Synthetic UDP-furanoses inhibit the growth of the parasite Leishmania

Article abstract of DOI:10.1016/j.carres.2010.02.020

The chemical synthesis of UDP-6-NHAc-6-deoxy-Galf was performed and it led to the isolation of both pure anomers. They were then evaluated together with the previously prepared UDP-furanoses for their anti-parasitic properties against Leishmania donovani promastigotes, one of the agents responsible for visceral leishmaniasis. Amongst them, the unnatural 1,2-trans UDP-6-NHAc-Galf demonstrated a high potency in inhibiting the growth of the parasite.

Full text of DOI:10.1016/j.carres.2010.02.020

Carbohydrate Research 345 (2010) 1299–1305
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthetic UDP-furanoses inhibit the growth of the parasite Leishmania
b
a,c
Rémy Dureau a,c, Florence Robert-Gangneux b, , Jean-Pierre Gangneux , Caroline Nugier-Chauvin
,
*
a,c,
a,c
Laurent Legentil a,c, , Richard Daniellou
, Vincent Ferrières
*
*
a Ecole Nationale Supérieure de Chimie de Rennes, CNRS, UMR 6226, Avenue du Général Leclerc, CS 50837, 35708 Rennes Cedex 7, France
b Laboratoire de Parasitologie-Mycologie, Centre Hospitalier Universitaire, EA SeRAIC 4427, IRSET, Université Rennes 1, 2, Avenue du Pr Léon Bernard, 35043 Rennes Cedex, France
c Université européenne de Bretagne, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 January 2010
Received in revised form 11 February 2010
Accepted 20 February 2010
Available online 24 February 2010
The chemical synthesis of UDP-6-NHAc-6-deoxy-Galf was performed and it led to the isolation of both
pure anomers. They were then evaluated together with the previously prepared UDP-furanoses for their
anti-parasitic properties against Leishmania donovani promastigotes, one of the agents responsible for vis-
ceral leishmaniasis. Amongst them, the unnatural 1,2-trans UDP-6-NHAc-Galf demonstrated a high
potency in inhibiting the growth of the parasite.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
UDP-furanoses
Leishmania
Hexofuranosides
Visceral leishmaniasis
1. Introduction
pensive drugs.2–4 Further basic studies are also needed to explore
various biological pathways than those already studied for new po-
Neglected Tropical Diseases (NTDs), as defined by the World
Health Organization (WHO, www.who.int), affect more than one
billion people and cause 550,000 deaths each year. Amongst them,
leishmaniasis and trypanosomiasis belong to the most NTDs be-
cause they are generally concentrated in the poorest rural areas.1
More than 20 species of the Leishmania genus are responsible for
2 million of new leishmaniasis cases per year for a potential of
350 million people at risk. These parasites, which are kinetoplastid
protozoa members of the family Trypanosomatidae, are responsi-
ble for a cluster of clinical manifestations, from localized and dis-
seminated cutaneous leishmaniasis and mucocutaneous
leishmaniasis to visceral leishmaniasis. They are endemic in nearly
90 countries, and present the highest rates of death amongst the
NTDs. This fact is correlated with a significant variation of the anti-
genic pattern. The parasite is transmitted to human beings by phle-
botomine sandflies but also canines have to be considered as
reservoir hosts. As a result, these diseases tend to spread world-
wide not only because of the parasite and its vector but also be-
cause of dogs that are thus a second family of mammals affected
by Leishmania. It also results from the variability of the antigens
that treatments still require chemotherapies while development
of vaccines is still challenging. Despite many efforts in recent years,
visceral leishmaniasis, which remains often fatal, demands further
research to design and produce efficient well-tolerated and inex-
tential drugs, immunomodulation, and/or vaccines, for human but
also veterinary health.
It is noteworthy that the survival of all species of Leishmania par-
asites and their infectivity is tightly connected to the integrity of the
glycocalyx, and more particularly to its major component, the lipo-
phosphoglycan (LPG). The core structure of the LPG, which is com-
mon to all Leishmania species, is depicted in Figure 1, and
variability according to these species depends on the polymeric
phosphoglycan linked to the primary hydroxyl of the
-Gal) entity d.5 This core oligosaccharide is also characterized by
the presence of a -galactofuranosyl ( -Galf) residue b whose ano-
D-galactose
(D
D
D
meric center is linked to OH-3 of a mannopyranosyl entity a, as in
Trypanosoma (for reviews on furanosides6,7). However, Leishmania’s
LPG varies from Trypanosoma’s GPI in that this D-Galf residue also
bears at position 3 a substituent, here a digalactopyranoside. The
biological importance of the LPG for the parasite infectivity and sur-
vival and its structural peculiarity make this molecular target of
great interest. Indeed, it was shown that alteration of the LPG struc-
ture leads to either attenuated virulence8 or death of the parasite.9
Hexofuranosides, such as D-Galf residue b present in the LPG of
Leishmania, are widespread in Nature and especially in numerous
pathogenic micro-organisms (Mycobacterium tuberculosis and
Aspergillus fumigatus, for example).4 Even more interestingly, they
are completely absent from mammals, which make them and the
enzymes involved in their biosynthesis of particular interest for
the development of original treatments.10,11 In fact, incorporation
* Corresponding authors. Tel.: +33 2 23 23 80 96; fax: +33 2 23 23 80 46 (R.D.).
E-mail address: richard.daniellou@ensc-rennes.fr (R. Daniellou).
of D-Galf entity in glycoconjugates implies at least two particular
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.02.020

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R. Dureau et al. / Carbohydrate Research 345 (2010) 1299–1305
OH
Varying polymeric phosphoglycan according to species
OH
O
d
HO
α-Glcp-(1,O)-P(O)O
OH
OH
O
O
OH
c
OH
HO
OH
O
OH
O
HO
a
O
O
O
O
H
O
O
O
O
O
O
O
H
NH2
b
O
O
P
HO
HO
HO
HO
OH
OAcyl
OAcyl
OH
OH
OH
HO
Figure 1. Structure of the lipophosphoglycan (LPG) of Leishmania.
kinds of enzymes namely the galactofuranosyltransferase (GlfT)
and the UDP-galactopyranosyl mutase (UGM), which require the
same UDP-Galf 1 as substrate (Fig. 2). To date, our knowledge in
this field has been limited mainly due to the lack of access to this
compound.12–16 Still many studies were performed related to its
activity (as well as those of few analogs) toward GlfT and UGM en-
zymes (for instance17–21). Recently, we have developed in the lab-
oratory a versatile and efficient chemical synthesis of this latter as
substituent at the C-6 position of the galactofuranosyl entity since
interesting effects were obtained with M. tuberculosis when using
compounds 2a–4a.26 The acetamido group was therefore first cho-
sen for multiple reasons: (i) its reasonable size, (ii) the chemical
stability of the amide linkage, and (iii) the acetyl moiety could be
easily replaced by longer fatty chains.
The synthesis of 5 (Scheme 1) started from D-galactose, which
was converted in two steps into the furanoside 6 using common
procedures developed in our laboratory.27 Next, 6 was benzoylated
on its two free hydroxyl positions to give 7 in 83% yield, an ester
protecting group being preferred to a benzyl ether for its easier re-
moval. The isopropylidene ring was hydrolyzed under acidic condi-
tions affording 8 (88%), without observing any benzoyl group
migration as confirmed by the chemical shifts of the corresponding
atoms in 1H and 13C NMR spectra. The resulting diol was converted
to a cyclic sulfite and then further oxidized to give the cyclic sulfate
derivative 9 in 88% yield for the last two steps. The sulfur-contain-
ing ring was selectively opened using sodium azide and the resid-
ual 5-sulfate was removed using aqueous sulfuric acid to give, in
85% yield, compound 10, which bore an azido function at C-6
and a free hydroxyl group at C-5. This latter compound was then
acetylated (11, 93%) and the anomeric octyl chain was removed
by acetolysis thus resulting in the formation of 12, in 79% yield,
as a mixture of anomers. Reduction of 12 under the Staudinger’s
conditions gave the primary amino function, which was directly
well as some UDP-furanose analogs (Fig. 2) in both pure a- and b-
configuration.22 This method was rapidly complemented by an
enzymatic procedure giving us in a stereoselective manner the
pure 1,2-cis UDP-sugars.23 The biological properties of all of them
were then evaluated against M. tuberculosis and especially the
1,2-cis UDP-6F-Galf 4a proved to be particularly efficient in inhib-
iting both the GlfT and UGM enzymes, thus leading to the death of
the mycobacteria.24,25
Herein, we report the activities against Leishmania promastigotes
observed when using these UDP-furanosides as well as the original
UDP-6-NHAc-6-deoxy-Galf 5, chemically synthesized during this
study.
2. Results and discussion
In the course of our ongoing research dealing with the chemical
preparation of analogs of 1, we were interested in introducing new
O
NH
HO
O
P
O-
O
P
O-
O
N
O
O
O
O
O
OH
HO
HO
OH OH
UDP-α-
D-Galf 1
O
N
O
NH
NH
O
P
O-
O
P
O-
HO
O
O
P
O-
O
P
O-
O
N
O
O
O
O
O
HO
O
O
O
O
OH
O
HO
R
OH OH
HO
OH OH
OH
R
R
2a
-Fucf 3a
-6F-Galf
R
2b
=H, UDP-β-
L
-Araf
D
=H, UDP-α-
L
-Araf
-Fucf 3b
4b
D-6F-Galf
R=CH3, UDP-α-
=CH2F, UDP-α-
R=CH3, UDP-β-D
R
=CH2F, UDP-β-
R
4a
D
Figure 2. Chemical structures of the previously prepared UDP-furanoses.

R. Dureau et al. / Carbohydrate Research 345 (2010) 1299–1305
1301
C8H17
O
C8H17
C8H17
BzO
O
O
O
BzO
HO
O
O
iii
ii
i
HO
HO
D-Gal
O
O
OBz
iv
OH
OBz
6
8
7
O
O
C8H17
C8H17
O
O
BzO
v
BzO
vii
O
BzO
O
O
OAc
RO
O
O2S
AcO
OBz
OBz
OBz
9
N3
O
12
N3
R=H, 10
, 11
vi
R
=OAc
viii
HN
R1
N
O
S
BzO
O
O
ix
OAc
R2
R1=Bz, R2=Ac, 14
R1=H, R2=H, 15
O
AcO
AcHN
OR1
x
OBz
13
AcHN
xi
O
O
N
NH
O
P
O-
O
P
O-
NH
N
O
HO
O
O
P
O-
O
P
O-
5a
O
O
O
O
O
HO
O
O
O
O
O
OH
HO
AcHN
OH OH
5b
HO
AcHN
OH
OH OH
Scheme 1. Reagents and conditions: (i) (a) octan-1-ol, FeCl3, CaCl2, THF, 33%; (b) dimethylacetal, CSA, acetone, 89%; (ii) BzCl, pyridine, rt, 5 h, 83%; (iii) TFA, H2O, CH2Cl2, rt,
5 h, 88%; (iv) (a) SOCl2, pyridine, CH2Cl2, rt, 20 min; (b) RuCl3, NaIO4, CH3CN, CH2Cl2, H2O, rt, 30 min, 86% for two steps; (v) (a) NaN3, DMF, rt, overnight; (b) H2SO4, THF, rt, 1 h,
85% for two steps; (vi) Ac2O, pyridine, rt, 24 h, 93%; (vii) Ac2O, H2SO4, CH2Cl2, rt, overnight, 79%; (viii) (a) PPh3, H2O, THF, CH2Cl2, rt; 2 h; (b) Ac2O, CH2Cl2, rt; overnight, 60% for
two steps; (ix) 2-mercaptobenzimidazole, BF3ꢀEt2O, CH2Cl2, rt, 24 h, 80%; (x) MeONa, MeOH, rt, 90%; (xi) UDP, DMF, 0 °C, 10 min, 28%.
protected in situ under the form of an acetamido group, leading to
the formation of 13 in 60% yield. Next, the pure b-anomer 14 was
obtained, thanks to a glycosylation reaction of 2-mercaptobenz-
imidazole with the help of the participating group at its C-2 posi-
tion. Final Zemplen transesterification produced the required
furanosyl thioimidate 15.
UDP-furanoses 2–5 were then evaluated for their biological
properties against Leishmania donovani (Fig. 3). Interestingly, an
inhibition of parasite growth was observed in vitro for several
furanosidic compounds, ranging from 50% to 94% for 100 or
1000 parasite burdens, compared to untreated parasites (A–E).
For higher parasite burdens, a 200 lg/mL drug concentration
With this compound in hand, UDP-furanose 5 was obtained by a
chemicalapproachpreviouslyreported byourgroup.22 Briefly, 5 was
synthesized by the direct reaction of 15 with a freshly prepared solu-
tion of UDP under its acidic form, in anhydrous DMF, for 10 min at
0 °C. Once again, neither ring extension nor UMP-furanoside was ob-
served. Preparativechromatographywasusedtopurifyandseparate
both anomers 5a and 5b, obtained in 0.7/1 mixture and 28% yield.
Their structures were finally unambiguously established using 1H
and 31P NMR and compared to those previously obtained for com-
pounds 1–4. On this basis, 5a, with its coupling constant J1,2 of
4.4 Hz was assessed to be the 1,2-cis derivative and 5b to be the
1,2-trans isomer because of its smaller J1,2 (1.2 Hz).
was necessary to obtain a significant effect. In addition, there
seemed to be a higher effect when some compounds were incu-
bated for 5 days rather than 2 days (A). There was usually a
dose-dependent effect, but a saturation was often observed for
the maximal concentration used, that is, 400 lg/mL (not shown),
that led us to abandon conditions with high concentrations (E).
Looking at the chemical structures, first experiments were per-
formed with an anomeric mixture of 3 which exhibited some
small anti-parasitic properties (A). Pure UDP-6F-Galf 4a and 4b
were further tested to evaluate the importance of the configura-
tion of the glycosidic linkage. In that particular case (6-fluoro
derivative), the 1,2-cis UDP-furanose seemed to be of greater

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R. Dureau et al. / Carbohydrate Research 345 (2010) 1299–1305
Figure 3. Parasite growth in the presence of UDP-furanoses (2d, 3d, and 5d refer to 2 days, 3 days, and 5 days, respectively).
effect than the 1,2-trans (B and C). However, 1,2-cis UDP-Araf 2a
and UDP-6-NHAc-Galf 5a were completely inefficient (not shown).
More interestingly, their 1,2-trans isomers proved to be the most
active molecules tested herein (D), with 5b being able to
completely inhibit the growth of the parasite (E). Finally, even
if questions arise from these studies about the mechanism of ac-
tion of these UDP-furanoses, these preliminary results showed
that synthetic well-designed UDP-furanoses are able to greatly
interfere with the course of parasite multiplication and thus
3. Experimental
3.1. General methods
Thin layer chromatography (TLC) analyses were conducted on E.
Merck 60 F254 Silica Gel non-activated plates and the compounds
were revealed using a 5% solution of H2SO4 in EtOH followed by
heating. For column chromatography, Geduran Si 60 (40–63 lm)
Silica Gel was used. 1H, 13C, and 31P spectra were recorded on a
Bruker ARX 400 spectrometer at 400 MHz for 1H, 100 MHz for
13C, 162 MHz for 31P and 376 MHz for 19F. Chemical shifts are given
in d units (ppm) measured downfield from Me4Si. HPLC purifica-
tions were performed on a Thermo SpectraSYSTEM P1000XR
instrument. The purifications were monitored with an ultraviolet
detector Thermo SpectraSYSTEM UV1000 at k = 280 nm. Optical
rotations were measured on a Perkin–Elmer 341 polarimeter. The
HRMS were measured at the CRMPO (University of Rennes 1,
France) with a MS/MS ZabSpec TOF Micromass using m-nitroben-
zylic alcohol as a matrix and accelerated cesium ions for ionization.
leading to the inhibition of
parasite multiplication,
probably through perturbation of enzymatic pathways of LPG
synthesis.
In conclusion, we performed the synthesis and the purifica-
tion of both anomers of the novel UDP-furanose 5, bearing an
acetamido group at the C-6 position, and in yields comparable
to those of analogs previously published. Moreover, the chemical
pathway designed is versatile enough to allow the incorporation
of numerous fatty acid chains. Among the panel of UDP-furanos-
es tested, it is noteworthy that many of them were able to inter-
fere with the growth of L. donovani promastigotes. Especially, 5b,
with the unnatural 1,2-trans configuration, proved to be the
more efficient compound in our study. Finally, even if the biolog-
ical interactions are not yet fully understood, this work paves
the way for further development of potential drugs based on
furanose structure.
3.2. n-Octyl 2,3-di-O-benzoyl-5,6-O-isopropylidene-b-D-
galactofuranoside (7)
To a cooled (0 °C) solution of the starting n-octyl 5,6-O-isopropyl-
idene-b- -galactofuranoside (6, 13.0 g, 39.1 mmol) in dry pyridine
D

R. Dureau et al. / Carbohydrate Research 345 (2010) 1299–1305
1303
(100 mL) was added dropwise benzoyl chloride (11.4 mL,
97.8 mmol). The mixture was stirred for 5 h at room temperature.
Pyridine was co-evaporated with toluene (3 ꢁ 100 mL). The residue
was dissolved into EtOAc (100 mL) and subsequently washed with
5% HCl (100 mL), saturated aq NaHCO3 solution (3 ꢁ 100 mL), and
brine (100 mL). The organic layer was dried over MgSO4, filtered,
and finally concentrated under reduced pressure. Purification of
the residue by flash chromatography on silica gel (100% CH2Cl2) gave
7 (17.6 g, 83%) as a colorless oil; Rf = 0.4 (9:1 cyclohexane–EtOAc);
and concentrated under reduced pressure. Purification of the resi-
due by flash chromatography on silica gel (100% CH2Cl2) afforded 9
(13.05 g, 88%) as a colorless oil; Rf = 0.4 (4:1 cyclohexane–EtOAc);
½
a 2D0
ꢂ
ꢃ10.4 (c 1.2, MeOH); 1H NMR (CDCl3): d 8.12ꢃ8.05 (m, 4H,
H
arom), 7.64ꢃ7.57 (m, 2H, Harom), 7.49ꢃ7.45 (m, 4H, Harom),
5.63ꢃ5.59 (m, 2H, H-2, H-5), 5.37 (br s, 1H, H-1), 5.34 (d, 1H,
J3,4 = 3.6 Hz, H-3), 4.84 (dd, 1H, J6a,6b = 8.8 Hz, J5,6a = 7.2 Hz, H-6a),
4.80 (dd, 1H, J5,6b = 7.2 Hz, H-6b), 4.32 (dd, J4,5 = 3.2 Hz, 1H, H-4),
3.74 (dt, 1H, 2J = 9.6 Hz, 3J = 6.8 Hz, OCH2CH2), 3.56 (dt, 1H,
3J = 6.4 Hz, OCH2CH2), 1.68ꢃ1.57 (m, 2H, OCH2CH2), 1.29ꢃ1.15
(m, 10H, octyl CH2), 0.85 (t, 3H, 3J = 6.8 Hz, CH3); 13C NMR (CDCl3):
d 166.3, 165.4 (COPh), 133.9, 133.7, 130.0, 129.9, 128.7, 128.6
(C6H5), 106.1 (C-1), 82.1 (C-4), 80.2 (C-2), 80.1 (C-5), 78.1 (C-3),
69.2 (C-6), 68.1 (OCH2CH2), 31.8, 29.5, 29.3, 29.2, 26.1, 22.6
((CH2)6), 14.0 (CH3); HRMS: calcd for C28H34O10SNa [M+Na]+ m/z
585.1770; found, m/z 585.1769.
½
a 2D0
ꢂ
ꢃ0.68 (c 1.1, MeOH); 1H NMR (CDCl3): d 8.08ꢃ8.06 (m, 4H, Har-
om), 7.62ꢃ7.57 (m, 2H, Harom), 7.48ꢃ7.44 (m, 4H, Harom), 5.43 (m, 1H,
H-3), 5.42 (d, 1H, J2,3 = 1.2 Hz, H-2), 5.26 (s, 1H, H-1), 4.50 (dt, 1H,
J4,5 = 6.0 Hz, J5,6a = J5,6b = 6.4 Hz, H-5), 4.29 (dd, 1H, J3,4 = 4.4 Hz, H-
4), 4.14 (dd, 1H, J6a,6b = 8.4 Hz, J5,6a = 6.4 Hz, H-6a), 3.98 (dd, 1H,
J5,6b = 6.4 Hz, H-6b), 3.79 (dt, 1H, 2J = 9.2 Hz, 3J = 6.8 Hz, OCH2CH2),
3.53 (dt, 1H, 3J = 6.0 Hz, OCH2CH2), 1.68ꢃ1.60 (m, 2H, OCH2CH2),
1.45, 1.41 (2s, 6H, OC(CH3)2), 1.35ꢃ1.25 (m, 10H, octyl CH2), 0.87
(t, 3H, 3J = 7.0 Hz, CH3); 13C NMR (CDCl3): d 165.6, 165.3 (COPh),
133.5, 129.9, 129.3, 128.5 (C6H5), 110.0 [OC(CH3)2], 105.7 (C-1),
83.3 (C-4), 81.7 (C-2), 77.6 (C-3), 75.7 (C-5), 67.6 (OCH2CH2), 65.7
(C-6), 31.8, 29.6, 29.4, 29.3, 26.2, 22.6 [(CH2)6], 26.4, 25.3 [OC(CH3)2],
14.1 (CH3).
3.5. n-Octyl 6-azido-6-deoxy-2,3-di-O-benzoyl-b-D-
galactofuranoside (10)
To a solution of 9 (2.0 g, 3.56 mmol) in dry DMF (35 mL) was
added NaN3 (0.46 g, 7.12 mmol). The mixture was stirred at room
temperature overnight. After concentration under reduced pres-
sure, the crude oil was diluted with THF (30 mL) before adding a
solution of concd H2SO4 (0.56 mL, 10.7 mmol) and water
(0.2 mL). The mixture was stirred at room temperature for 1 h,
then neutralized with Et3N, concentrated under reduced pressure,
and partitioned between EtOAc (35 mL) and brine (15 mL). The or-
ganic layer was washed with a saturated aq NaHCO3 solution
(3 ꢁ 25 mL). The aqueous layers were extracted with EtOAc
(2 ꢁ 5 mL). The combined organic layers were dried over MgSO4,
filtered, and concentrated under reduced pressure. Purification of
the residue by flash chromatography on silica gel (4:1 cyclohex-
ane–EtOAc) afforded 10 (1.59 g, 85%) as a colorless oil; Rf = 0.4
3.3. n-Octyl 2,3-di-O-benzoyl-b-D-galactofuranoside (8)
To a solution of 7 (16 g, 29.6 mmol) in CH2Cl2 (300 mL) was
added TFA containing 1% H2O (11 mL, 148 mmol). The mixture
was stirred for 5 h at rt. CH2Cl2 (200 mL) was further added and
the mixture was washed with a saturated aq NaHCO3 solution
(3 ꢁ 150 mL) and brine (150 mL). The organic layer was dried over
MgSO4, filtered, and concentrated under reduced pressure. Purifi-
cation of the residue by flash chromatography on silica gel (100%
CH2Cl2) afforded 8 (13.05 g, 88%) as a colorless oil; Rf = 0.2 (3:2
cyclohexane–EtOAc); ½a D20
ꢂ
ꢃ4.39 (c 1.2, MeOH); 1H NMR (CDCl3):
d 8.08ꢃ8.05 (m, 4H, Harom), 7.62ꢃ7.57 (m, 2H, Harom), 7.48ꢃ7.44
(m, 4H, Harom), 5.58ꢃ5.57 (m, 1H, H-3), 5.49 (d, 1H, J2,3 = 1.2 Hz,
H-2), 5.25 (s, 1H, H-1), 4.31 (dd, 1H, J4,5 = 4.4 Hz, J3,4 = 3.2 Hz, H-
4), 4.14ꢃ4.11 (m, 1H, H-5), 3.86 (dd, 1H, J6a,6b = 11.6 Hz,
J5,6a = 5.6 Hz, H-6a), 3.80 (dd, 1H, J5,6b = 4.4 Hz, H-6b), 3.74 (dt,
1H, 2J = 9.6 Hz, 3J = 6.8 Hz, OCH2CH2), 3.53 (dt, 1H, 3J = 6.4 Hz,
OCH2CH2), 2.75ꢃ2.70 (m, 1H, OH), 1.64ꢃ1.56 (m, 3H, OCH2CH2,
OH), 1.41ꢃ1.24 (m, 10H, octyl CH2), 0.86 (t, 3H, 3J = 6.8 Hz, CH3);
13C NMR (CDCl3): d 166.1, 165.3 (COPh), 133.6, 129.9, 129.1,
128.5 (C6H5), 105.7 (C-1), 84.2 (C-4), 81.3 (C-2), 78.0 (C-3), 70.8
(C-5), 67.7 (OCH2CH2), 64.4 (C-6), 31.8, 29.5, 29.4, 29.2, 26.1,
22.6, ((CH2)6), 14.1 (CH3).
(4:1 cyclohexane–EtOAc); ½a D20
ꢂ
ꢃ8.5 (c 1.1, MeOH); 1H NMR
(CDCl3): d 8.09ꢃ8.04 (m, 4H, Harom), 7.62ꢃ7.57 (m, 2H, Harom),
7.49ꢃ7.44 (m, 4H,
Harom), 5.54ꢃ5.53 (ddd, 1H, J3,4 = 4.6 Hz,
J2,3 = 1.2 Hz, 4J = 0.8 Hz, H-3), 5.50 (d, 1H, H-2), 5.26 (br s, 1H, H-
1), 4.27ꢃ4.26 (dt, 1H, J4,5 = 2.8 Hz, H-4), 4.23 (m, 1H, H-5), 3.74
(dt, 1H, 2J = 9.6 Hz, 3J = 6.8 Hz, OCH2CH2), 3.57 (dd, 1H,
J6a,6b = 12.8 Hz, J5,6a = 7.2 Hz, H-6a), 3.53 (dt, 1H, 3J = 6.4 Hz,
OCH2CH2), 3.44 (dd, 1H, J5,6b = 5.2 Hz, H-6b), 2.64 (d, 1H,
3J = 8.0 Hz, OH), 1.68ꢃ1.60 (m, 2H, OCH2CH2), 1.33ꢃ1.24 (m, 10H,
octyl CH2), 0.86 (t, 3H, 3J = 6.8 Hz, CH3); 13C NMR (CDCl3): d
166.1, 165.3 (COPh), 133.6, 129.9, 129.8, 129.0, 128.6, 128.5
(C6H5), 105.6 (C-1), 83.2 (C-4), 81.4 (C-2), 77.9 (C-3), 69.9 (C-5),
67.7 (OCH2CH2), 53.7 (C-6), 31.8, 29.5, 29.3, 29.2, 26.1, 22.6
((CH2)6), 14.1 (CH3); HRMS: calcd for C28H35N3O8K (M+K+) m/z
564.2112; found, m/z 564.2109.
3.4. n-Octyl 2,3-di-O-benzoyl-5,6-O-sulfonyl-b-D-
galactofuranoside (9)
To a cooled (0 °C) solution of 8 (13 g, 26 mmol) in dry CH2Cl2
(70 mL) were added dropwise thionyl chloride (3.95 mL, 54 mmol)
in dry CH2Cl2 (15 mL) and pyridine (8.8 mL). The mixture was stir-
red for 20 min at room temperature. CH2Cl2 (200 mL) was further
added and the mixture was washed with 5% HCl (2 ꢁ 100 mL), sat-
urated aq NaHCO3 solution (3 ꢁ 100 mL), and brine (100 mL). The
aqueous layers thus obtained were extracted with CH2Cl2
(2 ꢁ 20 mL). The combined organic layers were dried over MgSO4,
filtered, and concentrated under reduced pressure. The yellow oil
3.6. n-Octyl 5-O-acetyl-6-azido-6-deoxy-2,3-di-O-benzoyl-b-D-
galactofuranoside (11)
Acetylation of 10 (1.5 g, 2.85 mmol) was performed at room
temperature in dry pyridine (2.70 mL) using Ac2O (2.70 mL,
28.6 mmol) as acylating agent. After stirring for 48 h, concentration
under reduced pressure, and co-evaporation with toluene
(2 ꢁ 10 mL), the crude oil was diluted with EtOAc (60 mL), and suc-
cessively washed with 5% HCl (30 mL), saturated aq NaHCO3 solu-
tion (3 ꢁ 30 mL), and brine (50 mL). The resulting aqueous layers
were extracted with EtOAc (2 ꢁ 5 mL) and the combined organic
layers were dried over MgSO4, filtered, and concentrated under re-
duced pressure. Purification of the residue by flash chromatogra-
phy on silica gel (4:1 cyclohexane–EtOAc) gave 11 (1.51 g, 93%)
thus obtained was diluted in
a CH2Cl2–CH3CN–H2O mixture
(1:1:2, 80 mL) before adding NaIO4 (11.65 g, 54 mmol) and ruthe-
nium(III) chloride (47 mg). This solution was vigorously stirred at
rt for 30 min, and then diluted with CH2Cl2 (100 mL). After separa-
tion, the organic layer was washed with water (2 ꢁ 50 mL). The
combined aqueous layers were extracted with CH2Cl2 (3 ꢁ 5 mL).
The combined organic layers were dried over MgSO4, filtered,
as a colorless oil; Rf = 0.5 (4:1 cyclohexane–EtOAc); ½a D20
ꢃ8.8 (c
ꢂ
1.1, MeOH); 1H NMR (CDCl3):
d
8.08ꢃ8.06 (m, 4H, Harom),

1304
R. Dureau et al. / Carbohydrate Research 345 (2010) 1299–1305
7.62ꢃ7.57 (m, 2H, Harom), 7.49ꢃ7.43 (m, 4H, Harom), 5.46ꢃ5.42 (m,
2H, H-2, H-5), 5.39 (ddd, 1H, J3,4 = 4.8 Hz, J2,3 = 1.2 Hz, 4J = 0.8 Hz,
H-3), 5.26 (s, 1H, H-1), 4.44 (dd, 1H, J4,5 = 4.4 Hz , H-4), 3.76 (dt,
1H, 2J = 9.6 Hz, 3J = 6.8 Hz, OCH2CH2), 3.65 (dd, 1H, J6a,6b = 12.8 Hz,
J5,6a = 6.8 Hz, H-6a), 3.59 (dd, 1H, J5,6b = 6.0 Hz, H-6b), 3.52 (dt,
1H, 3J = 6.4 Hz, OCH2CH2), 2.07 (s, 3H, COCH3), 1.67ꢃ1.62 (m, 2H,
OCH2CH2), 1.42ꢃ1.26 (m, 10H, octyl CH2), 0.86 (t, 3H, 3J = 6.4 Hz,
CH3); 13C NMR (CDCl3): d 170.0 (CO(CH3)), 165.6, 165.3 (COPh),
133.6, 133.5, 130.0, 129.8, 129.1, 129.0, 128.5, 128.4 (C6H5),
105.5 (C-1), 81.7 (C-2), 80.9 (C-4), 77.1 (C-3), 70.5 (C-5), 67.7
(OCH2CH2), 50.5 (C-6), 31.8, 29.5, 29.3, 29.2, 26.1, 22.6 ((CH2)6),
20.8 (CO(CH3)), 14.1 (CH3); HRMS: calcd for C30H37N3O8Na
[M+Na]+ m/z 590.2478; found, m/z 590.2473.
H
arom), 7.64ꢃ7.59 (m, 2H, Harom), 7.50ꢃ7.45 (m, 4H, Harom), 6.54
(d, 1H, J1,2 = 4.4 Hz, H-1), 5.94 (dd, 1H, J3,4 = 6.4 Hz, H-3), 5.66
(dd, 1H, J2,3 = 7.2 Hz, H-2), 5.29ꢃ5.25 (m, 1H, H-5), 4.36 (dd, 1H,
J4,5 = 5.2 Hz, H-4), 3.89ꢃ3.83 (m, 1H, H-6a), 3.42 (dt, 1H,
J6a,6b = 14.4 Hz, J5,6b = J6b-NH = 5.6 Hz, H-6b), 2.13 (s, 3H, COCH3),
2.08 (s, 3H, COCH3), 1.96 (s, 3H, COCH3); 1H NMR (CDCl3) 13b: d
8.09ꢃ8.04 (m, 4H, Harom), 7.64ꢃ7.59 (m, 2H, Harom), 7.50ꢃ7.45
(m, 4H, Harom), 6.43 (s, 1H, H-1), 6.04ꢃ5.98 (m, 1H, NH), 5.59
(dd, 1H, J1,2 = 0.8 Hz, J2,3 = 1.6 Hz, H-2), 5.46 (ddd, 1H, J3,4 = 4.8 Hz,
4J = 0.8 Hz, H-3), 5.37 (dt, 1H, J4,5 = 4.0 Hz, J5,6a = J5,6b = 6.4 Hz, H-
5), 4.55 (dd, 1H, H-4), 3.82 (ddd, 1H, J6a,6b = 14.8 Hz, J6a-NH = 4.0 Hz,
H-6a), 3.52 (ddd, 1H, J
= 5.6 Hz, H-6b), 2.18 (s, 3H, COCH3), 2.04
(s, 3H, COCH3), 1.96 (s6,b-3NHH, COCH3); 13C NMR (CDCl3) 13a: d 170.7,
170.4 169.1 (COCH3), 165.6, 165.2 (COPh), 133.8, 133.7, 130.0
129.9, 128.8, 128.6 (C6H5), 93.0 (C-1), 81.0 (C-4), 75.8 (C-2), 74.0
(C-3), 70.7 (C-5), 40.2 (C-6), 23.1, 21.0 20.9 (COCH3); 13C NMR
(CDCl3) 13b: d 170.7, 170.4 169.1 (COCH3), 165.6, 165.2 (COPh),
133.9, 133.8, 130.0 129.9, 128.8, 128.6 (C6H5), 99.2 (C-1), 84.4 (C-
4), 80.8 (C-2), 77.1 (C-3), 70.7 (C-5), 40.9 (C-6), 23.2, 21.0 20.8
(COCH3).
3.7. 1,5-Di-O-acetyl-6-azido-6-deoxy-2,3-di-O-benzoyl-D-
galactofuranoside (12a and 12b)
Acetolysis of 11 (0.27 g, 0.47 mmol) was performed at room
temperature in dry CH2Cl2 (10 mL) by dropwise addition of Ac2O
(0.44 mL, 4.7 mmol) and concd H2SO4 (2.5 lL, 0.047 mmol). After
stirring overnight, the reaction was quenched by adding Et3N and
the mixture was concentrated under reduced pressure. Purification
of the residue by flash chromatography on silica gel (4:1 cyclohex-
3.9. 20-Benzimidazolyl 6-acetamido-5-O-acetyl-2,3-di-O-
benzoyl-6-deoxy-1-thio-b-D-galactofuranoside (14)
ane–EtOAc) afforded a 1:6 mixture of
a/b anomers 12a and 12b
(0.19 g, 79%); Rf = 0.2 (4:1 cyclohexane–EtOAc); 1H NMR (CDCl3)
12a: d 8.04ꢃ8.01 (m, 4H, Harom), 7.59ꢃ7.57 (m, 2H, Harom),
7.45ꢃ7.43 (m, 4H, Harom), 6.57 (d, 1H, J1,2 = 4.4 Hz, H-1), 5.96 (dd,
1H, J2,3 = 7.2 Hz, J3,4 = 6.0 Hz, H-3), 5.67 (dd, 1H, H-2), 5.33 (dt,
1H, J4,5 = 6.8 Hz, J5,6a = J5,6b = 5.2 Hz, H-5), 4.42 (dd, 1H, H-4),
3.66ꢃ3.58 (m, 2H, H-6a, H-6b), 2.14 (s, 3H, COCH3), 2.09 (s, 3H,
To a solution of a 1:7 anomeric mixture of 13a/13b (25 mg,
0.05 mmol) in dry CH2Cl2 (3 mL) were added 2-mercaptobenzimi-
dazole (23 mg, 0.15 mmol) and BF3ꢀEt2O (0.06 mL, 0.45 mmol). The
mixture was stirred for 24 h at room temperature and then diluted
with CH2Cl2 (25 mL) and washed with saturated aq NaHCO3 solu-
tion (3 ꢁ 25 mL) and water (2 ꢁ 25 mL). The aqueous layers thus
obtained were extracted with CH2Cl2 (2 ꢁ 5 mL). The combined or-
ganic layers were dried over MgSO4, filtered, and concentrated un-
der reduced pressure. Purification of the residue by flash
chromatography on silica gel (98:2 CH2Cl2–MeOH) afforded 14
COCH3); 1H NMR (CDCl3) 12b:
d
8.09ꢃ8.06 (m, 4H, Harom),
7.65ꢃ7.59 (m, 2H, Harom), 7.51ꢃ7.45 (m, 4H, Harom), 6.44 (s, 1H,
H-1), 5.60ꢃ5.59 (dd, 1H, J1,2 = 0.4 Hz, J2,3 = 1.4 Hz, H-2), 5.46 (ddd,
1H, J3,4 = 4.8 Hz, 4J = 0.8 Hz, H-3), 5.42 (dt, 1H, J4,5 = 4.4 Hz,
J5,6a = J5,6b = 6.0 Hz, H-5), 4.58 (dd, 1H, H-4), 3.66ꢃ3.58 (m, 2H, H-
6a, H-6b), 2.18 (s, 3H, COCH3), 2.04 (s, 3H, COCH3); 13C NMR (CDCl3)
12a: d 169.7, 169.3 (COCH3), 165.4, 165.1 (COPh), 133.8, 133.7,
129.9, 129.8, 128.7, 128.6 (C6H5), 93.1 (C-1), 79.5 (C-4), 76.2 (C-
2), 74.0 (C-3), 71.5 (C-5), 50.2 (C-6), 21.0, 20.7 (CO(CH3)); 13C
NMR (CDCl3) 12b: d 170.0, 169.0 (COCH3), 165.4, 165.1 (COPh),
133.9, 133.8, 129.9, 128.8, 128.7, 128.6 (C6H5), 99.1 (C-1), 83.0
(C-4), 81.0 (C-2), 76.7 (C-3), 70.4 (C-5), 50.3 (C-6), 21.0, 20.7
(COCH3); HRMS: calcd for C24H23N3O9Na [M+Na]+ m/z 520.1332;
found, m/z 520.1332.
(24 mg, 80%) as a white solid; Rf = 0.3 (95:5 CH2Cl2–MeOH); ½a D20
ꢂ
ꢃ95.7 (c 1.1, MeOH); 1H NMR (CDCl3): d 8.10ꢃ8.03 (m, 4H, Harom),
7.71ꢃ7.57 (m, 4H, Harom, H-50, H-80), 7.50ꢃ7.42 (m, 4H, Harom),
7.28ꢃ7.23 (m, 2H, H-60, H-70), 6.28 (dd, 1H, J6b-NH = 5.2 Hz, J6a-
NH = 8.8 Hz, NH), 6.03 (d, 1H, 4J = 0.8 Hz, H-1), 5.70 (t, 1H,
J2,3 = 1.6 Hz, H-2), 5.41 (ddd, 1H, 4J = 0.8 Hz, J3,4 = 5.2 Hz, H-3),
5.29 (ddd, 1H, J5,6a = 8.8 Hz, J5,6b = 4.0 Hz, J4,5 = 2.4 Hz, H-5), 4.75
(ddd, 1H, 4J = 0.8 Hz, H-4), 4.16 (dt, 1H, J6a,6b = 14.0 Hz, H-6a),
3.30 (ddd, 1H, H-6b), 2.04 (s, 3H, COCH3), 1.98 (s, 3H, COCH3);
13C NMR (CDCl3): d 171.2, 170.3 (COCH3), 165.7, 165.1 (COPh),
144.4 (C-20, C-40, C-90), 133.9, 133.8, 130.0 129.9, 128.7, 128.6,
128.5 (C6H5, C-50, C-80), 122.5 (C-60, C-70), 90.1 (C-1), 81.7 (C-2),
81.6 (C-4), 77.7 (C-3), 70.8 (C-5), 38.9 (C-6), 23.5, 20.7 (COCH3).
3.8. 6-Acetamido-1,5-di-O-acetyl-6-deoxy-2,3-di-O-benzoyl-D-
galactofuranoside (13a and 13b)
To
a
solution of
a
1:6 anomeric mixture of 12 (50 mg,
3.10. 20-Benzimidazolyl 6-acetamido-6-deoxy-1-thio-b-
D-
0.10 mmol) in THF–CH2Cl2 3:1 (2 mL) and water (0.25 mL) at 0 °C
was added triphenylphosphine (50 mg, 0.19 mmol). The mixture
was stirred at room temperature for 2 h, and then the reaction
was quenched by adding 5% HCl (1 mL). The mixture was diluted
with CH2Cl2 (50 mL) and washed with saturated aq NaHCO3 solu-
tion (3 ꢁ 50 mL), then brine (50 mL). The organic layer was dried
over MgSO4, filtered, and concentrated under reduced pressure.
The residue was dissolved with CH2Cl2 (2 mL) and pyridine
(0.5 mL). Ac2O was added dropwise (0.4 mL, 4.2 mmol) and the
mixture was stirred at room temperature overnight. The mixture
was then diluted with CH2Cl2 (50 mL) and washed with saturated
aq NaHCO3 solution (3 ꢁ 50 mL) and then brine (50 mL). The or-
ganic layer was dried over MgSO4, filtered, and concentrated under
reduced pressure. Purification of the residue by flash chromatogra-
phy on silica gel (4:1 cyclohexane–EtOAc) afforded a 1:7 mixture of
galactofuranoside (15)
To a solution of 14 (23 mg, 0.04 mmol) in dry MeOH (1 mL) was
added a 30 wt % solution of sodium methoxide in MeOH (0.7 L,
l
0.004 mmol). The mixture was stirred at room temperature until
no starting material was detected by TLC. Neutralization was then
carefully performed by adding Amberlite IR-120 (H+-form). The re-
sin was filtered off and the solvent was removed under reduced
pressure. Purification of the residue by flash chromatography on
silica gel (9:1 CH2Cl2–MeOH) afforded 15 (12.7 mg, 90%) as a white
solid; Rf = 0.1 (9:1 CH2Cl2–MeOH); ½a D20
ꢂ
ꢃ136.2 (c 1.3, MeOH); 1H
NMR (CD3OD): d 7.56ꢃ7.48 (m, 2H, H-50, H-80), 7.24ꢃ7.20 (m,
2H, H-60, H-70), 5.73 (d, 1H, J1,2 = 4.0 Hz, H-1), 4.15ꢃ4.10 (m, 2H,
H-2, H-3), 4.01 (ddd, 1H, J3,4 = 6.4 Hz, J4,5 = 3.2 Hz, H-4), 3.82
(ddd, 1H, J5,6b = 7.2 Hz, J5,6a = 5.6 Hz, H-5), 3.50 (dd, 1H,
J6a,6b = 13.6 Hz, H-6a), 3.20 (dd, 1H, H-6b), 1.93 (s, 3H, COCH3);
13C NMR (CD3OD): d 173.8 (COCH3), 149.3 (C-20, C-40, C-90), 124.0,
a
/b anomers 13a and 13b (31 mg, 60%) as a colorless oil; Rf = 0.3
(95:5 CH2Cl2–MeOH); 1H NMR (CDCl3) 13a: d 8.09ꢃ8.04 (m, 4H,

R. Dureau et al. / Carbohydrate Research 345 (2010) 1299–1305
1305
123.8 (C-50, C-80), 110.9 (C-60, C-70), 92.2 (C-1), 85.2 (C-4), 83.4 (C-
2), 78.0 (C-3), 69.9 (C-5), 43.6 (C-6), 22.6 (COCH3).
the suspensions were performed in 96-well microplates using
Schneider0s drosophila medium supplemented with 10% FCS. After
another incubation period of 5 days, the plates were examined
using an inverted microscope at a magnification of ꢁ200 or
ꢁ400. The viability was appreciated by the presence or absence
of mobile promastigotes in each well. The last dilution titer for
which the well contained at least one parasite was recorded and
compared to the titer obtained with untreated parasites. The re-
sults were expressed as the reduction percentage of parasite
growth.
3.11. Uridine 50-diphospho-6-acetamido-6-deoxy-
galactofuranose (5a and 5b)
D-
To a cooled (0 °C) solution of UDP disodium salt dihydrate
(30 mg, 0.07 mmol) in DMF (3 mL) was added Amberlite IR-120
(H+-form) until the UDP was completely dissolved. The resin was
filtered off and washed with DMF. The filtrate and washings were
freeze dried, dissolved in dry DMF (1 mL), and cooled to 0 °C. A
solution of 15 (10 mg, 0.028 mmol) in DMF (1 mL) was then added
dropwise. The mixture was stirred for 10 min, diluted with cold
water (1 mL) and 0.5 M NH4HCO3 (0.5 mL), and then centrifuged.
The resulting filtrate was freeze dried. The residue was purified
by semi-preparative HPLC on C-18 column with a 10 min isocratic
elution in (99:1 aqueous Et3NHꢀAcO (50 mM)–CH3CN) followed by
a 10-min isocratic elution in 98:2 and then a linear gradient from
Acknowledgments
R. Dureau is grateful to the Ministère de l0Enseignement Supéri-
eur et de la Recherche for fellowship. The authors also thank the
Bresmat network and the Université européenne de Bretagne for
financial support.
98:2 to 75:25 over 20 min. The pure UDP-6NHAc-b-
D
-Galf 5b with
Supplementary data
0.8 equiv of Et3NH+ (3.1 mg, 16%) and UDP-6NHAc-
a-D-Galf 5a
with 9 equiv of Et3NH+ (5 mg, 12%) were obtained with the reten-
tion time of 21.7 and 24.8 min, respectively; 1H NMR (D2O) 5a: d
7.95 (d, 1H, J5,6 = 8.0 Hz, H-5 uracil), 6.00ꢃ5.96 (m, 2H, H-1 Rib,
H-6 uracil), 5.64 (dd, J1,P = 5.6 Hz, J1,2 = 4.4 Hz, H-1 6NHAc-Gal),
4.40ꢃ4.36 (m, 2H, H-2 Rib, H-3 Rib), 4.31ꢃ4.18 (m, 5H, H-4 Rib,
H-5a Rib, H-5b Rib, H-2 6NHAc-Gal, H-3 6NHAc-Gal), 4.14 (ddd,
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2010.02.020.
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ium (Sigma) supplemented with 20% fetal calf serum (FCS) (InVit-
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effect of the UDP-furanoses 2–5, since their incorporation in the
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evaluated in vitro on promastigotes by a culture microtitration
method as previously described.28 Briefly, promastigote parasites
propagated at 27 °C in Schneider0s drosophila medium supple-
mented with 20% FCS were incubated with different concentra-
tions of UDP-furanose under sterile conditions. Various amounts
of parasites were used (100, 1000, or 10,000 parasites) and tested
in duplicate. After 2, 3, or 5 days of incubation, serial dilutions of
28. Buffet, P. A.; Sulahian, A.; Garin, Y. J.; Nassar, N.; Derouin, F. Antimicrob. Agents
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