitopes have been synthesized by "click chemistry" to study the effect of multivalency on glycosidase inhibition. Biological evaluation evidenced differences in the inhibition trends as a function of the enzyme nature. The results demonstrate that multivalency can be used in some case to modulate both the affinity and the selectivity of glycosidase inhibition.

Full text of DOI:10.1039/b815408b

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Multivalent iminosugars to modulate affinity and selectivity for glycosidases†
Jennifer Diot,a M. Isabel Garc´ıa-Moreno,b Se´bastien G. Gouin,*a Carmen Ortiz Mellet,b Karsten Hauptc and
Jose´ Kovensky*a
Received 3rd September 2008, Accepted 17th October 2008
First published as an Advance Article on the web 18th November 2008
DOI: 10.1039/b815408b
A series of mono-, di- and tri-valent iminosugars based on oligoethylene scaffolds and N-substituted
deoxynojirymicin epitopes have been synthesized by “click chemistry” to study the effect of
multivalency on glycosidase inhibition. Biological evaluation evidenced differences in the inhibition
trends as a function of the enzyme nature. The results demonstrate that multivalency can be used in
some case to modulate both the affinity and the selectivity of glycosidase inhibition.
iminocyclitols (iminosugars, azasugars)8 seem particularly ap-
Introduction
propriate for such goals, since they have been broadly used
Lectin–carbohydrate interactions have been intensively investi-
as affinity ligands for the purification of several glycosidases
gated, as they play a pivotal role in a host of biological events.1 In
after conjugation with a solid support, which actually implies
a multivalent presentation.9 We described herein the efficient
preparation of a series of mono- (1, 2), di- (3, 4) and tri-valent
(5; Fig. 1) derivatives of the iminosugar glycosidase inhibitor
1-deoxynojirimycin (1-DNJ).
order to overcome the weak binding affinity of monomeric sugar
ligands toward lectins, many groups have developed synthetic
glycoconjugates presenting multiple copies of the sugar epitopes.2
Affinity enhancements up to 6 orders of magnitude have been
reported for some multivalent synthetic glycoconjugates.3 Inter-
estingly, multivalency can also be put forward to modulate lectin
specificity.4
Although a strong multivalent or cluster effect is generally
associated with the interaction of receptors bearing multiple
recognition sites with multivalent sugar ligands, significant affinity
enhancements have also been observed for systems where the
receptor possesses a single binding centre available.5 Whereas the
chelate effect and intermolecular aggregative processes frequently
dominate in the first case, statistical rebinding and local concen-
tration effects may prevail in the last scenario.6 This situation
approaches that encountered in enzymes having a single catalytic
site. We reasoned that the affinity of an enzyme inhibitor could
be modulated in a similar manner by designing multivalent
derivatives of appropriate geometry, providing that the kinetic and
thermodynamic parameters of the individual binding phenomena
are appropriate.
While hundreds of multivalent glycomimetics have been synthe-
sized for studying carbohydrate-lectin interactions, evaluation of
multivalent inhibitor presentations on glycosidase inhibition has
been largerly disregarded.7 A main reason is, probably, the higher
difficulty of preparing functionalized azasugars regarding to
glycoconjugates and to graft them onto a scaffold. N-Substituted
aLaboratoire des Glucides UMR CNRS 6219, Institut de Chimie de
Picardie, Faculte´ des Sciences, Universite´ de Picardie Jules Verne, 33 rue
Saint-Leu, 80039, Amiens Cedex 1, France. E-mail: sebastien.gouin@u-
picardie.fr, jose.kovensky@u-picardie.fr; Fax: +33 03 22 82 75 60; Tel: +33
(0)3 22 82 75 69
Fig. 1 Structures of synthetic iminosugars.
bDepartamento de Qu´ımica Orga´nica, Facultad de Qu´ımica, Universidad de
Sevilla, Profesor Garc´ıa Gonza´lez 1, E-41012, Sevilla, Spain
The copper-catalyzed azide–alkyne cycloaddition (CuAAC),
described independently by Sharpless and Meldal,10 has been
implemented by using azide-functionalized 1-DNJ derivatives
and alkynyl-armed oligo(ethyleneglycol) (EG) scaffolds. The com-
pounds were designed to provide information on the structure-
activity relationships against a panel of glycosidase as a function of
cLaboratoire Ge´nie Enzymatique et Cellulaire, UMR CNRS -6022, Uni-
versite´ de Technologie de Compie`gne BP 20205, 60205, Compie`gne Cedex,
France
† Electronic supplementary information (ESI) available: 1H and 13C NMR
spectra of compounds 1–5, 9–13 and 16–20. See DOI: 10.1039/b815408b
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both the distance between the functional epitopes and the number
of active moieties.
Results and discussion
We decided to introduce the azido-propyl chain on the nitrogen of
DNJ because this position will not impair the binding and some
N-alkyl-DNJs are potent glycosidase inhibitors (i.e. N-butyl-1-
deoxynojirimycin).11 The synthesis of 9 was performed in five
steps from commercially available methyl-a-D-glucopyranoside
(Scheme 1).
Scheme 2
Scheme 1
4 were obtained in similar yields starting from the previously
described EG derivatives 14 and 15.19 In each case CuAAC was
highly regioselective, yielding 1,4-disubstitued triazole structures
Benzylation,12 acid hydrolysis of the methyl group13,14 and
quantitative reduction of the resulting hemiacetal with lithium
aluminium hydride afforded the known diol 6. Subsequent Swern
(12, 13 and 16, 17, respectively), identified by the large D(dC-4
C-5) values (around 22 ppm) observed in the corresponding 13C
NMR spectra for the triazole carbon atoms.20
-
◦
oxidation was conducted at -78 C to oxidize both alcohols.15
d
Compound 7 was too unstable to be isolated in pure form and
has been used in the next step without further purification.
Reductive amination in the presence of 4-azidopropylamine 816
under microwave irradiation at 50 ◦C allowed to obtain 9 in 46%
overall yield (two steps). Regarding to classical heating at the same
temperature, the reaction time was shorter (24 h to 1 h) and the
yield increased (13% to 46%). Exclusively the diastereomer having
the (R)-configuration at the C-5 position, analogous to that of
D-glucose, was detected.
The use of hydrophilic EG linkers to tether the 1-DNJ motifs
was expected to avoid water solubility problems. Moreover, EGs
have been shown to prevent non-specific adsorption of proteins.17
This last property is particularly relevant in our study in order
to avoid interferences due to interactions of the glycosidases
with the scaffold. The monovalent derivatives 1 and 2, with EG
substituents of different length, were first synthesized as reference
compounds. The required mono-functionalized derivatives 10 and
11 were obtained by reaction of the commercially available EGs
with propargyl bromide. CuAAC was performed with compounds
10 and 11 on iminosugar 9 in the presence of copper sulfate and
sodium ascorbate in a mixture of dioxane and water, affording the
cycloadducts 12 and 13 in 60% yield (Scheme 2).
Compound 5 was designed to incorporate spacer arms of similar
length as those in the mono- and divalent compounds 2 and 4.
Firstly, a tosyl group was introduced on 10 leading to 18 with
91% yield (Scheme 3). This EG was then grafted onto 1,1,1-
tris(hydroxymethyl)ethane in THF using sodium hydride as base
to give 19. CuAAC between 19 and the iminosugar 9 followed by
deprotection of the benzyl groups in the corresponding adduct 20
afforded the target trivalent 1-DNJ derivative 5 in 60% yield (two
steps). The unprotected inhibitors 1–5 were subjected to a final
preparative HPLC purification step in order to ensure that they
were not polluted by any salts before their biological evaluation.
The inhibition constants (Ki) of the synthetic azasugars 1–5
against several glycosidases are summarized in Table 1. Literature
data for 1-DNJ are included for comparative purposes. Generally,
the incorporation of the N-substituent had a deletorious effect
in the inhibitory potency (see data for 1 and 2) probably due
to steric clashes at the active site. Two significant exceptions
are b-glucosidase and a-mannosidase. For most glycosidases,
multivalent iminosugars do not bind more tightly regarding to
analogous monovalent derivatives. However, for amyloglucosi-
dase, although the presence of the triazolylpropyl substituent
causes a 10-fold decrease in the Ki value as compared with
1-DNJ, going from monovalent (1, 2) to divalent (3, 4) and trivalent
derivatives (5) results in a parallel increase in the inhibitory activity,
that is compatible with statistical rebinding. In the case of a-
mannosidase, the increase of inhibitory potency with increasing
Hydrogenolysis of the benzyl groups in 12 and 13 with Pd/C
or Pd(OH)2/C in methanol failed, probably due to poisoning of
the catalyst by amino group and/or the triazole moieties.18 Con-
ducting the reaction in acidic medium gave the fully deprotected
compounds 1 and 2 (quantitative). The divalent derivatives 3 and
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Table 1 Glycosidase Inhibitory Activities (Ki, mM) for 1-DNJ and the synthetic derivatives inhibitors 1–5 a
Enzyme
1-DNJ8
1
2
3
4
5
b-Galactosidase (bovine liver)
a-Galactosidase (green coffee beans)
b-Glucosidase (almonds, pH 7.3)
a-Glucosidase (bakers’ yeast)
b-Mannosidase (Helix pomatia)
a-Mannosidase (Jack bean)
42
—
47
25
—
270
11
950 20
n.i.
95
100
n.i.
n.i.
265 10
480 15
95
30
n.i.
215
70
n.i.
n.i.
120
n.i.
60
760 10
790 10
120
47
5
2
5
5
5
2
105
70
5
2
225
400 10
n.i.
n.i.
35
1440 20
5
n.i.
n.i.
230 10
n.i.
772 10
90
770 15
5
3
2
Isomaltase (bakers’ yeast)
1050 20
Naringinase (Penicillium decumbens)
Amyloglucosidase (Aspergillus niger)
—
2.1
80
28
3
1
5
2
3
1
55
20
2
2
75
11
3
1
17
a n.i. = no inhibition detected at 2 mM.
compatible with statistical effects. The multivalent effect observed
for a-mannosidase could be ascribed either to new interactions
of the additional epitopes close to the binding site or to a
sliding mechanism of the iminosugars in the primary binding
site as previously observed with multivalent glycoconjugates and
lectins.4,21 Though much more research is needed to understand
these phenomena, the present results demonstrate the suitability of
the synthetic approach to design multivalent glycosidase inhibitors
with defined architecture. If the glycosidase inhibitory activities
indicate that in general, multimeric iminosugars do not bind more
tightly, our results suggests that, in some cases, it is possible to use
this strategy to modulate the potency and the selectivity towards
different glycosidases.
Experimental
Synthesis
General procedure. All purchased materials were used without
further purification. Methylene chloride was distilled from calcium
hydride and tetrahydrofuran over sodium and benzophenone.
Analytical thin layer chromatography (TLC) was carried out on
Merck D.C.-Alufolien Kieselgel 60 F254. Flash chromatography
(FC) was performed on GEDURAN SI 60, 0.040–0.060 mm
Scheme 3
1
valency is stronger. The divalent and trivalent compounds 4 and
5 are 3 and 7.6-fold more potent than monovalent 2, meaning 1.5
and 2.5-fold in a molar basis, respectively.
Additionally, valency has a dramatic effect in the selectivity
profile of the inhibitors. Thus, going from monovalent 1 or 2
to trivalent 5 fully reverses the selectivity between b-glucosidase
and a-mannosidase. No significant changes were observed as a
function of the EG segment length.
pore size using distilled solvents. H, and 13C nuclear magnetic
resonance (NMR) spectra were recorded at 300 (75.5) MHz with
a Bruker AC-300 spectrometer, and chemical shifts are reported
in parts per million relative to tetramethylsilane or a residual
1
solvent peak (CHCl3: H: d 7.26, 13C: d 77.2). Peak multiplicity
is reported as: singlet (s), doublet (d), triplet (t), quartet (q),
pentet (p), sixtet (s), multiplet (m), and broad (br). High resolution
mass spectra HRMS were obtained by Electrospray Ionisation
(ESI) on a Micromass-Waters Q-TOF Ultima Global. Optical
rotations were measured on a 343 PERKIN ELMER at 20 ◦C
in a 1 cm cell in the stated solvent; [a]D values are given in
10-1 deg.cm2 g-1 (concentration c given as g/100 mL). Microwave
irradiation was performed in a CEM Discover apparatus (300 W).
Preparative reversed-phase HPLC was accomplished on a Waters
PREP LC 4000 chromatography system with a (DEDL) PL-ELS
1000 photodiode array detector. All HPLC samples were purified
on a preparative Prevail C-18 column (2.2 ¥ 25 cm). The mobile
phase was H2O (solvent A) and MeOH (solvent B). The gradient
consisted of 100% A for 5 min then 100% A to 100% B in 35 min
(22 mL/min flow rate).
Conclusion
At present it is difficult to rationalize the dependence of the
inhibitory activity towards valency for the different glycosidases.
The existence of a relatively deep catalytic site and the fact
that the formation of the enzyme-inhibitor complex frequency
involves relatively slow conformational changes probably preclude
sliding, local concentration effects and even statistical rebinding
for multivalent ligands in most cases. The catalytic site in
amyloglucosidase, an enzyme involved in the hydrolysis of large
polysaccharides, is probably comparatively more accessible, being
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∫
N-(3-Azidopropyl)-2,3,4,6-tetra-O-benzyl-1,5-dideoxy-1,5-imino-
D-glucitol (9). 7 was synthesized as described in litt.22 The crude
product 7 (2.00 g, 3.72 mmol) and 1-azido-3-amino propane
(8, 7.50 g, 73.4 mmol) were dissolved in MeOH (25 mL) and
the mixture was stirred at 0 ◦C for 20 min. Then, sodium
cyanoborohydride (937 mg, 14.8 mmol) was added and the
mixture was irradiated for 1 h at 50 ◦C (300 W) in a sealed
vessel. After evaporation of the organic solvent under reduced
pressure, the residue was dissolved in DCM (50 mL) and the
organic layer was washed with NaHCO3 satd. (2 ¥ 50 mL),
dried over Na2SO4 and filtered. The solvent was removed under
reduced pressure and the dry residue was purified by FC (97:3
cyclohexane-EtOAc) to give 9 (1.03 g, 46%) as a white solide. Rf =
0.43 (9:1 cyclohexane-EtOAc), [a]D +2 (c = 1, CH2Cl2); 1H NMR
(300 MHz, CDCl3): d = 7.36–7.29 (20 H, m, Har), 4.97–4.83 (3
H, 3 d, J 11.0 Hz, 11.1 Hz, 10.9 Hz, CH2Ph, CHHPh), 4.71–4.69
(2 H, m, CH2Ph), 4.50 (3 H, m, CH2Ph, CHHPh), 3.67–3.50 (4 H,
m, H-2, H-4, 2 H-6), 3.51 (1 H, t, J 8.9 Hz, H-3), 3.21 (2 H, ddd, J
12.3 Hz, 6.4 Hz, 5.9 Hz, CH2N3), 3.07 (1 H, dd, J1a,1b 11.0 Hz, J1,2
4.8 Hz, H-1a), 2.81 (1 H, ddd, J 6.3 Hz, 4.3 Hz, 3.2 Hz CHHN),
2.65 (1 H, ddd, CHHN), 2.35 (1H, d, J 9.2 Hz, H-5), 2.23 (1 H, t, J
11.0 Hz, H-1b), 1.68 (2 H, m, NCH2CH2CH2N3); 13C NMR (75.5
MHz, CDCl3): d = 139.1, 138.6, 137.8 (Car), 128.5, 128.4, 128.0,
127.8, 127.7, 127.6 (CHar), 87.4 (C-3), 78.7, 78.5 (C-2, C-4), 75.4,
75.3, 73.5, 72.9 (CH2Ph), 66.1 (C-6), 64.2 (C-5), 54.7 (C-1), 49.8
(CH2N3), 49.5 (CH2N), 24.2 (NCH2CH2CH2N3); HRMS (ESI+):
Found 629.3110 C37H42N4O4Na requires 629.3104.
H, br, OH), 2.42 (1 H, t, J 2.3 Hz, HC C); 13C NMR (75.5 MHz,
CDCl3): d = 79.7 (C-17), 74.6 (C-18), 72.7, 70.6, 70.4, 70.3, 69.1
(CH2), 61.7 (C-1), 58.4 (C-16); HRMS (ESI+): Found 299.1472
C13H24O6Na requires 299.1471.
4-[7-Hydroxy-2,5-dioxaheptyl]-1-[3-(2,3,4,6-tetra-O-benzyl-1,5-
dideoxy-1,5-imino-D-glucitol-N-yl)-propyl]-1H-[1,2,3]-triazole (12).
To a solution of the alkyne 10 (42 mg, 0.165 mmol) and the
azido-derivative 9 (100 mg, 0.165 mmol) in dioxane-H2O (5 mL,
4:1) copper sulfate (42 mg, 0.264 mmol) and sodium ascorbate
(83 mg, 0.528 mmol) were added and the mixture was irradiated
for 30 min at 80 ◦C in a sealed vessel. The mixture was poured
into an NH4Cl satd. solution (20 mL) and extracted with EtOAc
(3 ¥ 20 mL). The organic layer was dried (Na2SO4), filtered and
the solvent removed under reduced pressure. The residue was
purified by FC (1:19 MeOH-EtOAc) to give 12 (50 mg, 60% yield)
as a colourless oil. Rf = 0.17 (1:19 MeOH-EtOAc); [a]D +3 (c = 1,
CH2Cl2); 1H NMR (300 MHz, CDCl3): d = 7.47 (1 H, s, CCHN),
7.33–7.16 (20 H, m, Har), 4.97–4.79 (3 H, 3 d, J 11.0 Hz, 10.9
Hz, 10.6 Hz, CH2Ph, CHHPh), 4.68–4.61 (4 H, m, CH2Ph, H-6),
4.45–4.40 (3 H, m, CH2Ph), 4.29–4.15 (2 H, m, CH2NN), 3.73–
3.66 (10 H, m, CH2), 3.59–3.56 (2 H, m, H-2, H-4), 3.49 (1 H, t, J
7.2 Hz, H-3), 3.02 (1 H, dd, J 11.0 Hz, J 4.7 Hz, H-1a), 2.79 (1 H,
m, CHHN), 2.54 (1 H, m, CHHN), 2.35 (1 H, d, J 8.3 Hz, H-5),
2.16 (1 H, t, J 11.0 Hz, H-1b), 1.99 (2 H, m, NCH2CH2CH2NN);
13
=
C NMR (75.5 MHz, CDCl3): d = 144.9 (NC CH), 138.9, 138.4,
137.8 (Car), 128.5, 128.4, 128.3, 128.0, 127.9, 127.8, 127.7, 127.58
=
(CHar), 122.0 (NCC H), 87.1 (C-3), 78.5, 78.3 (C-2, C-4), 75.4,
3,6-Dioxanon-8-yn-1-ol(10). Bis(2-hydroxyethyl)ether (500 mg,
4,71 mmol) and sodium hydride (170 mg, 7.08 mmol) were
dissolved in anhydr DMF (40 mL) and the mixture was stirred at
0 ◦C for 30 min. Then, propargyl bromide (0.60 mL, 7.08 mmol)
was added dropwise and the resulting mixture stirred for 6 h at rt.
Methanol (5 mL) was added and the mixture was evaporated under
reduced pressure and the residue dissolved in ethyl acetate (20 mL).
The organic layer was washed with saturated ammonium chloride
(20 mL), the aqueous layer was extracted with ethyl acetate (2 ¥
20 mL), and the combined organic layers were evaporated under
vacuum. The dry residue was purified by FC (1:4 cyclohexane-
EtOAc) to give 10 (407 mg, 60%) as a colourless oil. Rf = 0.49
(EtOAc); 1H NMR (300 MHz, CDCl3): d = 4.18 (2 H, d, J 2.4 Hz,
75.3, 73.4, 73.2 (CH2Ph), 72.9, 70.5 (CH2), 66.3 (OCH2C), 64.6
(C-6), 64.5 (C-5), 61.5 (CH2OH), 54.4 (C-1), 49.2 (CH2NN),
48.4 (CH2N), 26.0 (NCH2CH2CH2N); HRMS (ESI+): Found
773.3869 C44H54N4O7Na requires 773.3890.
4-[16-Hydroxy-2,5,8,11,14-pentaoxahexadecyl]-1-[3-(2,3,4,6-
tetra-O-benzyl-1,5-dideoxy-1,5-imino-D-glucitol-N-yl)-propyl)]-
1H-[1,2,3]-triazole (13). To a solution of the alkyne 11 (52 mg,
0.165 mmol) and the azido-derivative 9 (100 mg, 0.165 mmol)
in dioxane-H2O (5 mL, 4–1) copper sulfate (42 mg, 0.26 mmol)
and sodium ascorbate (83 mg, 0.528 mmol) were added and the
mixture was irradiated at 80 ◦C for 30 min in a sealed vessel. The
mixture was poured into a NH4Cl satd. solution (20 mL) and
extracted with ethyl acetate (3 ¥ 20 mL). The organic layer was
dried (Na2SO4), filtered and the solvent removed under reduced
pressure. The residue was purified by FC (1:19 MeOH-EtOAc) to
give 12 (87 mg, 60%) as a colourless oil. Rf = 0.37 (1:9 MeOH-
∫
∫
CH2C C), 3.71–3.64 (8 H, m, CH2), 2.41 (1 H, t, J 2.4 Hz, HC C);
13C NMR (75.5 MHz, CDCl3): d = 79.5 (C-8), 74.8 (C-9), 72.6,
70.2, 69.1 (CH2), 61.7 (C-1), 58.4 (C-7); HRMS (ESI+): Found
167.0677 C7H12O3Na requires 167.0684.
1
3,6,9,12,15-Pentaoxaoctadec-17-yn-1-ol (11). Pentaethyleneg-
lycol (500 mg, 2.09 mmol) and sodium hydride (75 mg, 3.13 mmol)
were dissolved in anhydr. DMF (40 mL) and the mixture was
stirred at 0 ◦C for 30 min. Then, propargyl bromide (240 mL,
3.13 mmol) was added dropwise and the resulting mixture stirred
for 6 h at rt. MeOH (5 mL) was added and the mixture was
evaporated under reduced pressure, and the residue dissolved in
EtOAc (20 mL). The organic layer was washed with saturated
ammonium chloride (20 mL). Aqueous layer was extracted with
EtOAc (2 ¥ 20 mL), the organic layers were combined and
evaporated under vacuum. The dry residue was purified by FC
(2:98 MeOH-EtOAc) to give 11 (346 mg, 60%) as a colourless oil.
Rf = 0.2 (49:1 EtOAc-MeOH); 1H NMR (300 MHz, CDCl3): d =
EtOAc); [a]D +3 (c = 1, CH2Cl2); H NMR (300 MHz, CDCl3):
d = 7.50 (1 H, s, CCHN), 7.32–7.14 (20 H, m, Har), 4.97–4.78 (3 H,
3d, J 11.1 Hz, 11.0 Hz, 10.8 Hz, CH2Ph, CHHPh), 4.67–4.61 (4 H,
m, CH2Ph, H-6), 4.45–4.39 (3 H, m, CH2Ph, CHHPh), 4.28–4.17
(2 H, m, CH2NN), 3.71–3.64 (22 H, m, CH2), 3.59–3.56 (2 H, m,
H-2, H-4), 3.49 (1 H, t, J 7.5 Hz, H-3), 3.02 (1 H, dd, J 11.0 Hz,
J 4.7 Hz, H-1a), 2.80 (1 H, m, CHHN), 2.54 (1 H, m, CHHN),
2.30 (1 H, d, J 8.5 Hz, H-5), 2.15 (1 H, t, J 11.0 Hz, H-1b), 1.99
(2 H, m, NCH2CH2CH2NN); 13C NMR (75.5 MHz, CDCl3): d =
=
145.0 (NC CH), 138.9, 138.4, 137.7 (Car), 128.4, 128.3, 128.2,
127.9, 127.8, 127.7, 127.6, 127.5 (CHar), 122.9 (NC CH), 87.0
(C-3), 78.5, 78.2 (C-2, C-4), 75.3, 75.2, 73.3, 73.2 (CH2Ph), 72.8
(CH2), 70.4 (CH2), 70.1, 69.6 (CH2), 66.2 (OCH2C), 64.6 (C-
6), 64.4 (C-5), 61.5 (CH2OH), 54.3 (C-1), 49.1 (CH2NN), 48.4
=
∫
4.17 (2 H, d, J 2.4 Hz, CH2C C), 3.63 (20 H, m, CH2), 3.03 (1
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(CH2N), 25.9 (NCH2CH2CH2N); HRMS (ESI+): Found 905.4700
C50H66N4O10Na requires 905.4677.
CHHN), 2.34 (2 H, d, J 7.6 Hz, H-5), 2.16 (2 H, t, J, H-1b), 1.99
(4 H, m, NCH2CH2CH2NN); 13C NMR (75.5 MHz, CDCl3): d =
=
=
145.0 (NC CH), 145.1 (NC CH), 139.0, 138.5, 137.8 (Car), 128.5,
128.4, 128.3, 127.97, 128.0, 127.7, 127.6 (CHar), 122.8 (NCHC),
87.1 (C-3), 78.6, 78.3 (C-2, C-4), 75.4, 75.3, 73.3, 72.9 (CH2Ph),
70.6, 69.8 (CH2), 66.3 (OCH2C), 64.7 (C-6), 64.5 (C-5), 54.5 (C-1),
49.2 (CH2NN), 48.4 (CH2N), 26.0 (NCH2CH2CH2NN); HRMS
(ESI+): Found 1417.7256 C84H98N8O11Na requires 1417.7253.
1-[3-(1,5-Dideoxy-1,5-imino-D-glucitol-N-yl)-propyl)]-4-[7-hy-
droxy-2,5-dioxaheptyl]-1H-[1,2,3]-triazole
(1). Conventional
catalytic hydrogenation of compound 12 (100 mg, 0.133 mmol)
was carried out with Pd(OH)2 (90 mg, 0.641 mmol) in MeOH-1M
aq. HCl solution (8 mL, 1:1) at 1 atm for 15h. Then, the catalyst
was filtered over celite and the solvent removed under reduced
pressure to give 1 (quant) as a hygroscopic white solid purified
by preparative HPLC (tR = 12.03 min); [a]D -6 (c = 0.2, H2O);
1H NMR (300 MHz, D2O): d = 8.05 (1 H, s, CCHN), 4.68 (2
H, s, H-6), 4.44 (2 H, t, J 7.6 Hz, CH2NN), 3.79–3.68 (8 H, m,
CH2), 3.63–3.59 (2 H, m, CH2), 3.50 (1 H, dt, J 9.6 Hz, J 4.9 Hz,
H-2), 3.36 (1 H, t, J 9.6 Hz, H-4), 3.23 (1 H, t, H-3), 2.94 (1 H,
dd, J 11.3 Hz, H-1a), 2.69 (2 H, m, CH2N), 2.26 (2 H, m, H-1b,
H-5), 2.09 (2 H, m, NCH2CH2CH2NN); 13C NMR (75.5 MHz,
Bis-1,18-[1-(3-(2,3,4,6-tetra-O-benzyl-1,5-dideoxy-1,5-imino-D-
glucitol-N-yl)-propyl)triazol-4-yl]-2,5,8,11,14,17-hexaoxaoctadec-
ane (17). To a solution of the alkyne 14 (24 mg, 0.0825 mmol)
and the azido-derivative 9 (100 mg, 0.165 mmol) in dioxane-H2O
(5 mL, 4:1), copper sulfate (41 mg, 0.264 mmol) and sodium
ascorbate (81 mg, 0.528 mmol) were added and the mixture was
irradiated at 80 ◦C for 45 min in a sealed vessel. The mixture was
poured into a NH4Cl satd. solution (20 mL) and extracted with
ethyl acetate (3 ¥ 20 mL). The organic layer was dried (Na2SO4),
filtered and the solvent removed under reduced pressure. The
residue was purified by FC (1:49 MeOH-EtOAc) to give 17
(69 mg, 55%) as a colourless oil. Rf = 0.33 (1:49 MeOH-EtOAc);
[a]D +4 (c = 1, CH2Cl2); 1H NMR (300 MHz, CDCl3): d = 7.46
(2 H, s, CCHN), 7.32–7.13 (40 H, m, Har), 4.98–4.79 (6 H, 3
d, J 11.1 Hz, 11.0 Hz, 10.9 Hz, CH2Ph, CHHPh), 4.68–4.62
(8 H, m, CH2Ph, H-6), 4.45–4.36 (6 H, m, CH2Ph, CHHPh),
4.29–4.15 (4 H, m, CH2NN), 3.67–3.59 (24 H, m, CH2), 3.50
(2 H, t, J 7.5 Hz, H-3), 3.01 (2 H, dd, J 11.1 Hz, J 4.5 Hz,
H-1a), 2.79 (2 H, m, CHHN), 2.54 (2 H, m, CHHN), 2.34 (2
H, d, J 8.5 Hz, H-5), 2.16 (2 H, t, J 11.1 Hz, H-1b), 1.99 (4
H, m, NCH2CH2CH2NN); 13C NMR (75.5 MHz, CDCl3): d =
=
=
D2O): d = 143.9 (NC CH), 125.1 (NC CH), 78.2 (C-3), 71.7
(CH2), 70.0 (C-4), 69.4, 69.0 (CH2), 68.8 (C-2), 64.7 (C-5), 63.0
(C-6), 60.3 (CH2OH), 57.4 (OCH2C), 55.2 (C-1), 48.6 (CH2NN,
CH2N), 23.7 (NCH2CH2CH2N); HRMS (ESI+): Found 413.2007
C16H30N4O7Na requires 413.2012.
1-[3-(1,5-Dideoxy-1,5-imino-D-glucitol-N-yl)-propyl)]-4-[16-hy-
droxy-2,5,8,11,14-pentoxahexadecyl]-1H-[1,2,3]-triazole(2). Con-
ventional catalytic hydrogenation of 13 (100 mg, 0.113 mmol) was
carried out with Pd(OH)2 (76 mg, 0.541 mmol) in MeOH-1M aq.
HCl solution (8 mL, 1:1) at 1 atm for 15 h. Then, the catalyst was
filtered over celite and the solvent removed under reduced pressure
to give 2 (quant) as colourless oil purified by preparative HPLC
1
(tR = 16.52 min); [a]D -4 (c = 0.1, H2O); H NMR (300 MHz,
=
=
145.2 (NC CH), 138.9, 138.4, 137.8 (NC CH), 128.5, 128.4,
128.3, 127.9, 127.8, 127.6, 127.5 (CHar), 122.8 (NCHC), 87.1
(C-3), 78.5, 78.3 (C-2, C-4), 75.4, 75.2, 73.3, 72.9 (CH2Ph), 70.6
(3CH2), 69.8, 66.3 (CH2), 64.7 (C-6), 64.5 (C-5), 54.4 (C-1), 49.2
(CH2NN), 48.4 (CH2N), 26.0 (NCH2CH2CH2N); HRMS (ESI+):
Found 1549.8036 C90H110N8O14Na requires 1549.8039.
CDCl3): d = 8.16 (1 H, s, CCHN), 4.78 (2 H, s, H-6), 4.57 (2 H, t,
J 7.7 Hz, CH2NN), 3.86–3.71 (22 H, m, CH2), 3.65 (1 H, dt, J 9.8
Hz, J 4.8 Hz, H-2), 3.49 (1 H, t, J 9.8 Hz, H-4), 3.36 (1H, t, H-3),
3.10 (1H, dd, J 11.3 Hz, H-1a), 2.86 (2 H, m, CH2N), 2.46 (2 H,
m, H-1b, H-5), 2.25 (4 H, m, NCH2CH2CH2NN); 13C NMR (75.5
=
=
MHz, CDCl3): d = 143.9 (NC CH), 125.3 (NC CH), 78.0 (C-3),
71.7 (CH2), 70.3 (C-4), 69.6, 69.5, 69.4, 68.9 (CH2), 68.5 (C-2), 64.7
(C-5), 63.0 (C-6), 60.3 (CH2OH), 57.1 (OCH2C), 55.0 (C-1), 48.7
(CH2NN), 48.4 (CH2N), 23.7 (NCH2CH2CH2N); HRMS (ESI+):
Found 545.2798 C22H42N4O10Na requires 545.2799.
Bis-1,9-[1-(3-(1,5-dideoxy-1,5-imino-D-glucitol-N-yl)-propyl)tri-
azol-4-yl]-2,5,8-trioxanonane (3). Conventional catalytic hy-
drogenation of 16 (100 mg, 0.0717 mmol) was carried out with
Pd(OH)2 (97 mg, 0.689 mmol) in MeOH-1M aq. HCl solution
(4 mL, 1:1) at 1 atm for 15 h. Then, the catalyst was filtered over
celite and the solvent removed under reduced pressure to give 3
(quant) as a white solid purified by preparative HPLC (tR = 15.42
Bis-1,9-[1-(3-(2,3,4,6-tetra-O-benzyl-1,5-dideoxy-1,5-imino-D-
glucitol-N-yl)-propyl)triazol-4-yl]-2,5,8-trioxanonane (16). To a
solution of the alkyne 14 (15 mg, 0.083 mmol) and the azido-
derivative 9 (100 mg, 0.165 mmol) in dioxane-H2O (5 mL, 4:1),
copper sulfate (41 mg, 0.265 mmol) and sodium ascorbate (81 mg,
0.531 mmol) were added and the mixture was irradiated at 80 ◦C
for 45 min in a sealed vessel. The mixture was poured into saturated
ammonium chloride (20 mL) and extracted with ethyl acetate
(3 ¥ 20 mL). The organic layer was dried (Na2SO4), filtered and the
solvent removed under reduced pressure. The residue was purified
by FC (1:49 MeOH-EtOAc) to give 16 (69 mg, 60%) as a colourless
1
min); [a]D -11 (c = 0.1, H2O); H NMR (300 MHz, D2O): d =
8.01 (2 H, s, CCHN), 4.66 (4 H, s, H-6), 4.41 (4 H, t, J 7.5 Hz,
CH2NN), 3.73–3.65 (12 H, m, CH2), 3.51 (2 H, dt, J 10.4 Hz,
J 4.9 Hz, H-2), 3.32 (2 H, t, J 9.3 Hz, H-4), 3.22 (2 H, t, J 9.3
Hz, H-3), 2.93 (2 H, dd, J 11.3 Hz, H-1a), 2.67 (4 H, m, CH2N),
2.24 (4 H, m, H-1b, H-5), 2.10 (4 H, m, NCH2CH2CH2NN); 13
C
=
=
NMR (75.5 MHz, D2O): d = 143.9 (NC CH), 125.1 (NC CH),
78.2 (C-3), 69.9 (C-4), 69.5 (CH2), 69.2 (CH2), 68.4 (C-2), 64.7
(C-5), 63.0 (C-6), 57.3 (OCH2C), 55.2 (C-1), 48.6 (CH2NN),
48.5 (CH2N), 23.7 (NCH2CH2CH2N); HRMS (ESI+): Found
697.3495 C28H50N8O11Na requires 697.3497.
1
oil. Rf = 0.33 (EtOAc); [a]D +5 (c = 1, CH2Cl2); H NMR (300
MHz, CDCl3): d = 7.43 (2 H, s, CCHN), 7.33–7.14 (40 H, m, Har),
4.98–4.79 (6 H, 3 d, J 11.1 Hz, 11.0 Hz, 10.8 Hz, CH2Ph, CHHPh),
4.68–4.62 (6 H, m, CH2Ph, H-6), 4.42 (6 H, m, CH2Ph, CHHPh),
4.29–4.15 (4 H, m, CH2NN), 3.69–3.63 (14H, m, CH2, H-2), 3.57
(2 H, d, J 1.9 Hz, H-4), 3.50 (2 H, t, J 7.8 Hz, H-3), 3.02 (2 H, dd,
J 10.9 Hz, J 4.6 Hz, H-1a), 2.78 (2 H, m, CHHN), 2.54 (2 H, m,
Bis-1,18-[1-(3-(1,5-dideoxy-1,5-imino-D-glucitol-N-yl)-propyl)-
triazol-4-yl]-2,5,8,11,14,17-hexaoxaoctadecane
(4). Conven-
tional catalytic hydrogenation of 17 (100 mg, 0.0655 mmol) was
carried out with Pd(OH)2 (88 mg, 0.627 mmol) in MeOH-1M
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aq. HCl solution (4 mL, 1:1) at 1 atm for 15 h. Then, the
catalyst was filtered over celite and the solvent removed under
reduced pressure to give 4 (quant) as a hygroscopic white solid
purified by preparative HPLC (tR = 18.78 min); [a]D -5 (c = 0.1,
(3 ¥ 30 mL). The organic layer was dried over Na2SO4, filtered
and the solvent removed under reduced pressure. The residue
was purified by FC (1:49 MeOH-EtOAc) to give 20 (91 mg, 50%
yield) as a colourless oil. Rf = 0.57 (8:2 cyclohexane-EtOAc);
1
1
H2O); H NMR (300 MHz, D2O): d = 8.05 (2 H, s, CCHN),
[a]D +5 (c = 0.5, CH2Cl2); H NMR (300 MHz, CDCl3): d =
4.68 (4 H, s, H-6), 4.46 (4 H, t, J 6.4 Hz, CH2NN), 3.75–3.67
(26 H, m, CH2), 3.53 (2 H, dt, J 10.0 Hz, J1a,2 4.9 Hz, H-2),
3.34 (2 H, t, J 9.1 Hz, H-4), 3.23 (2 H, t, J 9.1 Hz, H-3), 2.95
(2 H, dd, J 11.4 Hz, H-1a), 2.81–2.59 (4 H, m, CH2N), 2.27
(4 H, m, H-1b, H-5), 2.12 (4 H, m, NCH2CH2CH2NN); 13C
7.42 (3 H, s, CCHN), 7.31–7.12 (60 H, m, Har), 4.97–4.78 (9 H,
3 d, J 11.1 Hz, 11.0 Hz, 10.8 Hz, CH2Ph, CHHPh), 4.67–4.61
(12 H, m, CH2Ph, H-6), 4.45–4.39 (9 H, m, CH2Ph, CHHPh),
4.27–4.17 (6 H, m, CH2NN), 3.65–3.46 (36 H, m, CH2, H-2, H-3,
H-4), 3.29 (6H, s, CCH2O), 3.03 (3 H, dd, J 11.2 Hz, J1a,2 4.5 Hz,
H-1a), 2.79 (3H, m, CHHN), 2.54 (3H, m, CHHN), 2.32 (3H,
d, J 8.3 Hz, H-5), 2.14 (3 H, t, J 11.2 Hz, H-1b), 1.99 (6 H, m,
NCH2CH2CH2NN), 0.91 (3 H, s, CH3); 13C NMR (75.5 MHz,
=
=
NMR (75.5 MHz, D2O): d = 144.0 (NC CH), 125.1 (NC CH),
78.2 (C-3), 69.9 (C-4), 69.6 (CH2), 68.9 (CH2), 68.7 (C-2), 64.7
(C-5), 63.0 (C-6), 57.4 (OCH2C), 55.2 (C-1), 48.6 (CH2NN),
48.5 (CH2N), 23.7 (NCH2CH2CH2N); HRMS (ESI+): Found
829.4293 C34H62N8O14Na requires 829.4283.
=
CDCl3): d = 145.1 (NC CH), 139.0, 138.5, 137.8 (Car), 128.6,
128.5, 128.4, 128.39, 128.0, 127.8, 127.7, 127.6 (CHar), 122.8
=
(NC CH), 87.2 (C-3), 78.6, 78.4 (C-2, C-4), 75.5, 75.3 (CH2Ph),
1-O-Tosyl-3,6-dioxanon-8-yne (18). Compound 10 (475 mg,
1.71 mmol) was dissolved in anhydr. DCM (20 mL), Et3N (1.2 mL,
8.54 mmol), DMAP (5 mg, 41 mmol) and tosyl chloride (980 mg,
5.15 mmol) were added and the mixture was stirred under Ar
for 15h. The mixture was washed with satd NaHCO3 (50 mL) and
water (50 mL). Organic layer was dried over sodium sulfate, filtered
and the solvent removed under reduced pressure. The residue was
purified by FC (4:1 cyclohexane-EtOAc) to give 18 (469 mg, 92%
yield) as a colourless oil. Rf = 0.26 (7:3 cyclohexane-EtOAc); 1H
NMR (300 MHz, CDCl3): d = 7.78 (2 H, d, J 7.9 Hz, Har),
74.0 (CH2), 73.4, 72.9 (CH2Ph), 71.1, 70.6, 70.5, 69.9 (CH2), 66.3
(OCH2C), 64.7 (C-6), 64.5 (C-5), 54.5 (C-1), 49.2 (CH2NN), 48.4
(CH2N), 41.1 (Cq), 26.0 (NCH2CH2CH2N), 17.5 (CH3); HRMS
(ESI+): Found 2340.2329 C137H168N12O21Na requires 2340.2345.
1,1,1-Tris[9-(1-(3-(1,5-dideoxy-1,5-imino-D-glucitol-N-yl)-propyl)-
triazol-4-yl)-2,5,8-trioxanonyl]ethane (5). Conventional catalytic
hydrogenation of 20 (70 mg, 0.0302 mmol) was carried out with
Pd(OH)2 (61 mg, 0.434 mmol) in MeOH-1M aq. HCl solution
(4 mL, 1:1) at 1 atm for 15h. Then, the catalyst was filtered over
celite and the solvent removed under reduced pressure to give 5
(quant) as a hygroscopic white solid purified by preparative HPLC
∫
7.33 (2 H, d, J 7.9 Hz, Har), 4.16–4.11 (4 H, m, CH2C CH2,
CH2OTs), 3.67 (2 H, m, CH2), 3.61 (8 H, m, CH2), 2.42 (4 H, s,
CH2CCH, CH3); 13C NMR (75.5 MHz, CDCl3): d = 144.9, 133.0
(Car), 129.9, 128.1 (CHar), 79.6 (Cq), 74.7 (C-8), 70.7, 69.3, 69.1,
68.8 (CH2), 58.5 (CH2CCH), 21.2 (CH3); HRMS (ES+): Found
321.0773 C14H18O5SNa requires 321.0773.
1
(tR = 20.39 min); [a]D -21 (c = 0.1, H2O); H NMR (300 MHz,
D2O): d = 8.03 (3 H, s, CCHN), 4.66 (6 H, s, H-6), 4.47 (6 H, t, J
6.8 Hz, CH2NN), 3.80–3.76 (6 H, m, CH2), 3.70 (12 H, m, CH2),
3.67–3.56 (9 H, m, CH2, H-2), 3.43 (3 H, t, J 9.3 Hz, H-4), 3.37
(6 H, s, CH2), 3.31 (3 H, t, J 9.3 Hz, H-3), 3.08 (3 H, dd, J 11.5
Hz, J 4.9 Hz, H-1a), 2.83 (6 H, m, CH2N), 2.47 (6 H, m, H-1b,
H-5), 2.18 (6 H, m, NCH2CH2CH2NN), 1.90 (3 H, s, CH3); 13C
1,1,1-Tris(2,5,8-trioxaundec-10-ynyl)ethane (19). 1.1.1-Tris-
(hydroxymethyl)ethane (20 mg, 0.166 mmol) was dissolved in
anhydr. THF (5 mL) and the solution was cooled at 0 ◦C. Sodium
hydride (80 mg, 3.33 mmol) and 18 (198 mg, 0.666 mmol) in
anhydr THF (3 mL) were added dropwise to the solution and the
mixture was stirred for 16 h at r t. Methanol was added (5 mL) and
the solvent removed under reduced pressure. The crude residue
was dissolved in ethyl acetate (15 mL) and the solution extracted
with water (2 ¥ 15 mL). Organic layer was dried (Na2SO4), filtered
and the solvent removed under reduced pressure. The residue was
purified by FC (3:1 cyclohexane-EtOAc) to give 19 (41 mg, 50%)
as a colourless oil. Rf = 0.43 (1:1 cyclohexane-EtOAc); 1H NMR
=
=
NMR (75.5 MHz, D2O): d = 144.0 (NC CH), 125.1 (NC CH),
77.6 (C-3), 73.4, 70.4, 69.6, 69.5, 69.2 (CH2), 69.0 (C-4), 68.0
(C-2), 64.9 (C-5), 63.1 (C-6), 56.5 (OCH2C), 54.7 (C-1), 48.9
(CH2NN), 48.2 (CH2N), 40.3 (Cq), 23.6 (NCH2CH2CH2N),
16.8 (CH3); HRMS (ESI+): Found 1259.6740 C53H96N12O21Na
requires 1259.6711.
Inhibition assay
Materials. The glycosidases a-glucosidase (from yeast), b-
glucosidase (from almonds), b-glucosidase/b-galactosidase (from
bovine liver, cytosolic), isomaltase (from yeast), a-galactosidase
(from green coffee beans), a-mannosidase (from Jack beans), and
amyloglucosidase (from Aspergillus Niger) used in the inhibition
studies, as well as the corresponding o- and p-nitrophenyl glycoside
substrates were purchased from Sigma Chemical Co.
∫
(300 MHz, CDCl3): d = 4.19 (6 H, d, J 2.4 Hz, CH2C C), 3.66
(6 H, s, CH2), 3.57 (18 H, m, CH2), 3.30 (6 H, s, H2C-9), 2.42 (3
H, t, J 2.4 Hz, HC-11), 0.91 (3 H, s, CH3); 13C NMR (75.5 MHz,
CDCl3): d = 79.8 (C), 74.6 (C-11), 74.0 (C-9), 71.1, 70.5, 69.3,
58.5 (CH2C), 41.1 (CH3C), 17.5 (CH3); HRMS (ESI+): Found
521.2712 C26H42O9Na requires 521.2727.
1,1,1-Tris[9-(1-(3-(2,3,4,6-tetra-O-benzyl-1,5-dideoxy-1,5-imino-
D-glucitol-N-yl)-propyl)triazol-4-yl)-2,5,8-trioxanonyl]ethane (20).
To a solution of the alkyne 19 (39 mg, 0.0783 mmol) and
azido-derivative 9 (166 mg, 0.274 mmol) in dioxane-H2O (5 mL,
4:1), copper sulfate (60 mg, 0.376 mmol) and sodium ascorbate
(119 mg, 0.752 mmol) were added and the mixture was irradiated
at 80 ◦C for 45 min in a sealed vessel. The mixture was poured into
a NH4Cl satd solution (30 mL) and extracted with ethyl acetate
General procedure. Inhibitory potencies were determined by
spectrophotometrically measuring the residual hydrolytic activi-
ties of the glycosidases against the respective o- (for b-glucosidase
from bovine liver) or p-nitrophenyl a- or b-D-glycopyranoside, in
the presence of the corresponding iminosugar derivative. Each
assay was performed in phosphate buffer or phosphate-citrate
buffer (for a-mannosidase and amyloglucosidase) at the optimal
pH for each enzyme. The reactions were initiated by addition of
362 | Org. Biomol. Chem., 2009, 7, 357–363
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enzyme to a solution of the substrate in the absence or presence
of various concentrations of inhibitor. After the mixture was
incubated for 10–30 min at 37 ◦C, the reaction was quenched by
addition of 1 M Na2CO3. The absorbance of the resulting mixture
was determined at 405 nm. The Ki value and enzyme inhibition
mode were determined from the slope of Lineweaver-Burk plots
and double reciprocal analysis using a Microsoft Office Excel 2003
program.
5 J. J. Lundquist and E. J. Toone, Chem. Rev., 2002, 102, 555.
6 L. L. Kiessling, J. E. Gestwicki and L. E. Strong, Angew. Chem. Int.
Ed., 2006, 45, 2348.
7 As far as we know, only two studies on multivalent glycosidase
inhibitors have been reported, although no systematic studies on the
effect of valency on the biological activity were included. See: (a) A.
Lohse, K. B. Jensen, K. Lundgren and M. Bols, Bioorg. Med. Chem.,
1999, 7, 1965; (b) B. A. John and C. R. Johnson, Tetrahedron Lett.,
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8 For a recent monograph, see: Iminosugars: From Synthesis to Therapeu-
tic Applications P. Compain, O. R. Martin, Eds., Wiley-VCH,
Weinheim, Germany, 2007.
9 A. De Raat, C. Ekhart, G. Legler, A. E. Stu¨tz, In Iminosugars as
Glycosidase Inhibitors, A. Stu¨tz, Ed.; Wiley-VCH, Weinheim, Germany,
1999, p. 207.
10 (a) V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B. Sharpless,
Angew. Chem. Int. Ed., 2002, 41, 2596; (b) C. W. Tornøe, C. Christensen
and M. Meldal, J. Org. Chem., 2002, 67, 3057; (c) For a recent review
see: V. D. Bock, H. Hiemstra and J. H. van Maarseveen, Eur. J. Org.
Chem., 2006, 51.
11 A. Borges de Melo, A. da Silveira Gomes and I. Carvalho, Tetrahedron,
2006, 62, 10277.
12 R. J. Tennant-Eyles, B. G. Davis and A. J. Fairbanks, Tetrahedron:
Asymmetry, 2000, 11, 231.
Acknowledgements
This work was carried out with financial support from the Centre
National de la Recherche Scientifique, the Ministe`re De´le´gue´a`
l’Enseignement Supe´rieur et a` la Recherche, the Conseil Re´gional
de Picardie, the Ministerio de Ciencia y Tecnolog´ıa (contract
number CTQ2007-61180/PPQ) and the Junta de Andaluc´ıa.
The authors thank Jose´ Manuel Garc´ıa Ferna´ndez for helpful
discussions.
13 I. Damager, C. E. Olsen, B. L. Møller and M. S. Motawia, Carbohydr.
Res, 1999, 320, 19.
14 P. Arya, A. Barkley and K. D. Randell, J. Comb. Chem., 2002, 4, 193.
15 C. R. R. Matos, R. S. C. Lopes and C. C. Lopes, Synthesis, 1999, 4,
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16 Y. Ju, D. Kumar and R. S. Varma, J. Org. Chem., 2006, 71, 6697.
17 (a) K. L. Prime and G. M. Whitesides, Science, 1991, 252, 1164; (b) K.
L. Prime and G. M. Whitesides, J. Am. Chem. Soc., 1993, 115, 10714.
18 Y. Le Merrer, L. Poitout, J.-C. Depezay, I. Dosbaa, S. Geoffroy and
M.-J. Foglietti, Bioorg. Med. Chem., 1997, 5, 519.
19 (a) M. M. McPhee and S. M. Kerwin, J. Org. Chem., 1996, 61, 9385;
(b) Z.-J. Yao, H.-P. Wu and Y.-L. Wu, J. Med. Chem., 2000, 43, 2484.
20 (a) A. Marra, A. Vecchi, C. Chiappe, B. Melai and A. Dondoni, J. Org.
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21 T. K. Dam and C. F. Brewer, Chem. Rev., 2002, 102, 387.
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The Royal Society of Chemistry 2009
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