Thiodisaccharides with galactofuranose or arabinofuranose as terminal units: Synthesis and inhibitory activity of an exo β-d-galactofuranosidase

Article abstract of DOI:10.1016/j.bmc.2009.02.045

Thiodisaccharides having β-d-Galf or α-l-Araf units as non-reducing end have been synthesized by the SnCl₄- or MoO₂Cl₂-promoted thioglycosylation of per-O-benzoyl-d-galactofuranose (1), its 1-O-acetyl analogue 4, or per-O-

Full text of DOI:10.1016/j.bmc.2009.02.045

Bioorganic & Medicinal Chemistry 17 (2009) 2703–2711
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Thiodisaccharides with galactofuranose or arabinofuranose as terminal
units: Synthesis and inhibitory activity of an exo b-D-galactofuranosidase
*
Evangelina Repetto, Carla Marino, M. Laura Uhrig, Oscar Varela
CIHIDECAR-CONICET, Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria,
1428-Buenos Aires, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
Thiodisaccharides having b-
SnCl4- or MoO2Cl2-promoted thioglycosylation of per-O-benzoyl-
analogue 4, or per-O-acetyl-
D
-Galf or
a
-
L
-Araf units as non-reducing end have been synthesized by the
-galactofuranose (1), its 1-O-acetyl
-arabinofuranose (16) with 6-thioglucose or 6-thiogalactose derivatives.
After convenient removal of the protecting groups, the free thiodisaccharides having the basic structure
b- -Galf(1?6)-6-thio- -Glcp-OMe (5) or b- -Galf(1?6)-6-thio- -Galp-OMe (15) were obtained. The
respective -Araf analogues 18 and 20 were prepared similarly from 16. Alternatively, b- -Galf(1?4)-4-
thio-3-deoxy- -Xylp-OiPr was synthesized by Michael addition to a sugar enone of 1-thio-b- -Galf
Received 22 December 2008
Revised 18 February 2009
Accepted 20 February 2009
Available online 26 February 2009
D
a
-L
D
a
-
D
D
a-D
a-
L
D
Keywords:
Thiodisaccharides
Furanose
a
-L
D
derivative, generated in situ from the glycosyl isothiourea derivative of 1. The free S-linked disaccharides
were evaluated as inhibitors of the b-galactofuranosidase from Penicillium fellutanum, being 15 and 20 the
more active inhibitors against this enzyme.
Furanosidase inhibition
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
microorganisms. Arabinofuranose is found in soil bacteria, fungi
and plants,8 and galactofuranose occurs in glycoconjugates of many
Carbohydrates play important roles in recognition events that
initiate immunological responses to bacterial and viral infections
and in signaling processes that occur in inflammation and cancer
metastasis.1 Carbohydrate mimetics, which are useful to investi-
gate these processes,2 are usually prepared by replacing oxygen
atoms in a sugar with carbon atoms or other heteroatoms. These
modified molecules generally show altered binding properties or
increased stability toward enzyme degradation.3
pathogenic bacteria, fungi, and protozoan parasites and are highly
immunogenic.9 Bothsugars arepresentinthemycolycacid-arabino-
galactan-peptidoglycan (mAGP) complex from Mycobacterium
tuberculosis.10 The fact that Galf is essential for the survival or viru-
lence of various pathogenic bacteria,9c,11 but is absent in higher
eukaryotes, hasattractedincreasinginterestinitsbiosyntheticpath-
ways.11,12 Degradation of Galf containing glycoconjugates is pro-
moted in some microorganisms by a b-
D
-galactofuranosidase. The
Oligosaccharide analogues that are resistant toward enzymatic
hydrolysis may act as glycosidase inhibitors. In this regard, thiogly-
cosides are particularly valuable, as they are tolerated by most
biological systems and they are usually resistant to metabolic pro-
cesses.4 The potential of thiooligosaccharides as therapeutics
prompted the investigation about their synthesis and the study
of their biological activities.4,5 In recent years, we have directed
our efforts toward the synthesis and inhibitory activities of this
type of molecules,6 in particular thiodisaccharides that possess a
furanose unit as non-reducing end. Thus, we have recently
reported successful approaches to prepare S-disaccharides of
1-thiopentofuranoses.7
best known is the specific exo b-
D-galactofuranosidase first purified
from the culture medium of Penicillium fellutanum13 and later
described in Helminthosporium sacchari14 and Penicillium and Aper-
gillius species.15,16 The role of this enzyme in P. fellutanum is the
release of galactose as carbon source by depolymerization of an
extracellular glycopeptide during the fungus growth.13
The b-D-galactofuranosidase is an interesting target considering
that during differentiation of Trypanosoma cruzi from the invasive
stage to the infective one, the amount of glycoconjugates contain-
ing Galf units is dramatically diminished.17 We have previously
described the synthesis of substrates,18 inhibitors19 and an affinity
purification method20 of the exo b-
D
-galactofuranosidase from P.
fellutanum. These developments, in combination with other tools,
allowed the detection for the first time of b- -galactofuranosidase
activity in T. cruzi.21 As inhibitors we have synthesized aryl and
heteroaryl 1-thio-b- -galac-
-galactofuranosides.19 Alkyl 1-thio-b-
Among the furanoses, arabinofuranose and galactofuranose are
widespread in nature. Polysaccharides containing galactofuranosyl
and arabinofuranosyl residues are key components of many
D
D
D
tofuranosides and the corresponding sulfones were also prepared
* Corresponding author. Fax: +5411 4576 3346.
E-mail address: varela@qo.fcen.uba.ar (O. Varela).
and tested as inhibitors of mycobacteria growth.22 Furthermore,
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.02.045

2704
E. Repetto et al. / Bioorg. Med. Chem. 17 (2009) 2703–2711
in P. fellutanum, 4-aminophenyl thiogalactofuranoside proved to
induce dramatic modifications during the mycelia growth.23
In line with these studies, we decided to investigate about the
synthesis and potential inhibitory activity of thiodisaccharides
containing a Galf unit, as such compounds have not been previ-
ously prepared. Therefore, we report here the synthesis of thiodi-
saccharides having terminal b-D-Galf or a-L-Araf units. These
compounds have been evaluated as inhibitors of the b-galactofura-
nosidase from P. fellutanum.
linkage, as established by the NMR data of 3. Thus, the small cou-
pling constant value between H-1 and H-2 (J1,2 < 1.0 Hz) and the
low field resonance of C-1,18 confirmed the b anomeric configura-
tion for the furanose moiety of 3.
As SnCl4 promotes successfully glycosylations18a,19,27 or acetoly-
sis of 1, this Lewis acid was employed for thioglycosylations as an
alternative to the neutral species MoO2Cl2. As expected, reaction 1
with 2, in the presence of SnCl4, led to the thiodisaccharide 3, in a
good yield. Similarly, the SnCl4-promoted condensation of the glyco-
syl donor 4 with 2 led to 3 in a yield comparable to that of the previ-
ous reaction. Because of the anchimeric assistance of the benzoyloxy
group at C-2 of 1 or 4, the reaction was highly diasteroselective in
favor of the b anomeric configuration for the S-linkage in 3.
The per-O-benzoyl-S-linked disaccharide 3 was O-debenzoylat-
ed with a mixture of MeOH/Et3N/H2O (3:1:3) to give the free
2. Results and discussion
2.1. Synthesis and chemical characterization
The per-O-benzoyl derivative of Galf (1), readily prepared by high
thiodisaccharide 5. This is the thioanalogue of the motif b-D-
temperaturebenzoylation ofD
-galactose,24 wasemployedasprecur-
Galf(1?6)-D-Glcp found in the lipopolysaccharide O-antigen of
Escherichia coli K-12.9d,28 The Galf containing glycoconjugates
appeared to be essential for virulence in pathogenic Gram-negative
bacteria.9d
sor of the galactofuranosyl non-reducing end of the thiodisaccha-
rides. The thiosugar component used as thiol donor was, in first
instance, methyl 2,3,4-tri-O-benzoyl 6-thio-a-D-glucopyranoside
(2).25 The thioglycosylation of 1 with the thiosugar 2, using MoO2Cl2
as neutral catalyst, led to low yields of the expected thiodisaccharide
3 (Scheme 1). This type of reaction has been successfully applied for
analogous thioglycosylations of 1-O-acetyl pyranoses26 or 1-O-acet-
The thiodisaccharide b-
D-Galf-S-(1?6)-6-thio-D-Galp (9) was
interesting as the b- -Galf(1?6)-
D
D-Galp unit was found in nature
as a constituent of the exopolysaccharide of Lactobacillus rhamno-
sus29 and of Streptococcus thermophilus.30 This disaccharide has also
been obtained by partial hydrolysis of immunological polysaccha-
rides from Mycoplasma mycoides31 and M. tuberculosis.32 Further-
yl-2,3,5-tri-O-benzoyl-b-D
-ribofuranose.7 The failure in the
MoO2Cl2-catalyzed reaction between 1 and 2 was attributed to the
fact that 1 possesses a benzoyloxy group as anomeric substituent,
instead of an acetyloxy, as in the cited examples. Therefore, the syn-
thesis of the 1-O-acetyl-per-O-benzoyl-Galf from 1 was attempted.
For the activation of the anomeric center of 1, tin(IV) chloride was
employed. This Lewis acid proved to be adequate for the in situ ano-
meric activation of per-O-acyl furanoses, which underwent further
glycosylation with conveniently protected aldonolactones,18a,27 or
with aryl or heteroaryl thiols.19 Thus, compound 1 was treated with
SnCl4 and, upon addition of acetic anhydride, the expected 1-O-acet-
yl derivative 4 was obtained (ꢀ80% yield). The NMR spectra of the
crude product indicated that the b anomer was the major compo-
nent. Integration of the area of the H-1 signal (b anomer:
more, the thiodisaccharide
9 was useful to investigate the
influence of the change of configuration of the reducing end, with
respect to 5, on the inhibition of the b-galactofuranosidase.
For the synthesis of 9 (Scheme 2) the Galf derivative 4 was
allowed to react with methyl 2,3:4,5-di-O-isopropylidene-6-thio-
a-D
-galactose (6).7 The MoO2Cl2-catalyzed reaction between 4
and 6 led to the stereoselective formation of the thioglycosidic
bond of b configuration to produce 7. In this case SnCl4 could not
be employed, as Lewis acids promote the removal of the isopropyl-
idene protecting groups of 6.
The thiodisaccharide 7 was O-debenzoylated to afford the par-
tially protected derivative 8, which was treated with TFA for the
hydrolysis of the acetal groups. However, this deprotection could
not be successfully accomplished because the acidic conditions
required for the hydrolysis of the isopropylidene functions produced
partial degradation of the interglycosidic S-linkage. This hydrolysis
could not be prevented, even under smooth conditions, such as ace-
tic acid/THF/water 5:1:1 at 65 °C, or under acetolysis reaction condi-
tions in which thioglycosidic bonds between pyranose units were
stable.33 It is known that furanosides are much more labile to acids
than their pyranoside analogues.34 As the purification of 9 from
the hydrolysis products was rather difficult and low yielding, a thio-
sugar with protecting groups labile to alkali was used.
6.52 ppm, J1,2 < 1.0 Hz;
5:1 b:
ratio. A similar relationship was determined from the 13C
NMR spectrum, which showed in the anomeric region the resonance
of C-1 at 99.4 ppm (b anomer) and 93.4 ppm ( anomer). Recrystal-
a anomer: 6.66 ppm, J1,2 = 4.8 Hz) gave a
a
a
lization of the crude product from EtOH afforded the b anomer as a
single diastereoisomer.
With the Galf derivative 4 in hand, the reaction with 2 was con-
ducted using MoO2Cl2 as catalyst. In this case, the thiodisaccharide
3 was obtained in a good yield. As previously observed,26 the
MoO2Cl2-promoted thioglycosylation took place under diastero-
control with selective formation of the 1,2-trans-thioglycosidic
OMe
OBz
BzO
OBz
O
O
O
S
BzO
BzO
OBz
OBz
SnCl4
OBz
SnCl4
4
BzO OBz
OBz
BzO
SH
O
2
1
3
BzO
BzO
OMe
OBz
MeOH / Et3N / H20
Ac2O,
SnCl4
2
1) MoO2Cl2
2)
2
OMe
OH
O
OBz
BzO
O
O
HO
BzO
OH
HO OH
S
OAc
OBz
OH
HO
4
5
Scheme 1. Synthesis of thiodisaccharide 5.

E. Repetto et al. / Bioorg. Med. Chem. 17 (2009) 2703–2711
2705
Me
O
OH
OR
RO
OH
HO
Me
O
O
O
O
RO
HO
O
S
OH
HO OH
S
1) MoO2Cl2
SH
TFA
H2O
4
O
Me
O
Me
OR
OH
O
2)
Me
O
O
Me
Me
O
O
7 R = Bz
8 R = H
9
Me
MeOH/Et3N/
H2O
6
OH
O
OTs
O
R
O
1) TsCl, C5H5N
2) BzCl
HO
OMe
BzO
OMe
KSCN
DMF
BzO
OMe
HO OH
BzO
OBz
BzO OBz
12 R = SCN
10
11
Zn, AcOH
13 R = H
OMe
OMe
OBz
O
OH
O
S
O
O
HO
BzO
SnCl4, 13
S
MeOH/Et3N/H2O
OH
HO OH
OBz
BzO OBz
1
OH
OBz
HO
BzO
15
14
Scheme 2. Synthesis of thiodisaccharides 8 and 15.
In view of the successful results in the synthesis of 5, the
thiosugar 13 (the galacto analogue of 2) was selected as thiol
donor. As compound 9 has not been previously described, it was
donors in the reaction with commercially available tetra-O-acet-
yl- -Araf (16). Thus, activation of 16 with SnCl4 or MoO2Cl2 fol-
a
-L
lowed by addition of thiosugar 2 afforded the thiodisaccharide
17. Analogous coupling of 16 with the 6-thiosugar derivative 13,
in the presence of SnCl4, led to the thiodisaccharide 19. The config-
uration for the anomeric center of the Araf unit in compounds 17
prepared by a short route starting from methyl a-D-galactopyran-
oside (10). Selective tosylation of the primary hydroxyl group of
10,35 followed by benzoylation, afforded the tosyl derivative 11.
Treatment of 11 with potassium thiocyanate in DMF at 120 °C
led to the thiocyano derivative 12 (55%) and unreacted 11 (24%).
The low reactivity of this tosylate towards substitution may be
attributed to the fact that the incoming nucleophile is hindered
by the C-4 substituent in an axial disposition, an effect commonly
observed for 6-O-sulfonates of galactosides.36 Reduction of 12 with
Zn in acetic acid gave the 6-thiohexopyranoside 13. Reaction of 4
with 13, in the presence of SnCl4, led to the thiodisaccharide 14.
As previously observed, the glycosylation took place with exclusive
diasterocontrol in favor of the b anomer. Debenzoylation of 14 with
and 19 was established as
similar to the b- -Galf analogues, small J1 ,2 values (J1 ,2 < 1 Hz),
a
, since their 1H NMR spectra showed,
0
0
0
0
D
characteristic of 1,2-trans thiofuranosides. Removal of the protect-
ing groups of 17 and 19 with MeOH/Et3N/H2O (3:1:3) afforded the
free thiodisaccharides 18 and 20.
Starting from the per-O-benzoyl Galf derivative 1, a different
methodology for the construction of the thioglycosidic linkage
was employed. This methodology involves the 1-thioGalf deriva-
tive 22 as an intermediate (Scheme 4). However, we have observed
that the procedures that are successful for the synthesis of 1-thio-
pyranoses derivatives usually fail for furanoses, which undergo
self-condensation to the disulfide and other side reactions.7 To
overcome this difficulty 1-thiosugars like 22, were generated from
the respective S-glycosyl isothiourea derivatives and reacted in situ
with a sugar enone to yield the corresponding thiodisaccharides.
Thus, the S-glycosyl isothiourea 21 was synthesized by reaction
MeOH/Et3N/H2O (3:1:3) gave the target thiodisaccharide b-
S-(1?6)-6-thio- -Galp-OMe (15).
The synthesis of thiodisaccharides containing a-L-Araf as non
D-Galf-
a-D
reducing end was carried out, since this sugar has the same stereo-
chemistry for the substituents of the five-membered ring as
b-D-Galf. Thiosugars 2 and 13 (Scheme 3) were employed as thiol
OMe
OBz
OMe
OH
MeOH/Et3N/H2O
O
O
O
O
S
AcO
S
MoO2Cl2, 2
or
HO
BzO OBz
OAc
HO OH
AcO
OH
HO
SnCl4, 2
17
18
O
OAc
OAc
AcO
AcO
16
OMe
OH
SnCl4, 13
OMe
O
MeOH/Et3N/H2O
O
O
O
S
S
OBz
HO
AcO
BzO OBz
HO OH
OH
OAc
19
Scheme 3. Synthesis of thiodisaccharides 18 and 20.
HO
AcO
20

2706
E. Repetto et al. / Bioorg. Med. Chem. 17 (2009) 2703–2711
OBz
BzO
OBz
BzO
NH
OBz
BzO
O
O
O
BzO
BzO
(NH2)CS
F3B.OEt2
BzO
OBz
OBz
S
C
Et3N
SH
OBz
NH2
OBz
21
1
22
OAc
O
OiPr
OAc
O
OAc
O
O
OBz
OBz
BzO
O
O
23
BzO
BzO
BzO
S
OBz
25
OiPr
S
OiPr
OiPr
22
24
+
O
Et3N
O
BzO
OBz
24
(3:1)
+
OAc
O
OAc
O
OBz
BzO
OBz
BzO
NaBH4
O
O
BzO
S
S
OiPr
OH
OH
OBz
26
OBz
27
(6:1)
MeOH/Et3N/H2O
OH
O
OH
HO
O
HO
S
OiPr
OH
OH
28
Scheme 4. Synthesis of thiodisaccharide 28.
of 1 with thiourea in the presence of F3BꢁOEt2. Crude compound 21
was treated with Et3N to promote the conversion of the isothiourea
into the thiosugar 22. Michael addition of 22 with the hex-3-eno-
pyranosid-2-ulose 23 gave the thiodisaccharide 24 as major prod-
uct, accompanied with the diastereoisomeric S-linked disaccharide
25. The addition took place diasteroselectively from the face of the
enone opposite to the anomeric substituent (ratio 24:25 > 3:1).
Thiodisaccharides 24 and 25 were readily separated by column
chromatography. However, they were slightly contaminated by
the starting enone 23, as the retro-Michael reaction seems to take
place during the chromatographic purification, as we have previ-
ously observed.7 The configuration for the C-4 stereocenter was
established as R (the thio group axially oriented), since the 1H
NMR spectrum of 24 showed only small coupling constant values
of H-4 with the adjacent methylene protons (J3ax,4 = 5.0,
J3eq,4 = 2.4 Hz) and with H-5 (J4,5 = 2.4 Hz). Furthermore, the small
and benzoyl protecting groups of 26 by hydrolysis with MeOH/
Et3N/H2O (3:1:3) afforded the free thiodisaccharide 28.
2.2. Evaluation of the inhibitory activity
The inhibitory activity of thiodisaccharides 5, 8, 15, 18, 20 and
28 against the exo b-
was evaluated in vitro following a previous protocol.19a 4-Nitro-
phenyl b-
-galactofuranoside18d was used as substrate and
-galactono-1,4-lactone (29) as
D-galactofuranosidase from P. fellutanum
D
D
a reference inhibitor (IC50
0.02 mM).19a The enzymatic reaction was performed in the pres-
ence of thiodisaccharides 5, 8, 15, 18 20 and 28 at concentrations
ranging from 0.2 to 1.0 mM. The amount of 4-nitrophenol released
from 4-nitrophenyl b-D-galactofuranoside was determined as a
measure of galactofuranosidase activity (Fig. 1).
value for the coupling between H-10 and H-20 (J1 ,2 < 1 Hz) was
indicative of the b- -Galf configuration, as described for the analo-
0
0
D
100
90
80
70
60
50
40
30
20
10
0
18
gous thiodisaccharides reported here.
28
The carbonyl group of compound 24 was reduced with sodium
borohydride to afford the mixture of epimeric thiodisaccharides 26
and 27 (ꢀ6:1 ratio). The selectivity observed suggests that the
approach of the hydride is controlled by the anomeric substituent
adjacent to the carbonyl. The configuration for the C-2 and C-4 ste-
reocenters of the reducing-end was readily established on the basis
5
8
20
15
of the 1H NMR data. Thus, the major diasteroisomer 26 has the
a-D-
xylo configuration, as H-2 showed small coupling constant values
with H-1 (J1,2 = 3.7 Hz) and H-3eq (J2,3eq = 4.3 Hz), whereas the
J2,3ax value was large (11.5 Hz), indicating that the HO-2 is equa-
torially disposed. The fact that the H-4 signal appeared as a broad
singlet, with small coupling constants with H-3ax, H-3eq and H-5
confirms the configuration already assigned for C-4 in 24. A similar
analysis for 27, which showed only small coupling constant values
for H-2 and H-4 with the respective coupled protons, indicated the
29
0
0.2
0.4
0.6
0.8
1
Figure 1. Effect of concentration of thiodisaccharides on the enzymatic activity of
exo b-D-galactofuranosidase from Penicillium fellutanum. Each point is the mean
obtained from three replicate experiments.
a-D-lyxo configuration for the reducing end. Removal of the acetyl

E. Repetto et al. / Bioorg. Med. Chem. 17 (2009) 2703–2711
2707
The thiodisaccharide 18 was not active, and compounds 8 and
15, although the more active thiodisaccharides, exhibited only a
weak inhibitory activity (IC > 1 mM) compared with lactone 29
(IC50 0.02 mM). Thus, the thiodisaccharides constituted by b-D-Galf
S-linked to galactose in the pyranose form (15) or even substituted
by isopropylidene groups (8) are able to interact with the enzyme.
stirred at 0 °C for 6 h. TLC showed the conversion of the starting
material into 4 (Rf = 0.48, toluene/EtOAc, 9:1). The solution was
diluted with Cl2CH2, extracted with satd aq NaHCO3 (3 ꢂ 20 mL)
and with brine. The extract was dried (MgSO4) and concentrated,
and the residue was purified by flash chromatography (toluene/
EtOAc, 98:2) to give compound 4 as a colorless oil (0.29 g, 78%).
Probably for this reason, the thiodisaccharide
a-
L-Araf(1?6)-6-
It was recrystallized from EtOH, mp 115 °C; ½a D20
ꢄ5.7 (c 0.9,
ꢃ
thio- -Galp-OMe (20), structurally related to 15, exhibits a sim-
a-
D
CHCl3); 1H NMR (500 MHz, CDCl3): d 8.07–7.28 (m, 20H, H-aro-
matic), 6.52 (br s, 1H, H-1), 6.08 (ddd, 1H, J4,5 = 3.6, J5,6 = 4.0,
ilar inhibitory activity as 15 and 8. In contrast, thiodisaccharide 18,
the analogue of 20 having a Glcp unit replacing Galp in the reduc-
ing end, was not active.
0
J5,6 = 7.3 Hz, H-5), 5.71 (dd, 1H, J2,3 = 1.0, J3,4 = 4.4 Hz, H-3), 5.60
0
(d, 1H, J2,3 = 1.0 Hz, H-2), 4.80 (dd, 1H, J5,6 = 4.0, J6,6 = 12.0 Hz, H-
Thiodisaccharide 5, with a structure similar to that of 15 and 18,
showed an inhibitory activity intermediate between that of 15 and
6), 4.79 (dd, 1H, J4,5 = 3.6, J3,4 = 4.4 Hz, H-4), 4.72 (dd, 1H,
J5,6 = 7.3, J6,6 = 12.0 Hz, H-60), 2.19 (s, 3H, CH3CO); 13C NMR
(50 MHz, CDCl3): d 169.0 (MeCO), 166.0, 165.7, 165.5, 165.2
(PhCO), 133.7–128.2 (C-aromatic), 99.4 (C-1), 83.6 (C-4), 81.3 (C-
2), 77.4 (C-3), 76.1 (C-5), 63.5 (C-6), 21.0 (CH3CO). Anal. Calcd for
C36H30O11: C, 67.71; H, 4.73. Found: C, 67.78; H, 4.55.
0
0
18. Therefore, the presence of
decreases the inhibitory activity of these molecules. Compound
28, in which Galf is S-linked to 3-deoxy-4-thio- -xylo-pyranoside
a Glcp unit as reducing-end
D
is also a weak inhibitor of the enzyme. These results are in accor-
dance with the high specificity previously observed for the
enzyme.13
4.2.2. General procedure for the thioglycosylation of Galf
derivatives
4.2.2.1. Method A: SnCl4-Promoted thioglycosylation of 1 or
3. Conclusion
4. 1,2,3,5,6-Penta-O-benzoyl-a,b-D-galactofuranoside (1, 0.10 mmol)
or 1-O-acetyl 2,3,5,6-tetra-O-benzoyl-b-
D
-galactofuranoside (4,
The synthesis of thiodisaccharides constituted by Galf or Araf as
non-reducing end has been successfully accomplished. The 1-O-
acetyl-per-O-benzoyl derivative of Galf (4) was synthesized and
employed for the SnCl4 and MoO2Cl2-promoted thioglycosylations,
and showed to be more reactive than the per-O-benzoyl analogue
1, in particular for reactions catalyzed by MoO2Cl2. This catalyst
was very useful when the thiosugar precursor contained acid labile
protecting groups. Additionally, the glycosyl isothiourea derivative
0.10 mmol) dissolved in anhydrous CH2Cl2 (ꢀ1 mL), and cooled
externally to 0 °C, was treated with SnCl4 (0.11–0.12 mmol). After
10 min, the corresponding thiosugar (0.12 mmol) was added and
the mixture was stirred at 0 °C for 4 h. The reaction mixture was
extracted with satd aq NaHCO3 and then with brine. The organic
solution was dried (MgSO4) and concentrated. The residue was
subjected to column chromatography with solvent specified in each
individual case.
of Galf was prepared and used to generate in situ the 1-thio-D-Galf,
which was coupled to a sugar enone via a Michael addition
reaction.
4.2.2.2. Method B: MoO2Cl2-Catalyzed thioglycosylation of 4. To
a solution of MoO2Cl2 (0.007 mmol) in anhydrous CH2Cl2 (0.2 mL)
was added under Argon, a solution of compound 4 (0.10 mmol)
in CH2Cl2 (0.5 mL). The mixture was stirred at room temperature
(rt) for 10 min and a solution of the thiosugar (0.05–0.06 mmol)
in CH2Cl2 (0.5 mL) was added. The mixture turned blue and gradu-
ally changed to yellowish brown. After stirring at rt for 24 h, the
solution was diluted with CH2Cl2 (20 mL) and washed with satd
aq NaHCO3 and then with brine. The organic extract was dried
(MgSO4) and concentrated, and the resulting residue purified by
column chromatography with the solvent indicated in each case.
The free thiodisaccharides have been evaluated as inhibitors of
the exo b-
saccharides constituted by b-
played higher inhibitory activity than the others. Thiodisaccharides
formed by two -Galf units linked through sulfur are expected to
D-galactofuranosidase from P. fellutanum. Those thiodi-
D
-Galf or -Araf S-linked to Galp dis-
a-L
D
be active inhibitors and, in addition, will provide more information
about the galactofuranosidase specificity. The synthesis of this type
of compounds is under way.
4. Experimental
4.2.3. Methyl 2,3,4-tri-O-benzoyl-6-S-(2,3,5,6-tetra-O-benzoyl-
4.1. General methods
b-
D
-galactofuranosyl)-6-thio-
The SnCl4-promoted procedure (Method A) was followed start-
ing from 1 (60 mg, 0.086 mmol) in anhydrous CH2Cl2 (1 mL) and
SnCl4 (12 L, 0.10 mmol). After the addition of methyl 2,3,4-tri-
O-benzoyl-6-thio-
-glucopyranoside 225 (54 mg, 0.103 mmol)
a-D-glucopyranoside (3)
Analytical thin layer chromatography (TLC) was performed on
Silica Gel 60 F254 (Merck) aluminum supported plates (layer thick-
ness 0.2 mm) with solvent systems given in the text. Visualization
of the spots was effected by exposure to UV light and charring with
a solution of 5% (v/v) sulfuric acid in EtOH, containing 0.5% p-anis-
aldehyde. Column chromatography was carried out with Silica Gel
60 (230–400 mesh, Merck). Optical rotations were measured with
a Perkin–Elmer 343 digital polarimeter. Nuclear magnetic reso-
nance (NMR) spectra were recorded with a Bruker AC 200 or with
a Bruker AMX 500 instruments. Assignements of 1H and 13C were
assisted by 2D 1H-COSY and HSQC experiments.
l
a
-D
the mixture was stirred at 0 °C for 4 h and then processed as indi-
cated above. Purification by column chromatography (toluene/
EtOAc, 98:2) afforded thiodisaccharide
3
(58 mg, 61%);
½ ꢃ
a 2D0
ꢄ14.7 (c 1.1, CHCl3); 1H NMR (500 MHz, CDCl3): d 8.12–7.27 (m,
35H, H-aromatic), 6.12 (t, 1H, J2,3 = J3,4 = 9.8 Hz, H-3), 6.08 (ddd,
1H, J4 ,5 ꢀ J5 ,6 a = 4.0, J5 ,6 b = 6.9 Hz, H-50), 5.93 (br s, 1H, J1 ,2 < 1 Hz,
0
0
0
0
0
0
0
0
H-10), 5.68 (dt, 1H, J2 ,3 = 1.1, J3 ,4 = 5.0 Hz, H-30), 5.46 (d, 1H,
0
0
0
0
J2 ,3 = 1.1 Hz, H-20), 5.44 (t, 1H, J3,4 = J4,5 = 9.8 Hz, H-4), 5.22 (dd,
1H, J1,2 = 3.6, J2,3 = 9.8 Hz, H-2), 5.19 (d, 1H, J1,2 = 3.6 Hz, H-1),
0
0
4.82 (dd, 1H, J4 ,5 = 4.0, J3 ,4 = 5.0 Hz, H-40), 4.77 (dd, 1H,
4.2. Synthesis
0
0
0
0
J5 ,6 a = 4.4, J6 a,6 b = 11.9 Hz, H-60a), 4.74 (dd, 1H, J5 ,6 b = 6.9,
0
0
0
0
0
0
J6 a,6 b = 11.9 Hz, H-60b), 4.31 (ddd, 1H, J5,6b = 2.2, J4,5 = J5,6a = 9.8 Hz,
H-5), 3.49 (s, 3H, CH3O), 3.07 (dd, 1H, J5,6a = 9.8, J6a,6b = 14.6 Hz, H-
6a), 2.85 (dd, 1H, J5,6b = 2.2, J6a,6b = 14.6 Hz, H-6b); 13C NMR
(125.7 MHz, CDCl3): d 165.8, 165.7, 165.5 (PhCO), 133.6–128.3
(C-aromatic), 96.6 (C-1), 89.2 (C-10), 82.6 (C-20), 81.3 (C-40), 77.8
4.2.1. 1-O-Acetyl-2,3,5,6-tetra-O-benzoyl-b-
1,2,3,5,6-Penta-O-benzoyl- ,b-
-galactofuranoside24 (1, 0.46 g,
0.66 mmol) was dissolved in anhydrous Cl2CH2 (11 mL), cooled to
0 °C and SnCl4 (92 L, 0.77 mmol) was added. After 10 min acetic
anhydride (136 L, 1.44 mmol) was added and the mixture was
D-galactofuranoside (4)
0
0
a
D
l
l

2708
E. Repetto et al. / Bioorg. Med. Chem. 17 (2009) 2703–2711
(C-30), 72.3 (C-4), 72.1 (C-2), 71.5 (C-5), 70.2 (ꢂ2, C-3,50), 63.4 (C-
60), 55.5 (CH3O), 31.3 (C-6). Anal. Calcd for C62H52O17S: C, 67.61;
H, 4.76; S, 2.91. Found: C, 67.27; H, 4.83; S, 2.55.
The same procedure (method A) was applied for the reaction of 1-
O-acetyl-2,3,5,6-tetra-O-benzoyl-b-D-galactofuranose (4, 60 mg,
4.2.6. Methyl 2,3,4-tri-O-benzoyl-6-deoxy-6-thiocyano-a-D-
galactopyranoside (12)
Tosylate 11 (0.34 g, 0.51 mmol) was dissolved in anhydrous
DMF (4 mL) and heated with KSCN (0.29 g, 3.00 mmol) at 120 °C
for 48 h. TLC showed formation of a major product of Rf = 0.44 (tol-
uene/EtOAc, 17:1) and some starting material remaining
(Rf = 0.40). Column chromatography (toluene/EtOAc, 99:1) affor-
0.094 mmol) with 2 (59 mg, 0113 mmol), to produce 3 (61 mg, 59%).
Alternatively, thiodisaccharide 3 was synthesized using MoO2Cl2
as catalyst (Method B). To
a
solution of MoO2Cl2 (1.2 mg,
ded 12 (0.16 g, 55.2%) and 11 (0.08 g, 24%). Compound 12: ½a D20
ꢃ
0.006 mmol) in anhydrous CH2Cl2 (0.2 mL), was added under Argon
a solution of compound 4 (55 mg, 0.086 mmol) in CH2Cl2 (0.5 mL).
The mixture was stirred for 10 min at rt and methyl 2,3,4-tri-O-ben-
+182.5 (c 1.2, CHCl3); 1H NMR (200 MHz, CDCl3): d 8.11–7.20 (m,
15H, H-aromatic), 5.97 (dd, 1H, J3,4 = 3.4, J2,3 = 10.6 Hz, H-3), 5.89
(dd, 1H, J4,5 = 0.9, J3,4 = 3.4 Hz, H-4), 5.67 (dd, 1H, J1,2 = 3.5,
J2,3 = 10.6 Hz, H-2), 5.26 (d, 1H, J1,2 = 3.5 Hz, H-1), 4.24 (br t, 1H,
zoyl-6-thio-a-D
-glucopyranoside25 (2, 24 mg, 0.046 mmol) in
0
CH2Cl2 (0.5 mL) was added. After the usual work-up and purification
procedure, the thiodisaccharide 3 (28 mg, 56%) was obtained
(Rf = 0.59, toluene/EtOAC, 10:1). This product was identical to the
one previously described.
J5,6 ꢀ J5,6 ꢀ 7.1 Hz, H-5), 3.48 (s, 3H, CH3O), 3.13 (d, 2H,
J5,6 ꢀ J5,6 ꢀ 7.1 Hz, H-6,60); 13C NMR (50 MHz, CDCl3): d 166.1,
0
165.8, 165.4 (PhCO), 133.9–128.3 (C-aromatic), 112.0 (SCN), 97.7
(C-1), 70.3, 68.9, 68.1, 67.9 (C-2,3,4,5), 56.1 (CH3O), 34.6 (C-6).
Anal. Calcd for C29H25O8SN.1.5H2O: C, 60.62; H, 4.91; S, 5.58.
Found: C, 60.48; H, 4.90; S, 5.40.
4.2.4. 1,2:3,4-di-O-isopropylidene-6-S-(2,3,5,6-tetra-O-benzoyl–
D-galactofuranosyl)-6-thio-a-D-galactopyranose (7)
The MoO2Cl2-catalyzed procedure (Method B) was followed,
4.2.7. Methyl 2,3,4-tri-O-benzoyl-6-thio-a-D-galactopyranoside (13)
starting from
4
(160 mg, 0.251 mmol), MoO2Cl2 (2.7 mg,
The thiocyano derivative 12 (0.70 g, 1.28 mmol) was heated
under reflux with Zn (1.67 g, 25.6 mmol) in glacial acetic acid
(25 mL) for 24 h. The mixture was poured into water, and extracted
with CH2Cl2. The organic phase was washed successively with
NaCO3H, and brine, dried and concentrated. The resulting syrup
showed by TLC a main spot of Rf = 0.49 (toluene/EtOAc, 17:1). Purifi-
cationbycolumn chromatography(toluene/EtOAc, 99:1)afforded13
0.014 mmol) and 67,37 (35 mg, 0.127 mmol). After 24 h of stirring
at rt, TLC showed a main spot of Rf = 0.48 (toluene/EtOAc, 7:1). Thi-
odisaccharide 7 (40 mg, 37%) was purified by column chromatogra-
phy using (toluene/EtOAc, 97:3); ½a D20
ꢃ
ꢄ73.5 (c 0.9, CHCl3); 1H NMR
(500 MHz, CDCl3): d 8.10–7.10 (m, 15H, H-aromatic), 6.09 (ddd, 1H,
J5 ,6 a = 4.2, J4 ,5 = 4.8, J5 ,6 b = 7.3 Hz, H-50), 5.75 (t, 1H, J1 ,3 = 0.8,
0
0
0
0
0
0
0
0
J1 ,2 = 1.5 Hz, H-10), 5.67 (dddd, 1H, J1 ,3 = 0.8, J2 ,3 = 1.5, J3 ,4 = 4.8 Hz,
(0.52 g, 78.3%); ½a D20
ꢃ
+240.7 (c 1.1, CHCl3); 1H NMR (200 MHz, CDCl3):
0
0
0
0
0
0
0
0
H-30), 5.55 (t, 1H, J1 ,2 = J2 ,3 = 1.5 Hz, H-20), 5.52 (d, 1H, J1,2 = 5.0 Hz,
d 8.10–7.15 (m, 15H, H-aromatic), 5.99–5.93 (m, 2H, H-3,4), 5.64 (dd,
1H, J1,2 = 3.7, J2,3 = 10.7 Hz, H-2), 5.28 (d, 1H, J1,2 = 3.7 Hz, H-1), 4.26
0
0
0
0
H-1), 4.81 (t, 1H, J3 ,4 = J4 ,5 = 4.8 Hz, H-40), 4.78 (dd, 1H, J5 ,6 a = 4.2,
0
0
0
0
0
0
J6 a,6 b = 11.9 Hz, H-60a), 4.72 (dd, 1H, J5 ,6 b = 7.3, J6 a,6 b = 11.9 Hz, H-
60b), 4.60 (dd, J2,3 = 2.4, J3,4 = 8.0 Hz, H-3), 4.30 (m, 2H, H-2,4), 3.97
(ddd, 1H, J4,5 = 1.7, J5,6b = 6.3, J5,6a = 7.5 Hz, H-5), 3.06 (dd, 1H,
J5,6a = 7.5, J6a,6b = 13.7 Hz, H-6a), 2.89 (dd, 1H, J5,6b = 6.3,
J6a,6b = 13.7 Hz, H-6b), 4.49, 1.44, 1.32, 1.29 (4s, 12H, 2 ꢂ (CH3)2C);
13C NMR (125.7 MHz, CDCl3): d 166.0, 165.7, 165.5, 165.3 (PhCO),
133.5–128.3 (C-aromatic), 109.4, 108.6 (Me2C), 96.6 (C-1), 88.4
(C-10), 82.6 (C-20), 81.5 (C-40), 77.9 (C-30), 71.7, 70.5 (C-2,4), 70.8
(C-3), 70.3 (C-50), 68.8 (C-5), 63.5 (C-60), 30.5 (C-6), 26.0, 25.9, 24.9,
24.5 [2 ꢂ (CH3)2C]. Anal. Calcd for C46H46O14S: C, 64.61; H, 5.43.
Found: C, 64.94; H, 5.33.
(dd, 1H, J5,6 = 5.8, J5,6 = 7.9 Hz, H-5), 3.52 (s, 3H, CH3O), 2.81 (ddd,
0
0
0
0
0
0
0
0
0
1H, J5,6 ꢀ J6,SH = 7.9, J6,6 = 14.0 Hz, H-6), 2.64 (ddd, 1H, J5,6 = 5.8,
J6 ,SH = 9.5, J6,6 = 14.0 Hz, H-60), 1.76 (dd, 1H, J6,SH = 7.9, J6 ,SH = 9.5 Hz,
SH); 13C NMR (50 MHz, CDCl3): d 166.1, 165.8, 165.5 (PhCO), 133.6–
128.3 (C-aromatic), 97.6 (C-1), 71.0, 70.1, 69.4, 68.6 (C-2,3,4,5), 55.8
(s, 3H, CH3O), 24.8 (C-6). Anal. Calcd for C28H26O8S: C, 64.36; H, 5.01;
S, 6.13. Found: C, 64.41; H, 5.10; S, 6.18.
0
0
0
4.2.8. Methyl 2,3,4-tri-O-benzoyl-6-S-(2,3,5,6-tetra-O-benzoyl-
b-D
-galactofuranosyl)-6-thio-
The SnCl4-promoted thioglycosylation (Method A) was em-
ployed, starting from (79 mg, 0.11 mmol), SnCl4 (16 L,
a-D-galactopyranoside (14)
1
l
4.2.5. Methyl 2,3,4-tri-O-benzoyl-6-O-tosyl-
galactopyranoside (11)
a
-
D
-
0.13 mmol) and 13 (71 mg, 0.135 mmol). Column chromatography
(toluene/EtOAc, 98:2) afforded thiodisaccharide 14 (65 mg, 52.5%);
To a suspension of methyl
a-
D-galactopyranoside (10, 1.50 g,
Rf = 0.52 (toluene/EtOAc, 10:1); ½a D20
ꢃ
+65.0 (c 0.4, CHCl3); 1H NMR
7.73 mmol) in anhydrous pyridine (15 mL) was added tosyl chlo-
ride (2.2 g, 11.54 mmol) and the mixture was stirred for 4 days
at rt. Then, benzoyl chloride (4.5 mL, 38.7 mmol) was added and
the stirring was maintained at rt for 18 h. Then, the mixture was
poured into ice/water. Analysis by TLC (hexane/EtOAc, 2:1) showed
a major product of Rf = 0.44, together with a minor one of Rf = 0.59.
These products were isolated by column chromatography (hexane/
EtOAc, 9:1–3:1). The less polar component was spectroscopically
(500 MHz, CDCl3): d 8.13–7.22 (m, 35H, H-aromatic), 6.03 (ddd,
1H, J4 ,5 = J5 ,6 a = 4.3, J5 ,6 b = 6.8 Hz, H-50), 5.95 (dd, 1H, J3,4 = 3.4,
J2,3 = 10.7 Hz, H-3), 5.89 (br d, 1H, J4,5 < 1, J3,4 = 3.4 Hz, H-4), 5.89
0
0
0
0
0
0
(br s, 1H, J1 ,2 < 1 Hz, H-10), 5.70 (d, 1H, J3 ,4 = 5.0 Hz, H-30), 5.62
0
0
0
0
(dd, 1H, J1,2
=
3.6, J2,3 = 10.6 Hz, H-2), 5.48 (br s, 1H,
J1 ,2 ꢀ J2 ,3 < 1.0 Hz, H-20), 5.27 (d, 1H, J1,2 = 3.6 Hz, H-1), 4.79 (t,
0
0
0
0
1H, J4 ,5 = 4.3, J3 ,4 = 5.0 Hz, H-40), 4.71 (dd, 1H, J5 ,6 a = 4.5,
0
0
0
0
0
0
J6 a,6 b = 11.8 Hz, H-60a), 4.67 (dd, 1H, J5 ,6 b = 7.0, J6 a,6 b = 11.8 Hz,
H-60b), 4.46 (dd, 1H, J5,6b = 3.5, J5,6a = 9.5 Hz, H-5), 3.50 (s, 3H,
CH3O), 3.12 (dd, 1H, J5,6a = 9.5, J6a,6b = 14.3 Hz, H-6a), 2.76 (dd,
1H, J5,6b = 3.8, J6a,6b = 14.3 Hz, H-6b); 13C NMR (125.7 MHz, CDCl3):
d 166.1, 165.9, 165.7 (ꢂ2), 165.5 (ꢂ2), 165.3 (PhCO), 134.5–128.2
(C-aromatic), 97.4 (C-1), 89.0 (C-10), 82.6 (C-20), 81.3 (C-40), 77.7
(C-30), 70.8 (C-4), 70.4 (C-5), 70.2 (C-50), 69.3 (C-2), 68.5 (C-3),
63.2 (C-60), 55.7 (CH3O), 30.7 (C-6). Anal. Calcd for C62H52O17S: C,
67.61; H, 4.76. Found: C, 67.36; H, 4.76.
0
0
0
0
0
0
identified as methyl 2,3,4,6-tetra-O-benzoyl-a-D-galactopyrano-
side38 (1.05 g, 22.3%). From the following fractions from the col-
umn was isolated 11 (2.00 g, 39.2%); ½a D20
ꢃ
+164.1 (c 0.3, CHCl3);
1H NMR (200 MHz, CDCl3): d 8.00–7.10 (m, 19H, H-aromatic),
5.90 (dd, 1H, J3,4 = 3.5, J2,3 = 10.3 Hz, H-3), 5.85 (dd, 1H, J4,5 = 1.1,
J3,4 = 3.5 Hz, H-4), 5.57 (dd, 1H, J1,2 = 3.6, J2,3 = 10.3 Hz, H-2), 5.24
0
(d, 1H, J1,2 = 3.6 Hz, H-1), 4.49 (br t, 1H, J4,5 = 1.1, J5,6 = 5.7,
0
J5,6 = 7.0 Hz, H-5), 4.24 (dd, 1H, J5,6 = 7.0, J6,6 = 10.3 Hz, H-6), 4.09
(dd, 1H, J6,6 = 5.7, J5,6 = 7.0, J6,6 = 10.3 Hz, H-60), 3.45 (s, 3H,
CH3O), 2.33 (s, 3H, CH3Ar); 13C NMR (50 MHz, CDCl3): d 166.0,
165.8, 165.3 (PhCO), 127.9 (C-aromatic), 97.5 (C-1), 69.0, 68.7,
68.1, 67.3, 66.6 (C-2,3,4,5,6), 55.8 (CH3O), 21.6 (CH3Ar). Anal. Calcd
for C35H32O10S: S, 4.97. Found: S, 4.90.
0
0
0
4.2.9. Methyl 2,3,4-tri-O-benzoyl-6-S-(2,3,5-tri-O-acetyl-
arabinofuranosyl)-6-thio- -glucopyranoside (17)
Tetra-O-acetyl- -arabinofuranose (16, 50 mg, 0.16 mmol) and
2 (99 mg, 0.19 mmol) reacted in the presence of SnCl4 (20 L,
a-L-
a
-D
a-L
l

E. Repetto et al. / Bioorg. Med. Chem. 17 (2009) 2703–2711
2709
0.17 mmol) as described above (Method A) to give 17 (Rf = 0.34,
toluene/EtOAc, 5:1). Column chromatography (toluene/EtOAc,
95:5) afforded thiodisaccharide 17 (61 mg, 50%) as a yellowish syr-
was stirred at 0 °C for 3 h. TLC (toluene/EtOAc, 9:1) showed two
main products of Rf = 0.34 (major) and 0.16, respectively. After con-
centration and purification by flash chromatography (toluene/
EtOAc, 96:4), thiodissacharide 24 (145 mg, 52%) was obtained. This
product was slightly contaminated by the enone 23. 1H NMR
(500 MHz, CDCl3): d 8.10–7.16 (m, 20H, H-aromatic), 6.10 (dt,
up; ½a 2D0
ꢃ
ꢄ29.3 (c 0.6, CHCl3); 1H NMR (500 MHz, CDCl3): d 8.00–
7.28 (m, 15H, H-aromatic), 6.12 (t, 1H, J2,3 = J3,4 = 9.5 Hz, H-3),
5.61 (s, 1H, J1 ,2 < 1 Hz, H-10), 5.68 (t, 1H, J3,4 = J4,5 = 9.7 Hz, H-4),
5.23 (dd, 1H, J1,2 = 3.6, J2,3 = 9.5 Hz, H-2), 5.22 (d, 1H, J1,2 = 3.6 Hz,
0
0
1H, J4 ,5 ꢀ J5 ,6 a = 4.3, J5 ,6 b = 6.4 Hz, H-50), 5.70 (br d, 1H,
0
0
0
0
0
0
H-1), 5.08 (t, 1H, J1 ,2 = J2 ,3 = 1.7 Hz, H-20), 5.02 (dd, 1H, J2 ,3 = 1.7,
J2 ,3 < 1.0, J3 ,4 = 4.8 Hz, H-30), 5.69 (br s, 1H, H-10), 5.50 (br s, 1H,
H-20), 4.80–4.75 (m, 3H, H-40,5,60a), 4.79 (br s, 1H, J1,2 < 1.0 Hz, H-
0
0
0
0
0
0
0
0
0
0
J3 ,4 = 5.1 Hz, H-30), 4.39 (m, 2H, H-40,50a), 4.27 (m, 2H, H-5,50b),
3.52 (s, 3H, CH3O), 3.00 (dd, 1H, J5,6a = 9.7, J6a,6b = 14.5 Hz, H-6a),
2.81 (dd, 1H, J5,6b = 2.3, J6a,6b = 14.5 Hz, H-6b), 2.11, 2.09 (2s, 9H,
CH3CO); 13C NMR (125.7 MHz, CDCl3): d 170.6, 170.0, 169.5
(MeCO), 165.8, 165.7, 165.5 (PhCO), 133.5–128.3 (C-aromatic),
96.7 (C-1), 88.9 (C-10), 81.8 (C-20), 79.7 (C-40), 77.6 (C-30), 72.4 (C-
4), 72.1 (C-2), 71.2 (C-5), 70.1 (C-3), 62.9 (C-50), 55.5 (CH3O), 31.7
(C-6), 20.8, 20.7 (CH3CO). Anal. Calcd for C39H40O15S: C, 59.97; H,
5.17. Found: C, 60.07; H, 5.44.
0
0
1), 4.74 (dd, 1H, J5 ,6 a = 6.4, J6 a,6 b = 12.0 Hz, H-60b), 4.37 (dd, 1H,
J5,6a = 7.3, J6a,6b = 11.6 Hz, H-6a), 4.28 (dd, 1H, J5,6b = 4.9,
J6a,6b = 11.6 Hz, H-6b), 4.02 (m, 1H, J = 6.2 Hz, Me2CHO), 3.70 (dt,
0
0
0
0
J3eq,4 = J4,5 = 2.4, J3ax,4 = 5.0 Hz, H-4), 3.17 (dd, 1H, J3ax,4
=
5.0, J3ax,3eq = 15.2 Hz, H-3ax), 2.82 (dd, 1H, J3eq,4 = 2.4, J3ax,3-
eq = 15.2 Hz, H-3eq), 2.05 (s, 3H, CH3CO2), 1.28, 1.18 (2d, 6H,
J = 6.2 Hz, (CH3)2CHO); 13C NMR (125.7 MHz, CDCl3): d 198.7 (C-
2), 170.4 (MeCO), 166.1, 165.7, 165.5, 165.2 (PhCO), 133.6–128.4
(C-aromatic), 98.0 (C-1), 86.6 (C-10), 82.6, 82.1 (C-20,40), 77.8 (C-
30), 71.8, 70.2, 68.4 (Me2CHO, C-5,50), 64.6, 63.4 (C-6,60), 45.1 (C-
4), 42.6 (C-3), 23.3, 21.8 [(CH3)2CHO], 20.7 (CH3CO).
Alternatively, 17 (94 mg, 65%) was obtained by catalysis of
MoO2Cl2 (Method B), starting from 16 (100 mg, 0.31 mmol), 2
(97 mg, 0.19 mmol) and MoO2Cl2 (3.8 mg, 0.02 mmol).
Further fractions from the column afforded syrupy 25 (45 mg,
4.2.10. Methyl 2,3,4-tri-O-benzoyl-6-S-(2,3,5-tri-O-acetyl-
arabinofuranosyl)-6-thio- -galactoyranoside (19)
Tetra-O-acetyl- -arabinofuranose (16, 50 mg, 0.157 mmol)
and 13 (99 mg, 0.190 mmol) reacted under the presence of SnCl4
(20 L, 0.17 mmol) as described above (Method A). Purification by
a-L-
16%); 1H NMR (500 MHz, CDCl3): d 8.09–7.28 (m, 20H, H-aromatic),
a-
D
6.08 (dt, 1H, J4 ,5 = J5 ,6 a = 4.3, J5 ,6 b = 7.0 Hz, H-50), 5.72 (br s, 1H,
0
0
0
0
0
0
0
0
0
0
0
0
a-
L
J1 .2 < 1.0 Hz, H-10), 5.70 (br d, 1H, J2 ,3 < 1.0, J3 ,4 = 4.3 Hz, H-30),
5.47 (br s, 1H, H-20), 4.85 (t, 1H, J3 ,4 = J4 ,5 = 4.3 Hz, H-40), 4.79
0
0
0
0
0
0
l
(br s, 1H, J1,2 < 1.0 Hz, H-1), 4.78 (dd, 1H, J5 ,6 a = 4.3,
J6 a,6 b = 11.8 Hz, H-60a), 4.72 (dd, 1H, J5 ,6 b = 7.0, J6 a,6 b = 11.8 Hz,
H-60b), 4.59 (dd, 1H, J5,6a = 1.4, J6a,6b = 11.9 Hz, H-6a), 4.47 (dd,
1H, J5,6b = 5.0, J6a,6b = 11.9 Hz, H-6b), 4.43 (ddd, 1H, J5,6a = 1.4,
J5,6b = 5.0, J4,5 = 10.8 Hz, H-5), 3.98 (m, 1H, J = 6.2 Hz, Me2CHO),
3.42 (ddd, 1H, J3eq,4 = 5.0, J3ax,4 = 12.8 Hz, H-4), 3.02 (dd, 1H,
0
0
0
0
0
0
column chromatography (toluene/EtOAc, 95:5) afforded syrupy thi-
odisaccharide 19 (75 mg, 61%); Rf = 0.33 (toluene/EtOAc, 5:1); ½a D20
ꢃ
+67.5 (c 0.4, CHCl3); 1H NMR (500 MHz, CDCl3): d 8.10–7.15 (m,
15H, H-aromatic), 5.94 (dd, 1H, J3,4 = 3.5, J2,3 = 10.6 Hz, H-3), 5.90
(dd, 1H, J4,5 = 0.7, J3,4 = 3.5 Hz, H-4), 5.62 (dd, 1H, J1,2
= 3.6,
J2,3 = 10.6 Hz, H-2), 5.52 (br s, 1H, H-10), 5.28 (d, 1H, J1,2 = 3.6 Hz,
J3ax,4 = 12.8, J3ax,3eq = 14.6 Hz, H-3ax), 2.86 (dd, 1H, J3eq,4
=
H-1), 5.09 (t, 1H, J1 ,2 = J2 ,3 = 1.6 Hz, H-20), 5.03 (dd, 1H, J2 ,3 = 1.6,
5.0, J3ax,3eq = 14.6 Hz, H-3eq), 2.06 (s, 3H, CH3CO2), 1.25, 1.17 (2d,
6H, J = 6.2 Hz, (CH3)2CHO); 13C NMR (125.7 MHz, CDCl3): d 199.0
(C-2), 170.6 (MeCO), 166.0, 165.6, 165.4, 165.3 (PhCO), 133.7–
128.3 (C-aromatic), 97.7 (C-1), 87.9 (C-10), 82.7 (C-20), 81.8 (C-40),
77.6 (C-30), 71.8, 70.3, 70.2 (Me2CHO, C-5,50), 63.6, 63.2 (C-6,60),
42.9, 42.7 (C-3,4), 23.2, 21.7 [(CH3)2CHO], 20.7 (CH3CO).
0
0
0
0
0
0
J3 ,4 = 5.4 Hz, H-30), 4.41 (ddd, 1H, J4,5 = 0.7, J5,6b = 4.2, J5,6a = 9.2 Hz,
0
0
H-5), 4.34 (ddd, 1H, J4 ,5 a = 3.4, J3 ,4 = 5.4, J4 ,5 b = 6.1 Hz, H-40), 4.32
0
0
0
0
0
0
(dd, 1H, J4 ,5 a = 3.4, J5 a,5 b = 12.6 Hz, H-50a), 4.24 (dd, 1H,
0
0
0
0
J4 ,5 b = 6.1, J5 a,5 b = 12.6 Hz, H-50b), 3.04 (dd, 1H, J5,6a = 9.2,
J6a,6b = 14.1 Hz, H-6a), 2.74 (dd, 1H, J5,6b = 4.2, J6a,6b = 14.1 Hz, H-
6b); 13C NMR (125.7 MHz, CDCl3): d 170.5, 170.0, 169.5 (MeCO),
166.1, 165.7, 165.5 (PhCO), 133.5–128.2 (C-aromatic), 97.4 (C-1),
88.7 (C-10), 81.7 (C-20), 79.7 (C-40), 77.2 (C-30), 70.7 (C-4), 70.0 (C-
5), 69.3 (C-2), 68.4 (C-3), 62.7 (C-50), 55.7 (CH3O), 31.1 (C-6), 20.7
(CH3CO). Anal. Calcd for C39H40O15S: C, 59.97; H, 5.17. Found: C,
60.10; H, 5.19.
0
0
0
0
4.2.12. 2-Propyl 6-O-acetyl-3-deoxy-4-S-(2,3,5,6-tetra-O-benzoyl-
b-
propyl 6-O-acetyl-3-deoxy-4-S-(2,3,5,6-tetra-O-benzoyl-b-
galactofuranosyl)-4-thio- -lyxo-hexopyranoside (27)
D-galactofuranosyl)-4-thio-a-D-xylo-hexopyranoside (26) and 2-
D-
a-D
Compound 24 (163 mg, 0.19 mmol) was dissolved in THF (5 mL)
and treated with NaBH4 (50 mg, 1.31 mmol) at ꢄ18 °C. The mixture
was stirred for 30 min, neutralized with Dowex 50 (H+) resin, fil-
tered and concentrated, and evaporated with MeOH, the syrup
showed by TLC (hexane/EtOAc, 7:3) two products of Rf 0.60 (minor)
and 0.53 (major). Column chromatography (hexane/EtOAc, 69:31)
afforded first the less polar product 27 (10 mg, 6%) as a syrup and
the major thiodisaccharide 26 (65 mg, 41%) as a colorless oil. Com-
4.2.11. 2-Propyl 6-O-acetyl-3-deoxy-4-S-(2,3,5,6-tetra-O-benzoyl-
b-
and 2-propyl 6-O-acetyl-3-deoxy-4-S-(2,3,5,6-tetra-O-benzoyl-b-
galactofuranosyl)-4-thio- -erythro-hexopyranosid-2-ulose (25)
To a solution of 1,2,3,5,6-penta-O-benzoyl- ,b- -galactofura-
nose24 (1, 280 mg, 40 mmol) in MeCN (anhyd, 8 mL), was added
L,
D-galactofuranosyl)-4-thio-a-D-threo-hexopyranosid-2-ulose (24)
D-
a-D
a
D
thiourea (34 mg, 0.45 mmol), followed by F3BꢁOEt2 (260
l
pound 26: ½a 2D0
ꢃ
ꢄ24.4 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3): d
0
0
2.07 mmol). The mixture was heated under reflux for 2.5 h, when
8.17–7.23 (m, 20H, H-aromatic), 6.09 (br q, 1H, J4 ,5 = 4.2,
J5 ,6 a = J5 ,6 b = 5.0 Hz, H-50), 5.69 (br d, 1H, J2 ,3 < 1.0, J3 ,4 = 5.0 Hz,
0
0
0
0
0
0
0
0
TLC (toluene/EtOAc, 9:1) showed complete consumption of the
H-30), 5.65 (br s, 1H, J1 ,2 < 1.0, H-10), 5.50 (br s, 1H, H-20), 4.93 (d,
0
0
starting material. Evaporation of the solvent gave the b-
D
-galacto-
furanosyl isothiourea (21) as a syrup; 1H NMR (500 MHz, CDCl3): d
8.15–7.20 (m, H-aromatic + NH2), 6.36 (s, 1H, H-1), 6.09 (m, 1H, H-
5), 5.80 (d, 1H, J3,4 = 3.6 Hz, H-3), 5.55 (s, 1H, H-2), 4.95 (dd, 1H,
1H, J1,2 = 3.7 Hz, H-1), 4.79 (br t, 1H, J3 ,4 ꢀ J4 ,5 = 4.5 Hz, H-40),
4.74 (m, 2H, H-60a,60b), 4.28 (m, 2H, H-5,6a), 4.16 (dd, 1H,
J5,6b = 7.5, J6a,6b = 14.7 Hz, H-6b), 4.10 (tt, 1H, J1,2 = 3.7, J2,3eq = 4.3,
J2,3ax = J2,OH = 11.5 Hz, H-2) 3.95 (m, 1H, J = 6.2 Hz, Me2CHO), 3.33
(br s, 1H, H-4), 2.25 (ddd, 1H, J3eq,4 = 3.5, J2,3eq = 4.3, J3ax,3eq = 13.0 Hz,
H-3eq), 2.04 (ddd, 1H, J3ax,4 = 5.1, J2,3ax = 11.5, J3ax,3eq = 13.0 Hz, H-
3ax), 2.03 (s, 3H, CH3CO2), 1.85 (d, 1H, J2,OH = 11.5 Hz, HO), 1.27,
1.19 (2 d, 6H, J = 6.2 Hz, (CH3)2CHO); 13C NMR (125.7 MHz, CDCl3):
d 170.5 (MeCO), 166.1, 165.7, 165.5, 165.4 (PhCO), 133.7–128.4 (C-
aromatic), 96.9 (C-1), 87.6 (C-10), 82.8 (C-20), 81.7 (C-40), 77.8 (C-
30), 70.9 (Me2CHO), 70.2 (C-50), 67.8 (C-2), 65.4 (C-6), 64.3 (C-5),
0
0
0
0
0
J3,4 = 3.6, J4,5 = 4.1 Hz, H-4), 4.89 (dd, 1H, J5,6 = 4.7, J6,6 = 11.9 Hz,
H-6), 4.66 (dd, 1H, J5,6 = 6.5, J6,6 = 11.9 Hz, H-60); 13C NMR
(50 MHz, CDCl3): d 170.8, (SC(NH2)2), 166.2, 165.7, 165.6, 165.3
(PhCO), 133.4–128.3 (C-aromatic), 88.2 (C-1), 84.2, 81.7 (C-2,4),
77.0 (C-3), 69.8 (C-5), 63.0 (C-6).
0
0
To the original mixture, cooled to 0 °C, 2-propyl 6-O-acetyl-2,3-
dideoxy-
a
-D
-glycero-hex-3-enopyranosid-2-ulose
(23,
76 mg,
0.33 mmol) and Et3N (518
ll, 3.72 mmol) were added. The mixture

2710
E. Repetto et al. / Bioorg. Med. Chem. 17 (2009) 2703–2711
63.5 (C-60), 43.5 (C-4), 34.0 (C-3), 23.2, 22.0 [(CH3)2CHO], 20.8
(CH3CO). Anal. Calcd for C45H46O14S: C, 64.12; H, 5.50. Found: C,
64.19; H, 5.78.
J6a,6b = 13.7 Hz, H-6b), 1.51, 1.40, 1.34, 1.33 (4s, 12H, 2 (CH3)2C);
13C NMR (125.7 MHz, CDCl3): d 110.3, 109.9 (Me2C), 97.9 (C-1),
90.7 (C-10), 83.9 (C-20), 83.2 (C-40), 78.5 (C-30), 72.8 (C-4), 72.3 (C-
50), 72.2 (C-3), 71.9 (C-2), 69.8 (C-5), 64.6 (C-60), 31.3 (C-6), 26.4,
26.3, 25.1, 24.6 [(CH3)2C]. Anal. Calcd for C18H30O10S: C, 49.30; H,
6.90. Found: C, 50.00; H, 7.39.
Compound 27: ½a 2D0
ꢃ
ꢄ32.3 (c 0.9, CHCl3); 1H NMR (500 MHz,
CDCl3):
d
0
8.08–7.23 (m, 20H, H-aromatic), 6.08 (ddd, 1H,
J4 ,5 ꢀ J5 ,6 a = 4.2, J5 ,6 b = 6.4 Hz, H-50), 5.77 (br s, 1H, J1 ,2 < 1.0 Hz,
0
0
0
0
0
0
0
H-10), 5.71 (brd, 1H, J2 ,3 < 1.0, J3 ,4 = 5.0 Hz, H-30), 5.50 (br s, 1H,
H-20), 4.92 (br s, 1H, J1,2 < 1 Hz, H-1), 4.79 (m, 3H, H-40,5,60a),
0
0
0
0
4.3.3. Methyl 6-S-(b-D-galactofuranosyl)-6-thio-a-D-
4.76 (dd, 1H, J6 a,6 b = 11.9 Hz, H-60b), 4.45 (m, 1H, H-2), 4.38 (dd,
1H, J5,6a = 8.0, J6a,6b = 11.4 Hz, H-6a), 4.23 (dd, 1H, J5,6b = 4.0 Hz, H-
6b), 3.99 (m, 1H, J = 6.2 Hz, Me2CHO), 3.28 (br s, 1H, H-4), 2.31
(m, 2H, H-3ax,3eq), 2.05 (s, 3H, CH3CO2), 1.26, 1.20 (2d, 6H,
J = 6.2 Hz, (CH3)2CHO); 13C NMR (125.7 MHz, CDCl3): d 170.5
(MeCO), 166.0, 165.7, 165.5, 165.4 (PhCO), 133.7–128.4 (C-aro-
matic), 99.0 (C-1), 86.8 (C-10), 82.8, 82.0 (C-20,40), 77.8 (C-30),
70.2, 69.5, 67.9, 67.5 (C-2,5,50,Me2CHO), 65.2, 63.4 (C-6,60), 39.4
(C-4), 29.6 (C-3), 23.2, 21.5 [(CH3)2CHO], 20.8 (CH3CO). Anal. Calcd
for C45H46O14S: C, 64.12; H, 5.50. Found: C, 63.98; H, 5.51.
galactopyranoside (15)
0
0
O-Debenzoylation of 14 led to 15 (67%); Rf = 0.68 (MeOH); ½a D20
ꢃ
–13.5 (c 0.9, H2O); 1H NMR (500 MHz, CDCl3): d 5.12 (d, 1H,
J1 ,2 = 5.3 Hz, H-10), 4.71 (d, 1H, H-1), 4.03 (dd, 1H, J2 ,3 = 5.3,
0
0
0
0
J3 ,4 = 7.4 Hz, H-30), 3.93 (t, 1H, J1 ,2 = J2 ,3 = 5.3 Hz, H-20), 3.92–
0
0
0
0
0
0
3.86 (m, 2H, H-4,5), 3.88 (dd, 1H, J4 ,5 = 3.5, J3 ,4 = 7.5 Hz, H-40),
0
0
0
0
3.76 (ddd, 1H, J4 ,5 = 3.5, J5 ,6 a = 4.6, J5 ,6 b = 7.6 Hz, H-50), 3.71 (m,
0
0
0
0
0
0
2H, H-2,3), 3.60 (dd, 1H, J5 ,6 a = 4.6, J6 a,6 b = 11.6 Hz, H-60a), 3.55
0
0
0
0
(dd, 1H, J5 ,6 b = 7.6, J6 a,6 b = 11.6 Hz, H-60b), 3.35 (s, 3H, CH3O),
2.90 (dd, 1H, J5,6a = 8.9, J6a,6b = 14.0 Hz, H-6a), 2.74 (dd, 1H,
J5,6b = 4.8, J6a,6b = 14.0 Hz, H-6b); 13C NMR (125.7 MHz, CDCl3): d
99.5 (C-1), 89.1 (C-10), 81.3 (C-40), 81.0 (C-20), 76.0 (C-30), 70.9,
70.3, 70.1 (C-4,5,50), 69.5, 68.0 (C-2,3), 62.7 (C-60), 55.2 (CH3O),
31.5 (C-6). Anal. Calcd for C13H24O10S: C, 41.91; H, 6.50. Found:
C, 42.26; H, 6.70.
0
0
0
0
4.3. General procedure for the O-deacylation of
thiodisaccharides 3, 7, 14, 17, 19 and 26
The O-acylated thiodisaccharide (0.10 mmol) was suspended in
MeOH/Et3N/H2O, 3:1:3 (16 mL) and the mixture was stirred at
40 °C for 25–30 h. When TLC showed complete consumption of
the starting material, the mixture was concentrated and the resi-
due, dissolved in 1 mL of MeOH/water 1:1 (or in the case of com-
pound 8 in MeOH/acetone 1:1), was passed through a column
filled with Dowex MR-3C mixed bed ion-exchange resin. The elu-
ate was concentrated, dissolved in water, or MeOH/water 1:1,
and purified in an octadecyl C18 minicolumn. The pure fractions
according with TLC (in n-BuOH/EtOH/H2O, 2.5:1:1) or MeOH were
collected and concentrated to afford the free thiodisaccharide.
4.3.4. Methyl 6-S-(a-L-arabinofuranosyl)-6-thio-a-D-
glucopyranoside (18)
Removal of the ester protecting groups from 17 led to the free
thiodisaccharide 18 (68%); Rf = 0.57 (n-BuOH/EtOH/H2O, 2.5:1:1);
½
a 2D0
ꢃ
½
a 2D0
ꢃ
ꢄ13.2 (c 1, H2O); 1H NMR (500 MHz, CDCl3): d 5.17 (d,
1H, J1 ,2 = 4.8 Hz, H-10), 4.70 (d, 1H, J1,2 = 3.7 Hz, H-1), 3.96 (t, 1H,
0
0
J1 ,2 = J2 ,3 = 4.8 Hz, H-20), 3.95 (m, 1H, H-40), 3.89 (dd, 1H,
0
0
0
0
J2 ,3 = 4.8, J3 ,4
=
= 7.2 Hz, H-30), 3.73 (dd, 1H, J4 ,5 a = 2.9,
0
0
0
0
0
0
J5 a,5 b = 12.4 Hz, H-50a), 3.71 (m, 1H, H-5), 3.63 (dd, 1H,
0
0
J4 ,5 b = 5.3, J5 a,5 b = 12.5 Hz, H-50b), 3.53 (t, 1H, J2,3 = J3,4 = 9.5 Hz,
H-3), 3.48 (dd, 1H, J1,2 = 3.7, J2,3 = 9.5 Hz, H-2), 3.36 (s, 3H, CH3O),
3.23 (t, 1H, J3,4 = J4,5 = 9.5 Hz, H-4), 3.08 (dd, 1H, J5,6a = 2.0,
J6a,6b = 14.2 Hz, H-6a), 2.78 (dd, 1H, J5,6b = 8.8, J6a,6b = 14.2 Hz, H-
0
0
0
0
4.3.1. Methyl 6-S-(b-D-galactofuranosyl)-6-thio-a-D-
glucopyranoside (5)
The O-deacetylation procedure applied to 3 led to the free thi-
odisaccharide 5 (67%), Rf = 0.44 (n-BuOH/EtOH/H2O, 2.5:1:1); ½a D20
ꢃ
6b); C NMR (125.7 MHz, CDCl3): d 99.2 (C-1), 89.4 (C-10), 82.2,
13
ꢄ31.8 (c 0.5, H2O); 1H NMR (500 MHz, CDCl3): d 5.15 (d, 1H,
81.4 (C-20,40), 75.8 (C-30), 72.9 (C-3), 72.7 (C-4), 71.8 (C-5), 71.2
(C-2), 60.6 (C-50), 55.1 (CH3O), 32.9 (C-6). Anal. Calcd for
C12H22O9S.0.5H2O: C, 41.00; H, 6.60. Found: C, 41.36; H, 6.86.
J1 ,2 = 5.3 Hz, H-10), 4.70 (d, 1H, J1,2 = 3.7 Hz, H-1), 4.03 (dd, 1H,
0
0
J2 ,3 = 5.4, J3 ,4 = 7.5 Hz, H-30), 3.94 (t, 1H, J1 ,2 = J2 ,3 = 5.3 Hz, H-
0
0
0
0
0
0
0
0
20), 3.88 (dd, 1H, J4 ,5 = 3.5, J3 ,4 = 7.5 Hz, H-40), 3.75 (ddd, 1H,
0
0
0
0
J4 ,5 = 3.5, J5 ,6 a = 4.7, J5 ,6 b = 7.7 Hz, H-50), 3.70 (ddd, 1H, J5,6a = 2.2,
4.3.5. Methyl 6-S-(a-L-arabinofuranosyl)-6-thio-a-D-
0
0
0
0
0
0
0
0
J5,6b = 8.8, J4,5 = 9.0 Hz, H-5), 3.60 (dd, 1H, J5 ,6 a = 4.7,
galactopyranoside (20)
J6 a,6 b = 11.6 Hz, H-60a), 3.56 (dd, 1H, J5 ,6 b = 7.7, J6 a,6 b = 11.6 Hz,
H-60b), 3.54 (t, 1H, J2,3 = J3,4 = 9.8 Hz, H-3), 3.49 (dd, 1H, J1,2 = 3.7,
J2,3 = 9.8 Hz, H-2), 3.36 (s, 3H, CH3O), 3.23 (dd, 1H, J4,5 = 9.0,
J3,4 = 9.8 Hz, H-4), 3.07 (dd, 1H, J5,6a = 2.2, J6a,6b = 14.3 Hz, H-6a),
2.78 (dd, 1H, J5,6b = 8.8, J6a,6b = 14.3 Hz, H-6b); 13C NMR
(125.7 MHz, CDCl3): d 99.9 (C-1), 89.4 (C-10), 81.3 (C-40), 81.1 (C-
20), 76.0 (C-30), 72.9 (C-3), 72.6 (C-4), 71.9 (C-5), 71.2 (C-2), 70.4
(C-50), 62.8 (C-60), 55.2 (CH3O), 32.4 (C-6). Anal. Calcd for
C13H24O10S.0.5 H2O: C, 40.92; H, 6.61. Found: C, 40.82; H, 6.85.
O-Deacylation of 19 gave thiodisaccharide 20 (77%); Rf = 0.79
0
0
0
0
0
00
(MeOH); ½a 2D0
ꢃ
ꢄ44.4 (c 0.9, MeOH); 1H NMR (500 MHz, CDCl3): d
5.16 (d, 1H, J1 ,2 = 4.8 Hz, H-10), 4.72 (d, 1H, H-1), 3.97 (ddd, 1H,
0
0
J4 .5 a = 3.0, J4 ,5 b = 5.3, J3 ,4 = 7.3 Hz, H-40), 3.96 (t, 1H,
0
0
0
0
0
0
J1 ,2 = J2 ,3 = 4.8 Hz, H-20), 3.93 (br dd, 1H, J5,6b = 4.5, J5,6a = 9.0 Hz,
0
0
0
0
0
0
0
0
H-5), 3.91 (br s, 1H, H-4), 3.90 (dd, 1H, J2 ,3 = 4.8, J3 ,4 = 7.3 Hz, H-
30), 3.73 (dd, 1H, J4 ,5 a = 3.0, J5 a,5 b = 12.6 Hz, H-50a), 3.72 (m, 2H,
0
0
0
0
H-2,3), 3.64 (dd, 1H, J4 ,5 b = 5.3, J5 a,5 b = 12.6 Hz, H-50b), 3.35 (s,
3H, CH3O), 2.92 (dd, 1H, J5,6a = 9.0, J6a,6b = 14.0 Hz, H-6a), 2.76
(dd, 1H, J5,6b = 4.5, J6a,6b = 14.0 Hz, H-6b); 13C NMR (125.7 MHz,
CDCl3): d 99.4 (C-1), 89.2 (C-10), 82.2, 81.3 (C-20,40), 75.8 (C-30),
70.7, 70.2 (C-4,5), 69.4, 68.0 (C-2,3), 60.6 (C-50), 55.2 (CH3O), 31.4
(C-6). Anal. Calcd for C12H22O9S: C, 42.08; H, 6.48. Found: C,
41.58; H, 6.67.
0
0
0
0
4.3.2. 1,2:3,4-di-O-isopropylidene-6-S-(-D-galactofuranosyl)-6-
thio-a-D-galactopyranose (8)
Deprotection of 7 afforded 8 (91%); Rf = 0.64 (MeOH); ½a D20
ꢃ
ꢄ150.8 (c 1, H2O); 1H NMR (500 MHz, CDCl3): d 5.46 (d, 1H, J1,2
=
5.0 Hz, H-1), 5.12 (d, 1H, J1 ,2 = 4.3 Hz, H-10), 4.63 (dd, 1H,
J2,3 = 2.4, J3,4 = 7.9 Hz, H-3), 4.38 (dd, 1H, J4,5 = 1.8, J3,4 = 7.9 Hz, H-
0
0
4.3.6. 2-Propyl 3-deoxy-4-S-(b-
xylo-hexopyranoside (28)
D-galactofuranosyl)-4-thio-a-D-
4), 4.34 (dd, 1H, J2,3 = 2.4, J1,2
=
5.0 Hz, H-2), 4.05 (dd, 1H,
J2 ,3 = 4.8, J3 ,4 = 7.4 Hz, H-30), 3.96 (ddd, 1H, J4,5 = 1.6, J5,6b = 6.7,
O-Deacylation of 26 afforded syrupy 28 (92%); Rf = 0.58 (n-
0
0
0
0
J5,6a = 7.0 Hz, H-5), 3.94 (dd, 1H, J4 ,5 = 3.1, J3 ,4 = 7.4 Hz, H-40),
BuOH/EtOH/H2O, 2.5:1:1); ½a D20
ꢃ
ꢄ83.3 (c 0.8, MeOH); 1H NMR
0
0
0
0
3.90 (t, 1H, J1 ,2 = J2 ,3 ꢀ5.0 Hz, H-20), 3.73 (ddd, 1H, J4 ,5 = 3.1,
(500 MHz, CDCl3): d 5.12 (d, 1H, J1 ,2 = 4.7 Hz, H-10), 4.90 (d, 1H,
0
0
0
0
0
0
0
0
J5 ,6 a = J5 ,6 b = 6.8 Hz, H-50), 3.60 (m, 2H, H-60a,60b), 2.87 (dd, 1H,
J5,6a = 7.0, J6a,6b = 13.7 Hz, H-6a), 2.77 (dd, 1H, J5,6b = 6.8,
J1,2 = 3.7 Hz, H-1), 4.16 (m, 1H, H-5), 4.03 (dd, 1H, J2 ,3 = 4.7,
0
0
0
0
0
0
J3 ,4 = 7.4 Hz, H-30), 4.02 (dt, 1H, J1,2 = 3.7, J2,3eq = 4.6,
0
0

E. Repetto et al. / Bioorg. Med. Chem. 17 (2009) 2703–2711
2711
J2,3ax = 12.0 Hz, H-2), 3.97 (t, 1H, J1 ,2 = J2 ,3 = 4.7 Hz, H-20), 3.94 (m,
5. (a) Szilágyi, L.; Varela, O. Curr. Org. Chem. 2006, 10, 1745; (b) Pachamuthu, K.;
Schmidt, R. R. Chem. Rev. 2006, 106, 160.
0
0
0
0
1H, J = 6.2 Hz, Me2CHO), 3.90 (dd, 1H, J4 ,5 = 3.6, J3 ,4 = 7.4 Hz, H-40),
0
0
0
0
6. (a) Manzano, V. E.; Uhrig, M. L.; Varela, O. J. Org. Chem. 2008, 73, 7224; (b)
Uhrig, M. L.; Szilágyi, L.; Kovér, K. E.; Varela, O. Carbohydr. Res. 2007, 342, 1841;
(c) Uhrig, M. L.; Manzano, V. E.; Varela, O. Eur. J. Chem. 2006, 162.
7. Repetto, E.; Marino, C.; Uhrig, M. L.; Varela, O. Eur. J. Org. Chem. 2008, 540.
8. Minic, Z.; Do, C. T.; Rihouey, C.; Morin, H.; Lerouge, P.; Jounin, L. J. Exp. Bot.
2006, 57, 2339.
3.78 (ddd, 1H, J4 ,5 = 3.6, J5 ,6 a = 4.6, J5 ,6 b = 7.5 Hz, H-50), 3.62 (m,
0
0
0
0
0
0
2H, H-6a,6b), 3.61 (dd, 1H, J5 ,6 a = 4.6, J6 a,6 b = 11.6 Hz, H-60a),
0
0
0
0
3.56 (dd, 1H, J5 ,6 b = 7.5, J6 a,6 b = 11.6 Hz, H-60b), 3.30 (m, 1H, H-
4), 2.12 (ddd, 1H, J3ax,4 = 3.7, J2,3ax = 12.0, J3ax,3eq = 13.2 Hz, H-3ax),
2.03 (dddd, 1H, J3eq,4 = 3.5, J2,3eq = 4.6, J3ax,3eq = 13.2 Hz, H-3eq),
1.18, 1.11 (2d, 6H, J = 6.2 Hz, (CH3)2CHO); 13C NMR (125.7 MHz,
CDCl3): d 96.9 (C-1), 88.8 (C-10), 82.4, 82.2 (C-20,40), 77.1 (C-30),
71.3, 71.2, 71.1 (C-5,50,Me2CHO), 64.9 (C-2), 63.8, 63.5 (C-6,60),
44.3 (C-4), 33.4 (C-3), 23.2, 21.3 [(CH3)2CHO]. Anal. Calcd for
C15H28O9S.H2O: C, 44.76; H, 7.51; S, 7.97. Found: C, 45.25; H,
7.92; S, 7.78.
0
0
0
0
9.
(a) Lederkremer, R. M.; Colli, W. Glycobiology 1995, 5, 547; (b) Houseknecht, J.
B.; Lowary, T. L. Curr. Opin. Chem. Biol. 2003, 7, 677; (c) Pedersen, L. L.; Turco, S.
J. Cell. Mol. Life Sci. 2003, 60, 259; (d) Peltier, P.; Euzen, R.; Daniellou, R.; Nugier-
Chauvin, C.; Ferrières, V. Carbohydr. Res. 2008, 343, 1897.
10. Crick, D. C.; Mahaprata, S.; Brennan, P. J. Glycobiology 2001, 11, 107.
11. Kremer, L.; Dover, L. G.; Morehouse, C.; Hitchin, P.; Everett, M.; Morris, H. R.;
Dell, A.; Brennan, J.; MacNeil, M. R.; Flaherty, C.; Duncan, K.; Besra, G. S. J. Biol.
Chem. 2001, 276, 26430.
12. (a) Nassau, P. M.; Martin, S. L.; Brown, R. E.; Weston, A.; Monsey, D.; McNeil, M.
R.; Duncan, K. J. Bacteriol. 1996, 178, 1047; (b) Sanders, D. A. R.; Staines, A. G.;
McMahon, S. A.; McNeil, M. R.; Whitfield, C.; Naismith, J. H. Nat. Struct. Biol.
2001, 8, 858.
4.4. Enzymatic assays
13. Rietschel-Berst, M.; Jentoft, N. H.; Rick, P. D.; Pletcher, C.; Fang, F.; Gander, J. E.
J. Biol. Chem. 1977, 252, 3219.
14. Daley, L. S.; Ströbel, G. A. Plant Sci. Lett. 1983, 30, 145.
15. Cousin, M. A.; Notermans, S.; Hoogerhout, P.; Van Boom, J. H. J. Appl. Bacteriol.
1989, 66, 311.
16. Wallis, G.-L.; Hemming, F. W.; Pederby, J. F. Biochim. Biophys. Acta 2001, 1525, 19.
17. Golgher, D. B.; Colli, W.; Souto-Padron, T.; Zingales, B. Mol. Biochem. Parasitol.
1993, 60, 249.
18. (a) Marino, C.; Varela, O.; Lederkremer, R. M. Carbohydr. Res. 1989, 190, 65; (b)
Marino, C.; Cancio, M. J.; Varela, O.; Lederekremer, R. M. Carbohydr. Res. 1995,
276, 209; (c) Mariño, K.; Marino, C.; Lederkremer, R. M. Anal. Biochem. 2002,
301, 325; (d) Varela, O.; Marino, C.; Lederkremer, R. M. Carbohydr. Res. 1986,
155, 247.
The enzymatic activity was assayed using the filtered medium
of a stationary culture of P. fellutanum as source of b-
nosidase and 4-nitrophenyl b-
-galactofuranoside as substrate19a
The standard assay was conducted with 50 L of 66 mM NaOAc
buffer (pH 4.9), 15 L of a 5 mM solution of 4-nitrophenyl b-
galactofuranoside and 25 L (10 g protein) of the enzyme med-
ium, in a final volume of 250 L. The thiodisaccharides were incor-
D-galactofura-
D
l
l
D-
l
l
l
porated in the amounts needed to obtain a final concentration from
0.2 to 1.0 mM. The enzymatic reaction was stopped after 1.5 h of
incubation at 37 °C by addition of 0.5 mL of 0.1 M Na2CO3 buffer
(pH 9.0). The 4-nitrophenol released was measured spectrophoto-
metrically at 410 nm.
19. (a) Marino, C.; Mariño, K.; Miletti, L.; Manso Alves, M. J.; Colli, W.; Lederkremer,
R. M. Glycobiology 1998, 8, 901; (b) Mariño, K.; Marino, C. Arkivoc 2005, XII, 341.
20. Miletti, L. C.; Marino, C.; Mariño, K.; Lederkremer, R. M.; Colli, W.; Alves, M. J.
M. Carbohydr. Res. 1999, 320, 176.
21. Miletti, L. C.; Mariño, K.; Marino, C.; Colli, W.; Alves, M. J. M.; Lederkremer, R.
M. Mol. Biochem. Parasitol. 2003, 127, 85.
22. Davis, C. B.; Hartnell, R. D.; Madge, P. D.; Owen, D. J.; Thomson, R. J.; Chong, A.
K. J.; Coppel, R. L.; von Itzstein, M. Carbohydr. Res. 2007, 342, 1773.
23. Mariño, K.; Lima, C.; Maldonado, S.; Marino, C.; Lederkremer, R. M. Carbohydr.
Res. 2002, 337, 891.
Acknowledgments
Support of our work by the University of Buenos Aires (Project
X059), the National Research Council of Argentina (CONICET, Pro-
ject PIP 5011) and the National Agency for Promotion of Science
and Technology (ANPCyT, project 13922) is gratefully acknowl-
edged. O.V., C.M. and M.L.U. are Research Members of CONICET.
24. D0Accorso, N. B.; Thiel, I. M. E.; Schüller, M. Carbohydr. Res. 1983, 124, 177.
25. Sherry, B. D.; Loy, R. N.; Toste, F. D. J. Am. Chem. Soc. 2004, 126, 4510.
26. Weng, S.-S.; Lin, Y.-D.; Chen, C.-T. Org. Lett. 2006, 8, 5633.
27. du Mortier, C.; Varela, O.; Lederkremer, R. M. Carbohydr. Res. 1989, 189, 79.
28. Stevenson, G.; Neal, B.; Liu, D.; Hobbs, M.; Packer, N. H.; Batley, M.; Redmond, J.
W.; Lindquist, L.; Reeves, P. J. Bacteriol. 1994, 176, 4144.
Supplementary data
29. Lipinski, T.; Jones, C.; Lemercinier, X.; Korzeniowska-Kowal, A.; Strus, M.;
Rybka, J.; Gamian, A.; Heczko, P. B. Carbohydr. Res. 2003, 338, 605.
30. Faber, E. J.; van den Haak, M. J.; Kamerling, J. P.; Vliegenthart, J. F. G. Carbohydr.
Res. 2001, 331, 173.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.02.045.
31. Plackett, P.; Buttery, S. H. Biochem. J. 1964, 90, 201.
32. Vilkas, E.; Amar, C.; Markovits, J.; Vliegenthart, J. F. G.; Kamerling, J. P. Biochim.
Biophys. Acta 1973, 297, 423.
33. Witczak, Z. J.; Chhabra, R.; Chen, H.; Xie, X.-Q. Carbohydr. Res. 1997, 301, 167.
34. (a) Robyt, J. F. In Essentials of Carbohydrate Chemistry; Cantor, C. R., Ed.; Springer
Verlag: New York, 1998; pp 179–180; (b) Green, J. W. Adv. Carbohydr. Chem.
1966, 21, 95.
References and notes
1. (a) Varky, A. Glycobiology 1993, 3, 97; (b) Dwek, R. A. Chem. Rev. 1996, 96, 683;
(c) Dube, D. H.; Bertozzi, C. R. Nat. Rev. Drug Disc. 2005, 4, 477.
2. (a) Sears, P.; Wong, C.-H. Angew. Chem., Int. Ed. 1999, 38, 2300; (b) Marcaurelle,
L. A.; Bertozzi, C. R. Chem. Eur. J. 1999, 5, 1384.
3. (a) Robina, I.; Vogel, P. Synthesis 2005, 5, 675; (b) Renaudet, O.; Dumy, P.
Tetrahedron 2008, 58, 2127; (c) Yuan, X.; Linhardt, R. J. Curr. Top. Med. Chem.
2005, 5, 1393; (d) Arjona, O.; Gómez, A. M.; López, J. C.; Plumet, J. Chem. Rev.
2007, 107, 1919.
35. Lewis, B. A.; Smith, F.; Stephen, A. M. Methods Carbohydr. Chem. 1963, 2, 172.
36. Boons, G.-J.; Hale, K. J. In Organic Synthesis with Carbohydrates; Sheffield
Academic Press: Sheffield, 2000; pp 60–61.
37. Martins Alho, M. A.; D0Accorso, N. B.; Thiel, I. M. E. J. Heterocycl. Chem. 1996, 33,
1339.
38. Constantino, V.; Fattorusso, E.; Imperatore, C.; Mangoni, A. J. Org. Chem. 2004,
69, 1174.
4. (a) Driguez, H. ChemBioChem 2001, 2, 311; (b) Driguez, H. Top. Curr. Chem. 1997,
187, 85; (c) Defaye, J.; Gelas, J.. In Studies in Natural Product Chemistry; Attaur-
Rahman, Ed.; Elsevier: Amsterdam, 1991; Vol. 8, pp 315–357.