Synthesis of the (1→6)-linked thiodisaccharide of galactofuranose: Inhibitory activity against a β-galactofuranosidase

Article abstract of DOI:10.1016/j.bmc.2013.02.057

A new (1→6)-linked thiodisaccharide formed by two galactofuranosyl units has been synthesized. Methyl (methyl α,β-d-galactofuranosid) uronate was employed as the starting compound, which was per-O-silylated with TBSCl and reduced with LiAlH₄ to afford methyl 2,3,5-tri-O-tert- butyldimethylsilyl-β-d-galactofuranoside (2β) as a key precursor for the preparation of methyl per-O-tert-butyldimethylsilyl-6-thio-β-d- galactofuranoside (12). The free thiol group of 12 was glycosylated and the product O-deprotected to afford the target β-d-Galf-S-(1→6)-β-d- Galf-OMe (14). The conformations of this thiodisaccharide were preliminarily studied using combined theoretical calculations and NMR data. Furthermore, the glycomimetic 14 showed to be a competitive inhibitor of the β- galactofuranosidase from Penicillum fellutanum (K_i = 3.62 mM).

Full text of DOI:10.1016/j.bmc.2013.02.057

Bioorganic & Medicinal Chemistry 21 (2013) 3327–3333
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Bioorganic & Medicinal Chemistry
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Synthesis of the (1?6)-linked thiodisaccharide of galactofuranose:
Inhibitory activity against a b-galactofuranosidase
⇑
Evangelina Repetto, Carla Marino, Oscar Varela
CIHIDECAR-CONICET-UBA, Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires 1428, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
A new (1?6)-linked thiodisaccharide formed by two galactofuranosyl units has been synthesized.
Received 6 December 2012
Revised 8 February 2013
Accepted 18 February 2013
Available online 31 March 2013
Methyl (methyl
per-O-silylated with TBSCl and reduced with LiAlH4 to afford methyl 2,3,5-tri-O-tert-butyldimethylsi-
lyl-b- -galactofuranoside (2b) as a key precursor for the preparation of methyl per-O-tert-butyldimethyl-
silyl-6-thio-b- -galactofuranoside (12). The free thiol group of 12 was glycosylated and the product
O-deprotected to afford the target b- -Galf-S-(1?6)-b- -Galf-OMe (14). The conformations of this thiodi-
saccharide were preliminarily studied using combined theoretical calculations and NMR data. Further-
more, the glycomimetic 14 showed to be a competitive inhibitor of the b-galactofuranosidase from
Penicillum fellutanum (Ki = 3.62 mM).
a,b-D-galactofuranosid)uronate was employed as the starting compound, which was
D
D
D
D
Keywords:
Thiogalactofuranosyl disaccharide
6-Thiogalactofuranose
Conformational analysis
Ó 2013 Elsevier Ltd. All rights reserved.
exo b-D-Galactofuranosidase
Enzyme inhibition
1. Introduction
saccharides that contain
a
1-thiopentofuranose10 or 1-
thiohexofuranose11 moiety as non reducing end. Furthermore,
the synthesis of oligosaccharide analogues having the ring oxygen
atom of Galf replaced by sulfur has been reported.12
Carbohydrate mimetics are usually designed and prepared
introducing alterations in the core structure or modification of
the O-glycosidic linkage with respect to their natural analogues.
To overcome the problem of the lability of the glycosidic bond, sta-
ble mimetics have been synthesized.1 The development of modi-
fied linkages between monosaccharide units has risen
considerable interest due to the stability to enzyme degradation
and the resulting potential for glycosidase inhibition.2 The most
common modification is the replacement of the anomeric oxygen
atom by a sulfur, a nitrogen or a carbon atom to afford, respec-
tively, S-, N-, or C-glycosides.
The interest in thiodisaccharides of furanose sugars, and partic-
ularly of Galf, arises from the fact that this sugar is present in
numerous structures considered to be essential for virulence in
many pathogenic organisms.13,14 Thus, the motif b-
D-Galf-(1?6)-
Galf occurs in polyfuranosides of pathogenic Aspergillus, Cryphonec-
tria parasitica, Renibacterium salmoninarum and Mycobacterium,
and in many other microorganisms.13,14 For example, the arabino-
galactan of the cell wall complex of mycobacteria and other Actino-
mycetes15 comprises a linear chain of approximately 30 alternating
During the last years, we have been involved in the synthesis
and biological evaluation of thiooligosaccharides and we have re-
ported valuable procedures for the construction of the thioglycos-
idic linkage, that complement the approaches already described in
the literature.3,4 For example, the Michael addition5,6 and the ring-
opening reaction of epoxides7,8 or thiirane derivatives8,9 of sugars
have been employed as key steps in the synthesis of thiooligosac-
charides. However, the methodologies reported so far are mostly
referred to the construction of thioglicosidic linkages between
pyranose rings, while just a few examples of thiooligosaccharides
constituted by furanose rings have been reported. We have de-
scribed some straightforward strategies for the synthesis of thiodi-
b-(1?5) and b-(1?6)-linked D-Galf residues. The inhibition of the
enzymes involved in the metabolism of these polyfuranosides is
expected to prevent the proliferation of mycobacteria, including
the causative agent of tuberculosis, Mycobacterium tuberculosis.16
In addition, as such furanosyl residues are not found in mam-
mals, these enzymes can be considered interesting targets for the
design of new antituberculosis drugs. In fact, aryl and heteroaryl
1-thio-b-D-galactofuranosides have been evaluated as inhibitors
of a b-galactofuranosidase17 and 1-thioalkyl galactofuranosides
and their sulfones have been tested as inhibitors of mycobacteria
growth.18 However, thiodisaccharides formed by two Galf units
have not been synthesized.
In view of the previous considerations we wish to report here
the synthesis of the first dithiodisaccharide constituted by two
furanose units. This compound is the glycomimetic of the already
⇑
Corresponding author. Tel./fax: +54 11 45763352.
E-mail address: varela@qo.fcen.uba.ar (O. Varela).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.02.057

3328
E. Repetto et al. / Bioorg. Med. Chem. 21 (2013) 3327–3333
mentioned, natural and rather common b-Galf-(1?6)-
The free thiodisaccharide has been evaluated as inhibitor of the
b-galactosidase from Penicillium fellutanum.
D
-Galf unit.
The per-O-TBS derivatives of 1-thio-
pared as a
,b mixture of anomers,20 therefore this is not a conve-
nient precursor for our purposes as, in the case that the reaction
occurs, a mixture of and b S-linked disaccharides is expected.
D-Galf has been recently pre-
a
a
In contrast, we have described that the isothiouronium salt of Galf
6 releases, under alkaline conditions, the elusive 1-thio-Galf, which
was trapped in b-configuration by a sugar enone as Michael accep-
tor.10 Furthermore, it has been reported that 1-thioglycopyranoses
generated in situ from the corresponding isothiouronium salts re-
acted with halide or sulfonate derivatives to give thioglycosides.21
For these reasons, the glycosyl isothiourea 6 was prepared, and the
reaction with the sulfonates 4 and 5 was attempted under varied
conditions, using Et3N as catalyst and acetonitrile or DMF as sol-
vents. In all instances, the reaction was unsuccessful. In view of
these negative results, we decided to conduct an analogous reac-
tion using as nucleophile the thiol 8, the pyranose counterpart of
2. Results and discussion
2.1. Synthesis of b-Galf-S-(1?6)-D-Galf-OMe (14)
The preparation of the target thiodisaccharide constituted by
two galactofuranosyl units via a displacement reaction (SN2) re-
quired a fully protected Galf derivative having a good leaving group
at C-6. For the preparation of such a precursor we started from
methyl (methyl per-O-TBS-
readily prepared from
-galacturonic acid9 (Scheme 1). The reduc-
tion of 1
a,b-D-galactofuranosid)uronate (1a,b),
D
a
,b with a slight excess of LiAlH4 at ꢀ18 °C gave mainly 2
a,
2b and 3. The product distribution showed to be highly dependent
1-thio-D-Galf. Under the standard conditions that failed for the
on the reaction time. Thus, when the reduction was conducted for
furanose compound, the thiol 8 (via the sodium salt 9) reacted with
4 to give the expected thiodisaccharide 10 in 86% yield. The results
obtained seem to indicate that the concentration or nucleophilicity
of the glycosyl thiol generated in situ from the isothiourea 6 were
not sufficient for the substitution reaction. Therefore, an alterna-
tive approach was attempted, based in the glycosylation of a SH-
6 group in a Galf derivative, such as 12.
The sulfonyl derivatives 4 and 5 were appropriate precursors for
the synthesis of 12 (Scheme 2). Thus, nucleophilic displacement of
the sulfonate group in 4 or 5 with potassium thioacetate led to
good yields of the 6-S-acetyl derivative 11. Treatment of 11 with
NaOMe/MeOH produced the removal of the S-acetyl group to give
the 6-thio-Galf 12. The incorporation of a sulfur atom in C-6 of Galf
was evidenced by the 1H NMR spectrum, which showed an upfield
shifting for the signals of C-6 methylene group of 12 with respect
to the same signals in 2b. The 13C NMR spectrum of 12 showed also
short times, the expected 2,3,5-tri-O-silyl derivatives 2
a and 2b
were obtained, being the latter the major isomer. This compound
was conveniently protected for the activation of the OH-6 as sulfo-
nate. Thus, treatment of 2b with tosyl chloride or triflic anhydride
afforded the 6-O-sulfonyl derivatives 4 or 5, respectively. The 1H
NMR spectrum of these products showed the downfield shifting
of the C-6 methylene group, vicinal to the sulfonate, while the
chemical shift of the H-5 signal was much less affected. This result
indicates that sulfonylation of OH-6 took place and excludes the
possibility of 5?6 O-TBS migration, which we have observed at
higher temperatures.9
Sulfonyl derivatives of carbohydrates have been widely used as
glycosyl acceptors of 1-thioaldoses to form the thioglycosidic link-
age in thiooligosaccharides.3,4,19 This methodology takes advan-
tage of the high nucleophilicity of the thiol group. Unfortunately
the derivatives of 1-thio-Galf are highly unstable compounds.9–11
Scheme 1. Synthesis of 6-O-sulfonyl-b-
D-Galf-OMe 4 and 5 as precursors of thiodisaccharides.

E. Repetto et al. / Bioorg. Med. Chem. 21 (2013) 3327–3333
3329
Scheme 2. Synthesis of b-D-Galf-S-(1?6)-D-Galf-OMe (14).
the presence of a carbon bonded to sulfur (27.9 ppm) and the sig-
nals of C-1 (109.2 ppm), C-2 and C-4 (>80 ppm) characteristic of al-
the endocyclic and exocyclic oxygen atoms three rotamers: gg, gt
and tg are defined (Fig. 1). In the gg conformer H4ꢀH5 are trans
oriented, and this geometry does not justify the observed J4,5 and
kyl
b-D
-galactofuranosides.
Therefore,
the
b-furanosyl
0
0
configuration for 12 was maintained, and ring expansion or rear-
rangements, rather common in thiofuranoses,22 had not occurred.
The free thiol group of 12 was glycosylated using the trichloro-
acetimidate derivative 1323 as glycosyl donor to afford the target
thiodisaccharide 7. The structure of 7 was confirmed on the basis
of the NMR spectra which showed diagnostic data for the O- (dH-1
J4 ,5 values. In the tg conformer C6 is directed towards the furanose
ring and interacts with the ring substituents. The gg and tg rota-
mers present a repulsive gauche C5ꢀC6 interaction which is absent
in the gt form, which should be the preferred conformer. Further-
more, the NOESY spectrum of 14 showed an intramolecular NOE
contact between H3 and H5 (and H30, H50), as expected for the pro-
posed planar zigzag gt conformation for the C4ꢀC6 segment of 14.
In addition to some intraresidual cross peaks, such as H1ꢀH3,
H10ꢀH30, H2ꢀH4 and H20ꢀH40, other interresidue interactions
were observed in the NOESY spectrum of 14. These type of NOE
contacts are usually interpreted as the result of conformations
around of the glycosidic linkages that bring close in space protons
belonging to the two moieties of the disaccharide.25 Therefore we
tried to find some conformations that could explain the observed
interresidual NOE contacts. To explore preliminarily the conforma-
tional behavior of the thiodisaccharide 14 a combined theoretical
calculations and NMR approach was employed.
0
4.72, J1,2 1.9 Hz, dC-1 109.1) and S-galactofuranosides (dH-1 5.62,
J1 ,2 1.2 Hz, dC-1 88.1).9,10,20
0
0
0
Removal of the protecting groups of 7 was accomplished by
reaction with tetrabutylammonium fluoride (TBAF) for the O-desi-
lylation, followed by O-debenzoylation with NaOMe/MeOH. The
13C NMR spectrum of this product showed signals at 91.6 and
35.7 ppm, corresponding to C-10 and C-6, respectively, being diag-
nostics for the S-(1?6) inter Galf linkage.
2.2. Conformational analysis
The 1H NMR spectrum of the free thiodisaccharide 14, recorded
in methanol-d4, appeared quite well resolved and the conforma-
tion of the furanose rings could be evaluated according to the val-
ues of the coupling constants (J). The coupled protons in the non
reducing end ring of 14 showed larger J values than those corre-
sponding to the same couplings in the other ring. The observed
magnitudes for J suggested a 4E ꢀ 4T3 ꢀ E3 conformation for
the 1-thio Galf ring, whereas the conformational equilibrium
From the conformational point of view, the substitution of the
interglycosidic oxygen by sulfur in thiodisaccharides leds to popu-
late different states compared to the natural counterparts.12,25 This
fact is the result of the differences in the CꢀS bond lengh (1.78 Å)
and the CꢀSꢀC bond angle (99°) with respect to the values for CꢀO
(1.41 Å) and CꢀOꢀC (116°) and the magnitude of stereoelectronic
effects, in particular the anomeric effects. Thus, the conformation
of many thiodisaccharides formed by pyranose units was studied
using a combination of NMR spectroscopy and molecular mechan-
ics and dynamics calculations.26 The theoretical results were
experimentally confirmed by the interresidue NOEs that unequiv-
ocally characterize the minimum energy conformations deter-
mined by calculations.25 The same type of study applied to 14 is
far more complex as the two furanose rings are linked through four
bonds instead of the three bonds that link the pyranose rings in
(1?6)-disaccharides. In fact, the determination of the conforma-
tion for this type of disaccharides is more complex than that in
disaccharides involving glycosidic bonds to other positions of the
pyranose (C-2 to C-4) as, because of the C5ꢀC6 exocyclic bond, a
seems to be shifted towards the E0
ꢀ ꢀ
1T0 1E segment of the
pseudorotational itinerary for the furanose ring of the reducing
end. In this case, the anomeric substituent is quasiaxially oriented
satisfying the anomeric effect, which is stronger in O- than in S-
glycosides. A similar conformational behavior has been reported
for O- and S- galactofuranosides,17 and it was also observed by
the theoretical calculations described below. The possible rotamers
around the C4ꢀC5 linkage were also examined and the small val-
ues for the corresponding coupled protons (J4,5 = 1.7 and
0
0
J4 ,5 = 3.1 Hz) suggested that they adopt a ‘gauche’ relationship.
For simplification we have employed the notation used for the
dihedral angles formed by rotation of the hydroxymethyl group
in pyranosides.24 Thus, according to the relative orientation of
third angle (x) is needed to describe all the possible orientations
between the pyranose rings.27 For this preliminary study we per-
formed molecular mechanics (MM+) and the minimum energy
structures were refined using a semiempirical method (AM1). For-
tunately, and in agreement with above mentioned analysis based
on coupling constants, all the minimum energy structures found
for 14 showed exclusively the presence of the gt rotamer for the
C4ꢀC5 bond. The other three torsion angles that involve the link-
ages between the furanose rings were defined as follows:
U
= H10ꢀC10ꢀSꢀC6,
W
= C10ꢀSꢀC6ꢀC5 and
x
= SꢀC6ꢀC5ꢀC4. A
Figure 1. Notation for the dihedral angles around the C4ꢀC5 bond.

3330
E. Repetto et al. / Bioorg. Med. Chem. 21 (2013) 3327–3333
(A)
(B)
(C)
(D)
(E)
Figure 2. Depiction of the more stable conformers of 14. Experimental NOE contacts are shown.
schematic representation of these angles has been included in Sup-
plementary Figure S1. In addition, the sign of the rotation angles
b-D-galactofuranosidase from P. fellutanum. This is a model enzyme
frequently employed for galactofuranosidase activity, that we were
able to isolate in our laboratory from the culture broth of the fun-
gus.17a The natural substrate for this enzyme is the extracellular
glycopeptide called peptidophosphogalactomannan (pPGM),
has been illustrated for
x.
Among the calculated low energy conformations we selected
those that justify observed interresidue NOE contacts. Thus, for
the conformation A (Fig. 2) was detected a characteristic NOE be-
tween H-40 and pro(R) H-6 of the methylene at C-6. As an extension
of the nomenclature employed for common disaccharides,24 this
containing terminal (1?5)-linked b-
a
D
-Galf units, attached to an
-mannose core.28 For the evaluation of thiodisaccharide 14 as
in vitro inhibitor of the exo b-D-galactofuranosidase from P. felluta-
lower energy arrangement was labeled as anti
U
/syn
W
/anti
x
.
num was followed the protocol usually employed.11,17 4-Nitro-
The syn and anti rotamers, formed by rotation of the S-glycosidic
linkage, have been depicted in Fig. S2 of Supplementary data. A
rotation around the C5ꢀC6 bond leads to the following minimum
phenyl b-
D-galactofuranoside was used as substrate and
D-galactono-1,4-lactone (15) as the reference inhibitor. Compound
14 was subjected to the enzymatic reaction, in concentrations
ranging from 0.6 to 2.4 mM. Releasing of 4-nitrophenol was em-
ployed as a measurement of galactofuranosidase activity. The ef-
fect of the concentration of 14 on the activity of the enzyme is
shown in Figure 3.
energy conformer anti
U/syn W/syn x (B), that should also exhibit
the H40ꢀH6a,6b NOE cross-peak. The conformers C and D present a
syn disposition for the torsion angles
U
and x, whereas the agly-
conic dihedral angle
W
is found as anti
W
in C and syn W in D.
These two conformations, C and D, seem to be confirmed by the
The Lineweaver–Burk plot (Fig. 4) indicates that the thiodisac-
charide 14 is a competitive inhibitor of the enzyme (Ki = 3.62 mM),
weaker than D-galactono-1,4-lactone (Ki = 0.10 mM). The enzyme
respective observed NOE contacts H10ꢀH6a,6b and H10ꢀH5. The
following low energy rotamer was E, which has an anti
U
arrange-
ment, as that observed for A. However, opposite to this conformer,
E exhibits anti /syn geometries. The presence of E in the con-
activity showed to be highly dependent on the glycone and also
the aglycone structure, in agreement with previous work from this
W
x
formational equilibrium is justified by the NOE contact between
H-40 and pro(S) H-6.
Similar to O- and S-glycosides, the conformations of A–D
around
served for thiodisaccharides, compound 14 exhibits syn
conformers.25 The presence of a sulfur atom and a methylene
U
are stabilized by the exo-anomeric effect and also, as ob-
W
and anti
W
group in the chain that links the two furanose moieties confers,
as expected, a high degree of flexibility. This effect accounts for
the average values observed for J5,6a and J5,6b, which is a conse-
quence of the relative orientation that H-6a and H-6b adopt with
respect to H-5 by rotation of the C5ꢀC6 bond. It is worth to men-
tion that for flexible compounds one of the existing conformations
in solution, or even a distorted form of it, could be selected and
bound to the binding site of a protein without major energy
conflicts.25
2.3. Evaluation of the inhibitory activity
Figure 3. Effect of the concentration of thiodisaccharide 14 on the enzymatic
activity of the exo b-
D-galactofuranosidase from Penicillium fellutanum. 4-Nitrope-
nyl b- -galactofuranoside was used as substrate and
as reference inhibitor. Each point is the mean obtained from three replicate
experiments.
D
D
-galactono-1,4-lactone (15)
As neither endo nor exo galactofuranosidases are commercially
available, thiodisaccharide 14 was evaluated as inhibitor of the exo

E. Repetto et al. / Bioorg. Med. Chem. 21 (2013) 3327–3333
3331
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. Assignments of 1H and 13C were
assisted by 2D 1H COSY and HSQC experiments. MM+ and AM1 cal-
culations have been performed with Hyperchem Professional 8.0.3.
4.2. Synthesis
4.2.1. Methyl 2,3,5-tri-O-tert-butyldimethylsilyl-
galactofuranoside (2 ), methyl 2,3,5-tri-O-tert-
butyldimethylsilyl-b- -galactofuranoside (2b), and methyl 2,3-
di-O-tert-butyldimethylsilyl-b- -galactofuranoside (3)
a-D-
a
D
D
Fig. 4. Lineweaver–Burk reciprocal plot for the inhibition of the exo b-
uranosidase from Penicillium fellutanum by thiodisaccharide 14 at concentrations:
) 0.00, ( ) 0.60, ( ) 1.00 and ( ) 1.40 mM. Each point is the mean obtained from
D-galactof-
A solution of uronate 1 (564 mg, 1.00 mmol)9 in dry THF
(15 mL) was cooled to ꢀ18 °C and LiAlH4 (50 mg, 1.30 mmol) was
added. The reaction mixture was stirred for 30 min, and then
EtOAc (15 mL), MeOH (15 mL), and AcOH (to pH 7) were added
in sequence, and finally the mixture was concentrated. The solid
obtained was suspended in CH2Cl2 and centrifuged (4ꢀ5 times)
to eliminate Li and Al salts. Evaporation of the solvent afforded a
residue that showed by TLC (hexane/EtOAc, 10:1) spots corre-
(
three replicate experiments.
laboratory on mimetics of Galf, including thioglycosides,11,17 which
were tested as substrates or inhibitors of the enzyme. For example,
while 4-nitrophenyl b-D-Galf is an useful substrate for the mea-
surement of the enzyme activity (Km = 0.31 mM), the benzyl
(Km = 3.70 mM) and methyl (Km = 2.60 mM) analogues are less
effective.17b
sponding to 2
lated by column chromatography (toluene/EtOAc 60:1 ? EtOAc).
The following yields were obtained 2 (70 mg, 13%), 2b (230 mg,
a (Rf 0.47), 2b (Rf 0.43), and 3 (Rf 0.00) that were iso-
a
On the other hand, 4-aminophenyl b-D-1-thio-Galf
43%), and 3 (97 mg, 23%) and the compounds exhibited physical
and spectral data that were in accordance with those already
reported.9
(IC50 = 0.08 mM) is more effective as inhibitor than its precursor
4-nitrophenyl b-D-1-thio-Galf (IC50 = 0.60 mM), suggesting hydro-
gen bonding donation in the active site of the enzyme.17a Never-
theless, the best inhibitor found for this enzyme, although
4.2.2. Methyl 2,3,5-tri-O-tert-butyldimethylsilyl-6-O-tosyl-b-D-
galactofuranoside (4)
To a solution of 2b (110 mg, 0.20 mmol) in dry pyridine (2 mL)
was added tosyl chloride (100 mg, 0.52 mmol) and the mixture
was stirred at rt for 24 h. Addition of MeOH (5 mL) followed by
moderate, is D-galactono-1,4-lactone (Ki = 0.10 mM), which resem-
bles the transition state of the hydrolysis.17a
3. Conclusions
concentration led to
a syrup that was dissolved in CH2Cl2
Thiodisaccharide Galf-S-(1?6)-Galf-OMe was synthesized
starting from readily available galactofuanosyl precursors. The
conformational behavior of this thiodisaccharide was studied pre-
liminarily using combined theoretical calculations and NMR data.
For the low energy conformations detected, the thioglycosidic link-
age adopts the two rotamers that justify the exoanomeric effect
and the C4ꢀC6 segment is found predominantly in the planar zig-
zag conformation, while the SꢀC6 linkage is highly flexible. As far
as we know, this is the first example of a thiodisaccharide consti-
tuted by two furanose units. The new glycomimetic proved to be
a moderate competitive inhibitor (Ki = 3.62 mM) of the b-galactof-
uranosidase from P. fellutanum, an enzyme that has not been fully
characterized. The gen encoding the enzyme has not been identi-
fied, nor has the amino acid sequence been determined. Therefore,
the thiodisaccharide could be employed as a useful tool to study
the active site of the b-galactofuranosidase. As inhibitor of this en-
zyme, Galf-S-(1?6)-Galf-OMe showed a similar activity to that
(10 mL). The organic solution was washed with water (20 mL),
dried (MgSO4) and concentrated. Analysis by TLC (hexane/EtOAc,
10:1) revealed a major product of Rf 0.58, which was purified by
column chromatography (hexane/EtOAc, 25:1) to afford
4
(116 mg, 84%);
½
a 2D5
ꢁ
ꢀ14.4 (c 1.1, CHCl3); 1H NMR (CDCl3,
500 MHz) d 7.78, 7.33 (2d, 4H, J ꢂ 8.2 Hz, H-aromatic), 4.65 (d,
1H, J1,2 = 1.5 Hz, H-1), 4.12 (dd, 1H, J5,6 = 3.2, J6a,6b = 9.3 Hz, H-6a),
4.01 (dd, 1H, J2,3 = 2.7, J3,4 = 5.7 Hz, H-3), 3.98 (ddd, 1H, J4,5 = 3.7,
J5,6a = 3.2, J5,6b = 7.2 Hz, H-5), 3.95 (dd, 1H, J5,6b = 7.2, J6a,6b = 9.3 Hz,
H-6b), 3.94 (dd, 1H, J1,2 = 1.5, J2,3 = 2.7 Hz, H-2), 3.79 (dd, 1H,
J3,4 = 5.7, J4,5 = 3.7 Hz, H-4), 3.28 (s, 3H, CH3O), 2.44 (s, 3H, CH3Ph),
0.87 (ꢃ2), 0.85 (3s, 27H, (CH3)3CSiMe2), 0.08, 0.07 (ꢃ3), 0.05, 0.04
(6s, 18H, (CH3)2SiBut); 13C NMR (CDCl3, 125.7 MHz) d 144.7, 133.0,
129.8, 128.0 (C-aromatic), 109.3 (C-1), 84.4 (C-4), 84.1 (C-2), 79.4
(C-3), 71.3 (C-6), 70.5 (C-5), 54.9 (CH3O), 25.9, 25.7 (ꢃ2)
[(CH3)3CSiMe2], 21.6 (CH3Ph), 18.3, 17.9, 17.8 [(CH3)3CSiMe2],
ꢀ4.0, ꢀ4.1, ꢀ4.4, ꢀ4.6 (ꢃ2), ꢀ4.9 [(CH3)2SiBut]. HRMS (ESI) m/z
[M+Na]+ calcd for C32H62O8SSi3+Na 713.33709, found 713.33654.
determined for b-D-Galf-S-(1?6)-D-Galp-OMe. Therefore, the
inhibitory activity seems to rely on the presence of the non-reduc-
ing Galf unit, while the pyranose or furanose configuration for the
reducing Gal residue has a negligible effect.
4.2.3. Methyl 2,3,5-tri-O-tert-butyldimethylsilyl-6-O-triflyl-b-D-
galactofuranoside (5)
A solution of compound 2b (130 mg, 0.24 mmol) in dry CH2Cl2
4. Experimental
(10 mL) was cooled to 0 °C and 2,6-di-tert-butyl-4-methyl-pyridine
(195 mg, 0.95 mmol) and triflic anhydride (80 lL, 0.47 mmol) were
4.1. General methods
added under N2 atmosphere. The mixture was stirred for 1 h and
then was poured into ice/water and extracted with CH2Cl2. The or-
ganic extract was washed with water (10 mL), dried (MgSO4) and
concentrated. The residue obtained (Rf 0.78, hexane/EtOAc, 10:1),
Analytical thin layer chromatography (TLC) was performed on
Silica Gel 60 F254 (Merck) aluminum supported plates (layer thick-

3332
E. Repetto et al. / Bioorg. Med. Chem. 21 (2013) 3327–3333
was purified by column chromatography (hexane to hexane/EtOAc,
(CDCl3, 125.7 MHz) d 195.3 (SCOMe), 109.3 (C-1), 85.5 (C-
4), 84.2 (C-2), 79.8 (C-3), 71.7 (C-5), 55.0 (CH3O), 32.6 (C-
6), 30.4 (SCOCH3), 26.0, 25.7 (ꢃ2) [(CH3)3CSiMe2], 18.3,
17.9, 17.8 [(CH3)3CSiMe2], ꢀ3.9, ꢀ4.0, ꢀ4.4, ꢀ4.5, ꢀ4.6,
ꢀ4.9 [(CH3)2SiBut]. HRMS (ESI) m/z [M+Na]+ calcd for
60:1) to afford 5 (105 mg, 66%); ½a D25
ꢁ
ꢀ18.4 (c 1.2, CHCl3); 1H NMR
(CDCl3, 500 MHz) d 4.71 (br s, J1,2 <1.0 Hz, 1H, H-1), 4.62 (dd, 1H,
J5,6 = 3.3, J6a,6b = 10.1 Hz, H-6a), 4.37 (dd, 1H, J5,6b = 7.7,
J6a,6b = 10.1 Hz, H-6b), 4.14 (dddd, 1H, J4,5 ꢂ 4.6, J5,6a = 3.3,
J5,6b = 7.7 Hz, H-5), 3.99 (m, 2H, H-2, H-3), 3.92 (T, 1H,
J3,4 = J4,5 = 4.6 Hz, H-4), 3.34 (s, 3H, CH3O), 0.92, 0.89, 0.87 (3s,
27H, (CH3)3CSiMe2), 0.13, 0.10, 0.08 (3s, 18H, (CH3)2SiBut); 13C
NMR (CDCl3, 125.7 MHz,) 109.4 (C-1), 85.4 (C-4), 83.4 (C-2), 79.1
(C-3), 77.9 (C-6), 70.7 (C-5), 54.9 (CH3O), 25.8, 25.7 (ꢃ2)
[(CH3)3CSiMe2], 18.2, 17.9, 17.8 [(CH3)3CSiMe2], ꢀ4.0, ꢀ4.4, ꢀ4.5,
ꢀ4.6, ꢀ4.7, ꢀ4.9 [(CH3)2SiBut]. HRMS (ESI) m/z [M+Na]+ calcd for
C27H58O6SSi3+Na 613.3160, found 617.3154.
(b) Starting from methyl 2,3,5-tri-O-tert-butyldimethylsilyl-6-O-
triflyl-b- -galactofuranoside (5): To a solution compound 5
D
(100 mg, 0.15 mmol) in dry MeCN (10 mL), potassium thio-
acetate was added (75 mg, 0.66 mmol). The mixture was
stirred at 0 °C for 3 h when TLC (hexane/EtOAc, 10:1)
showed a main spot of Rf 0.60. The solvent was evaporated
and the residue was purified by column chromatography
(hexane/EtOAc, 90:1) to afford 11 (75 mg, 84%). Compound
11 showed the same physical and spectral properties as
the product obtained according to the procedure (a).
C26H55F3O8SSi3+Na 691.2775, found 691.2770.
4.2.4. Methyl 2,3,5-tri-O-tert-butyldimethylsilyl-6-S-(2,3,4,6-
tetra-O-acetyl-b- -galactopyranosyl)-6-thio-b-
galactofuranoside (10)
2,3,4,6-Tetra-O-acetyl-1-thio-galactopyranose
D
D-
8
(36 mg,
0.10 mmol) and NaH (5 mg, 0,21 mmol) dissolved in dry THF
(5 mL) were stirred at rt for 30 min. The solvent was evaporated
and the obtained salt was added to a solution of 4 (30 mg,
0.04 mmol) and 18-crown-6 (15 mg, 0.06 mmol) in DMF (2 mL).
The reaction proceeded at rt for 26 h and was concentrated. The
product (Rf 0.56, hexane/EtOAc, 2:1) was purified by column chro-
matography (hexane/EtOAc 20:1 ? 5:1) to afford 10 (33 mg, 86%);
4.2.6. Methyl 2,3,5-tri-O-tert-butyldimethylsilyl-6-thio-b-D-
galactofuranoside (12)
A solution of compound 11 (84 mg, 0.14 mmol) in CH2Cl2 (5 mL)
was cooled to ꢀ15 °C and a 0.5 M NaOMe/MeOH solution (5 mL)
was added. The mixture was stirred for 2 h, when TLC (hexane/
EtOAc, 10:1) showed a main spot of Rf 0.82. The solvent was evap-
orated and the resulting residue was dissolved in CH2Cl2 (15 mL)
and washed with water (2 ꢃ 15 mL), dried (MgSO4) and concen-
trated to a syrup, which was purified by column chromatography
½
a 2D5
ꢁ
ꢀ35.2 (c 1.2, CHCl3); 1H NMR (CDCl3, 500 MHz) d 5.44 (dd, 1H,
J3 ,4 = 3.4, J4 ,5 = 0.9 Hz, H-40), 5.24 (t, 1H, J1 ,2 = J2 ,3 = 10.0 Hz, H-20),
0
0
0
0
0
0
0
0
5.02 (dd, 1H, J2 ,3 = 10.0, J3 ,4 = 3.4 Hz, H-30), 4.70 (d, 1H,
(hexane/toluene 7:1 ? 1:1) to afford 12 (70 mg, 61%); ½a D25
ꢀ28.2
ꢁ
0
0
0
0
J1,2 = 2.0 Hz, H-1), 4.46 (d, 1H, J1 ,2 = 10.0 Hz, H-10), 4.15 (dd, 1H,
(c 1.0, CHCl3); 1H NMR (CDCl3, 500 MHz)
d 4.70 (d, 1H,
0
0
J5 ,6 a = 7.2, J6 a,6 b = 11.3 Hz, H-60a), 4.11 (dd, 1H, J5 ,6 b = 6.4,
J1,2 = 1.5 Hz, H-1), 4.08 (dd, 1H, J3,4 = 5.2, J4,5 = 4.4 Hz, H-4), 4.01
(dd, 1H, J2,3 = 2.5, J3,4 = 5.2 Hz, H-3), 3.98 (dd, 1H, J1,2 = 1.5,
J2,3 = 2.5 Hz, H-2), 3.80 (ddd, 1H, J4,5 = 4.4 Hz, H-5), 3.35 (s, 3H,
CH3O), 2.79 (dddd, 1H, J5,6a = 6.4, J6a,HS = 8.6, J6a,6b = 13.6 Hz, H-
6a), 2.58 (dddd, 1H, J5,6b = 5.8, J6b,HS = 8.6, J6a,6b = 13.6 Hz, H-6b),
1.48 (t, 1H, J6a,HS = J6b,HS = 8.6 Hz, HS), 0.92, 0.89, 0.88 (3s, 27H,
(CH3)3CSiMe2), 0.14, 0.13, 0.10, 0.09, 0.08, 0.07 (6s, 18H, (CH3)2Si-
But); 13C NMR (CDCl3, 125.7 MHz) d 109.2 (C-1), 85.0 (C-4), 84.2
(C-2), 79.8 (C-3), 74.4 (C-5), 54.9 (CH3O), 27.9 (C-6), 26.0, 25.7
(ꢃ2) [(CH3)3CSiMe2], 18.3 (ꢃ2), 17.8 [(CH3)3CSiMe2], ꢀ3.9, ꢀ4.0,
ꢀ4.1, ꢀ4.3, ꢀ4.4, ꢀ4.5, ꢀ4.8 [(CH3)2SiBut]. HRMS (ESI) m/z
[M+Na]+ calcd for C25H56O5SSi3+Na 575.3054, found 575.3049.
0
0
0
0
0
0
J6 a,6 b = 11.3 Hz, H-60b), 4.10 (dd, 1H, J2,3 = 3.1, J3,4 = 6.0 Hz, H-3),
4.02 (dd, 1H, J3,4 = 6.0, J4,5 = 2.9 Hz, H-4), 3.99 (dd, 1H, J1,2 = 2.0,
J2,3 = 3.1 Hz, H-2), 3.92 (m, 2H, H-5, H-50), 3.35 (s, 3H, CH3O), 2.93
(dd, 1H, J5,6a = 7.8, J6a,6b = 13.2 Hz, H-6a), 2.87 (dd, 1H, J5,6b = 6.0,
J6a,6b = 13.2 Hz, H-6b), 2.15, 2.05, 2.04, 1.99 (4s, 12H, CH3CO),
0.92, 0.88 (ꢃ2) (2s, 27H, (CH3)3CSiMe2), 0.16, 0.14, 0.10, 0.09
(ꢃ2), 0.07 (5s, 18H, (CH3)2SiBut); 13C NMR (CDCl3, 125.7 MHz) d
170.3, 170.2, 170.1, 169.4 (MeCO), 109.1 (C-1), 84.6 (C-2), 84.0
(C-4), 83.7 (C-10), 79.6 (C-3), 74.5, 71.8, 71.7 (C-30, 5, 50). 67.2,
67.0 (C-20, 40), 61.2 (C-60), 55.1 (CH3O), 32.4 (C-6), 26.0, 25.7 (ꢃ2)
[(CH3)3CSiMe2), 20.7 (ꢃ2), 20.6 (ꢃ2) (CH3CO), 18.2, 17.9 (ꢃ2)
[(CH3)3CSiMe2), ꢀ3.5, ꢀ4.0, ꢀ4.2, ꢀ4.3, ꢀ4.4, ꢀ4,9 [(CH3)2SiBut).
HRMS (ESI) m/z [M+Na]+ calcd for C39H74O14SSi3+Na 905.3999,
found 905.4026.
0
0
4.2.7. Methyl 2,3,5-tri-O-tert-butyldimethylsilyl-6-S-(2,3,5,6-
tetra-O-benzoyl-b-
D-galactofuranosyl)-6-thio-b-D-
galactofuranoside (7)
4.2.5. Methyl 2,3,5-tri-O-tert-butyldimethylsilyl-6-S-acetyl-6-
thio-b-D-galactofuranoside (11)
A suspension of the thiol 12 (92 mg, 0.17 mmol), the trichloro-
acetimidate 1323 (246 mg, 0.33 mmol), and freshly activated pow-
dered molecular sieves (4 Å) in dry CH2Cl2 (10 mL) was stirred at
room temperature for 1.5 h. The mixture was cooled to ꢀ18 °C,
(a) Starting from methyl 2,3,5-tri-O-tert-butyldimethylsilyl-6-O-
tosyl-b- -galactofuranoside (4): To a solution of compound
D
and TMSOTf (22
lL, 0.13 mmol) was added. After 40 min Et3N
4 (100 mg, 0.15 mmol) in DMF (4 mL), potassium thioacetate
was added (212 mg, 1.88 mmol). The mixture was stirred at
80 °C for 50 h when TLC (hexane/EtOAc, 10:1) showed a
main spot of Rf 0.60. The solvent was evaporated and the
dark residue was dissolved in CH2Cl2 (15 mL) and washed
(2 ꢃ 15 mL). The organic phase was dried (MgSO4) and con-
centrated. The residue was purified by column chromatogra-
(5 lL, 0.03 mmol) was added and the mixture was concentrated.
TLC (toluene/EtOAc, 15:1) showed a main product (Rf 0.65), which
was purified by column chromatography (toluene/EtOAc, 60:1) to
afford the thiodisaccharide 7 (118 mg, 61%); ½a D25
ꢀ38.8 (c 0.7,
ꢁ
CHCl3); 1H NMR (CDCl3, 500 MHz) d 8.09ꢀ7.28 (H-aromatic), 6.09
(m, 1H, H-50), 5.70 (dt, 1H, J3 ,4 = 4.3 Hz, H-30), 5.62 (d, 1H,
0
0
J1 ,2 = 1.2 Hz, H-10), 5.52 (t, 1H, J1 ,2 = J2 ,3 = 1.2 Hz, H-20), 4.83 (t,
0
0
0
0
0
0
phy (hexane/EtOAc, 40:1) to afford 11 (54 mg, 60%); ½a D25
ꢁ
1H, J3 ,4 = J4 ,5 = 4.3 Hz, H-40), 4.75 (m, 2H, H-60a, H-60b), 4.72 (d,
1H, J1,2 = 1.9 Hz, H-1), 4.11 (t, 2H, H-3, H-4), 4.00 (t, 2H, H-2, H-
5), 3.34 (s, 3H, CH3O), 2.98 (dd, 1H, J5,6a = 7.5, J6a,6b = 13.3 Hz, H-
6a), 2.92 (dd, 1H, J5,6b = 6.0, J6a,6b = 13.3 Hz, H-6b), 0.89, 0.87, 0.84
(3s, 27H, (CH3)3CSiMe2), 0.12, 0.11, 0.08 (ꢃ2), 0.07 (ꢃ2) (6s, 18H,
(CH3)2SiBut); 13C NMR (CDCl3, 125.7 MHz) d 166.0, 165.6, 165.4,
165.2 (PhCO), 133.5ꢀ128.3 (C-aromatic), 109.1 (C-1), 88.1 (C-10),
84.5 (C-4), 84.4 (C-2), 82.6 (C-20), 81.5 (C-40), 79.7 (C-3), 77.8 (C-
30), 71.5 (C-5), 70.3 (C-50), 63.5 (C-60), 55.0 (CH3O), 33.7 (C-6),
0
0
0
0
ꢀ28.0 (c 1.2, CHCl3); 1H NMR (CDCl3, 500 MHz) d 4.74 (d,
1H, J1,2 = 1.8 Hz, H-1), 4.10 (dd, 1H, J2,3 = 2.8, J3,4 = 5.3 Hz,
H-3), 4.00 (dd, 1H, J1,2 = 1.8, J2,3 = 2.8 Hz, H-2), 3.92 (dd, 1H,
J3,4 = 5.4, J4,5 = 4.1 Hz, H-4), 3.83 (ddd, 1H, J4,5 = 4.1 Hz, H-
5), 3.37 (s, 3H, CH3O), 3.15 (dd, 1H, J5,6a = 6.8, J6a,6b = 13.5 Hz,
H-6a), 3.06 (dd, 1H, J5,6b = 6.1, J6a,6b = 13.5 Hz, H-6b), 2.35 (s,
3H, SCOCH3), 0.94, 0.90, 0.88 (3s, 27H, (CH3)3CSiMe2), 0.18,
0.17, 0.11 (ꢃ2), 0.10, 0.09 (6s, 18H, (CH3)2SiBut); 13C NMR

E. Repetto et al. / Bioorg. Med. Chem. 21 (2013) 3327–3333
3333
26.0, 25.7 (ꢃ2), [(CH3)3CSiMe2], 18.2, 17.9, 17.8 [(CH3)3CSiMe2],
ꢀ3.7, ꢀ4.0, ꢀ4.3, ꢀ4.4 (ꢃ2), ꢀ4.9 [(CH3)2SiBut]. HRMS (ESI) m/z
[M+Na]+ calcd for C59H82O14SSi3+Na 1153.4625, found 1153.4656.
for financial support. C.M. and O.V. are Research Members of CON-
ICET. E.R. was supported by a fellowship from CONICET.
Supplementary data
4.2.8. Methyl 6-S-(b-
galactofuranoside (14)
Removal of the O-silyl groups of 7 (65 mg, 0.06 mmol) was per-
formed with 1 M TBAF (198 L, 0.68 mmol) in THF (2 mL). The
D-galactofuranosyl)-6-thio-b-D-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.02.057.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
l
solution was stirred at 0 °C and monitoring by TLC (toluene/EtOAc,
1:1) showed the gradual conversion of the starting compound 7 (Rf
0.95) into a more polar product (Rf 0.75). After 2 h the mixture was
concentrated and the yellow residue obtained was dissolved in
CH2Cl2. The solution was washed with water, dried (MgSO4) and
concentrated. The resulting syrup was dissolved in a (5 mM)
NaOMe/MeOH solution (30 mL) and the mixture was stirred at rt
for 4 h. Monitoring by TLC (n-BuOH/EtOH/H2O, 2.5:1:1) showed a
product with Rf 0.67, which was UV inactive. The solvent was evap-
orated and the residue was purified by column chromatography
References and notes
1. (a) Koester, D. C.; Holkenbrink, A.; Werz, D. B. Synthesis 2010, 19, 3217; (b)
Sears, P.; Wong, C.-H. Angew. Chem., Int. Ed. 1999, 38, 2300.
2. (a) Yuasa, H.; Izumi, M.; Hashimoto, H. Curr. Top. Med. Chem. 2009, 9, 76; (b)
Lillelund, V. H.; Jensen, H. H.; Liang, X.; Bols, M. Chem. Rev. 2002, 102, 515.
3. Pachamuthu, K.; Schmidt, R. R. Chem. Rev. 2006, 106, 160.
4. Szilágyi, L.; Varela, O. Curr. Org. Chem. 2006, 10, 1745.
5. Uhrig, M. L.; Manzano, V. E.; Varela, O. Eur. J. Org. Chem. 2006, 162.
6. Cagnoni, A. J.; Uhrig, M. L.; Varela, O. Bioorg. Med. Chem. 2009, 17, 6203.
7. Manzano, V. E.; Uhrig, M. L.; Varela, O. J. Org. Chem. 2008, 73, 7224.
8. Manzano, V. E.; Uhrig, M. L.; Varela, O. Org. Biomol. Chem. 2012, 10, 8884.
9. Repetto, E.; Manzano, V. E.; Uhrig, M. L.; Varela, O. J. Org. Chem. 2012, 77, 253.
10. Repetto, E.; Marino, C.; Uhrig, M. L.; Varela, O. Eur. J. Org. Chem. 2008, 540.
11. Repetto, E.; Marino, C. M.; Uhrig, M. L.; Varela, O. Bioorg. Med. Chem. 2009, 17,
2703.
12. Randell, K. D.; Johnston, B. D.; Lee, E. E.; Pinto, B. M. Tetrahedron: Asymmetry
2000, 11, 207.
13. Peltier, P.; Euzen, R.; Daniellou, R.; Nugier-Chauvin, C.; Ferrieres, V. Carbohydr.
Res. 2008, 343, 1897.
14. Marino, C.; Gallo-Rodriguez, C.; Lederkremer, R. M. Galactofuranosyl-
Containing Glycans: Occurrence, Synthesis and Biochemistry. In Glycans:
Biochemistry, Characterization and Applications; Mora-Montes, H. M., Ed.;
Nova Science Publisher: New York, 2012; pp 201–268.
15. Besra, G. S.; Khoo, K. H.; McNeil, M. R.; Dell, A.; Morris, H. R.; Brennan, P. J.
Biochemistry 1995, 34, 4257.
(EtOAc ? EtOAc/MeOH, 1:1) to afford 14 (20 mg, 90%);
½ ꢁ
a 2D5
ꢀ93.8 (c 1.1, MeOH); 1H NMR (CD3OD, 500 MHz) d 5.13 (d, 1H,
J1 ,2 = 4.4 Hz, H-10), 4.76 (d, 1H, J1,2 = 1.6 Hz, H-1), 4.07 (dd, 1H,
0
0
J2 ,3 = 4.9, J3 ,4 = 7.4 Hz, H-30), 4.00 (m, 2H, H-3, H-4), 3.98 (dd,
0
0
0
0
1H, J3 ,4 = 7.4, J4 ,5 = 3.1, Hz, H-40), 3.93 (m, 2H, H-2, H-20), 3.88
(ddd, 1H, J4,5 = 1.7, J5,6a = 7.6, J5,6b = 5.8 Hz, H-5), 3.74 (ddd, 1H,
0
0
0
0
J4 ,5 = 3.1,
J5 ,6 a = J5 ,6 b = 6.1 Hz,
H-50),
3.63
(d,
2H,
0
0
0
0
0
0
J5 ,6 a = J5 ,6 b = 6.1 Hz, H-60a, H-60b), 3.37 (s, 3H, CH3O), 2.89 (dd,
1H, J5,6a = 7.6, J6a,6b = 13.9 Hz, H-6a), 2.84 (dd, 1H, J5,6b = 5.8,
J6a,6b = 13.9 Hz, H-6b); 13C NMR (CD3OD, 125.7 MHz) d 110.4 (C-
1), 91.2 (C-10), 85.5 (C-4), 83.9, 83.3 (C-2, 20), 82.9 (C-40), 78.8 (C-
3), 78.4 (C-30), 72.1 (C-50), 71.7 (C-5), 64.4 (C-60), 55.3 (CH3O),
35.7 (C-6). HRMS (ESI) m/z [M+Na]+ calcd for C13H24O10S+Na
395.0988, found 395.0976.
0
0
0
0
16. Barry, C. E. Biochem. Pharmacol. 1997, 54, 1165.
17. (a) Marino, C.; Mariño, K.; Miletti, L.; Manso Alves, M. J.; Colli, W.; Lederkremer,
R. M. Glycobiology 1998, 8, 901; (b) Mariño, K.; Maino, C. Arkivoc 2005, XII, 341;
(c) Mariño, K.; Lima, C.; Maldonado, S.; Marino, C.; Lederkremer, R. M.
Carbohydr. Res. 2002, 337, 891.
4.3. Enzymatic assays
18. 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.
19. Driguez, H. ChemBioChem 2001, 2, 311.
The enzymatic activity was assayed using the filtered medium
of a stationary culture of P. fellutanum as source of exo b-
tofuranosidase and 4-nitrophenyl b- -galactofuranoside as sub-
strate.17a The standard assay was conducted with 50
of
66 mM NaOAc buffer (pH 4.6), 15 L of a 5 mM solution of 4-nitro-
phenyl b- -galactofuranoside and 20 L (4 g protein) of the en-
zyme medium, in a final volume of 250 L. Compound 14 was
D-galac-
20. Baldoni, L.; Marino, C. Carbohydr. Res. 2012, 362, 70.
D
21. (a) Ibatullin, F. M.; Selivanov, S. I.; Shavva, A. G. Synthesis 2001, 3, 419; (b)
Ibatullin, F. M.; Shabalin, K. A.; Jänis, J. V.; Selivanov, S. I. Tetrahedron Lett. 2001,
42, 4565.
lL
l
22. (a) Hughes, N. A.; Wood, C. J. J. Chem. Soc., Perkin Trans. 1 1986, 695; (b) Hughes,
N. A.; Munkombwe, N. M.; Todhunyer, N. D. Carbohydr. Res. 1991, 216, 119.
23. (a) Choudhury, A. K.; Roy, N. Carbohydr. Res. 1998, 308, 207; (b) Gallo-
Rodriguez, C.; Gandolfi, L.; de Lederkremer, R. M. Org. Lett. 1999, 1, 245.
24. Yuasa, H.; Hashimoto, H. Trends Glycosci. Glyc. 2001, 13, 31.
25. (a) Montero, E.; García-Herrero, A.; Asensio, J. L.; Hirai, K.; Ogawa, S.; Santoyo-
González, F.; Cañada, F. J.; Jiménez-Barbero, J. Eur. J. Org. Chem. 2000, 1945; (b)
García-Herrero, A.; Montero, E.; Muñoz, J. L.; Espinosa, J. F.; Vián, A.; García, J.
L.; Asensio, J. L.; Cañada, F. J.; Jiménez-Barbero, J. J. Am. Chem. Soc. 2002, 124,
4804.
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D
l
l
l
incorporated in the amounts required to obtain a final concentra-
tion of 0.6 to 2.4 mM. The enzymatic reaction was stopped after
1.5 h of incubation at 37 °C by addition of 1 mL of 0.1 M Na2CO3
buffer (pH 9.0). The 4-nitrophenol released was measured spectro-
photometrically at 410 nm. Km and Ki values were determined by
the Lineweaver–Burk plot.
Acknowledgments
The authors are indebted to the University of Buenos Aires
(UBACYT 200 201001 00536), Agencia Nacional de Promoción
Científica y Tecnológica (ANPCyT-PICT 2007-0291) and the Na-
tional Research Council of Argentina (CONICET-PIP 2008-00064)