Synthesis of 5-deoxy-β-d-galactofuranosides as tools for the characterization of β-d-galactofuranosidases

Article abstract of DOI:10.1016/j.bmc.2010.05.038

Derivatives of 5-deoxy-β-d-galactofuranose (5-deoxy-α-l-arabino- hexofuranose) have been synthesized starting from d-galacturonic acid. The synthesis of methyl 5-deoxy-α-l-arabino-hexofuranoside (14α) was achieved by an efficient strategy previously optimized, involving a photoinduced electron transfer (PET) deoxygenation. Compound 14α was converted into per-O-acetyl-5-deoxy-α,β-l-arabino-hexofuranoside (16), an activated precursor for glycosylation reactions. The SnCl₄-promoted glycosylation of 16 led to 4-nitrophenyl (19α), and 4-methylthiophenyl 5-deoxy-α-l-arabino-hexofuranosides (20α). The oxygenated analog 4-methylphenyl 1-thio-β-d-galactofuranoside (23β) was also prepared. The 5-deoxy galactofuranosides were evaluated as inhibitors or substrates of the exo-β-d-galactofuranosidase from Penicillium fellutanum, showing that the absence of HO-5 drastically diminishes the affinity for the protein.

Full text of DOI:10.1016/j.bmc.2010.05.038

Bioorganic & Medicinal Chemistry 18 (2010) 5339–5345
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of 5-deoxy-b-D-galactofuranosides as tools for the characterization
of b-D-galactofuranosidases
*
Andrea Bordoni, Rosa M. de Lederkremer, Carla Marino
CIHIDECAR-CONICET, Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. Pabellón II,
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:
Derivatives of 5-deoxy-b-
D
-galactofuranose (5-deoxy-
a
-
L
-arabino-hexofuranose) have been synthesized
-arabino-hexofuranoside (14
was achieved by an efficient strategy previously optimized, involving a photoinduced electron transfer
(PET) deoxygenation. Compound 14 was converted into per-O-acetyl-5-deoxy- ,b- -arabino-hexofur-
anoside (16), an activated precursor for glycosylation reactions. The SnCl4-promoted glycosylation of
16 led to 4-nitrophenyl (19 ), and 4-methylthiophenyl 5-deoxy- -arabino-hexofuranosides (20 ).
The oxygenated analog 4-methylphenyl 1-thio-b- -galactofuranoside (23b) was also prepared. The 5-
deoxy galactofuranosides were evaluated as inhibitors or substrates of the exo-b- -galactofuranosidase
Received 13 April 2010
Revised 13 May 2010
Accepted 14 May 2010
Available online 20 May 2010
starting from
D
-galacturonic acid. The synthesis of methyl 5-deoxy-
a
-
L
a)
a
a
L
a
a
-
L
a
Keywords:
5-Deoxy-D-galactofuranosides
PET deoxygenation
Galactofuranosidase substrates
Galactofuranosidase inhibition
D
D
from Penicillium fellutanum, showing that the absence of HO-5 drastically diminishes the affinity for
the protein.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
sacchari.14 The best known is the specific exo-b-
D-galactofuranosi-
dase first purified from the culture medium of Penicillium felluta-
num.15 The natural substrate for the enzyme is the extracellular
glycopeptide peptidophosphogalactomannan (pPGM), containing
The glycobiology of galactofuranose is a field of great interest,
since b-D-galactofuranosyl residues are constituents of infectious
microorganisms, such as the bacteria Mycobacterium tuberculosis
and Mycobacterium leprae,1,2 trypanosomatids like Trypanosoma
cruzi and Leishmania,3–7 and fungi like Paracoccidioides brasilensis,8
but are absent in mammalian cells. Therefore, the metabolic path-
ways involved in the biosynthesis and degradation of these micro-
bial glycoconjugates, are attractive targets for the development of
therapeutic agents. The enzymes involved in the biosynthesis of
Galf-containing molecules are the UDP-galactopyranosylmutase
(EC 5.4.99.9),9 which catalyzes the interconversion of UDP-Galp
into the donor of Galf residues UDP-Galf, and the galac-
tofuranosyltransferases, which are responsible for the incorpora-
tion of the sugar into the glycoconjugates.10 The galactofuranosyl
content of these glycoconjugates may vary. For example, the pres-
terminal 1?5 linked b-D-Galf units. This glycoconjugate is a re-
serve source of phosphate, choline, and/or carbohydrates, which
are released by enzyme-catalyzed depolymerization.16 Among
other hydrolases, the exob-D-galactofuranosidase is involved in this
degradation. To date, the gene encoding this enzyme has not been
identified, nor has the aminoacid sequence been determined.
There have been significant efforts directed towards the devel-
opment of galactofuranosyl containing molecules as tools for the
Galf-processing enzymes.17 Our laboratory has also been involved
in studies on the glycobiology of
D-Galf, particularly for the
exob- -galactofuranosidase from P. fellutanum, which has become
D
a model enzyme. We have described the synthesis of substrates
for these enzymes (Fig. 1),18–23 inhibitors (Fig. 2),25–28 and deoxy-
genated analogs of the substrates (Fig. 3).28–32 We have contrib-
uted to the synthesis of galactofuranosyl precursors32,33 and the
development of and new glycosylation methodologies.28,33 We
ence of b-D-Galf units in the mucins of the parasite T. cruzi depends
on the strain.11 Also in T. cruzi, one of the differences in the surface
carbohydrates between the epimastigote form and the infective
trypomastigote form, is the Galf content of the glycoinositolphosp-
holipids (GIPLs), the most abundant cell-surface molecules in
epimastigotes.12 These structural variations could be attributed
*
*
have also synthesized radioactive substrates (4 and 5 ) which
were used for studies on the galactofuranosidase21,23 and on the
galactofuranosyltransferase.22
to the action of b-
D
-galactofuranosidases.
The activity of the exob-D-galactofuranosidase is dependent on
exob-
D
-Galactofuranosidases (EC 3.2.1.146) were characterized
the aglycone structure, both in substrates and inhibitors. For exam-
ple, while compound 8 is a moderate inhibitor (IC50 0.08 mM),
compound 7, its synthetic precursor, is much less effective (IC50
0.60 mM). We demonstrated that lactone 6 is an efficient inhibitor
of the enzyme (IC50 0.02 mM),24 and we developed an affinity
in Penicillium and Aspergillius species13 and in Helminthosporium
* Corresponding author. Fax: +54 11 4576 3346.
E-mail address: cmarino@qo.fcen.uba.ar (C. Marino).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.038

5340
A. Bordoni et al. / Bioorg. Med. Chem. 18 (2010) 5339–5345
CH3
O
The glycone specificity of the exob-
high and it was first evidenced by the fact that
D
-galactofuranosidase is also
-arabinofurano-
a-L
sides, the pentosyl homologs, were not hydrolyzed by the en-
zyme.15 Deoxy sugars, analogs of the substrates, are useful in
defining the importance of each specific hydroxyl group for the
interaction between carbohydrates and proteins. They also play a
significant role in many active compounds, such as antibiotics,
antiviral drugs, and glycosylation inhibitors. The development of
strategies for the synthesis of deoxy sugars as glycobiological tools
has become an important field of research, and they have been syn-
thesized as inhibitors of glycosidases, and glycosyltransferases.36
O
O
NO2
O
O
OH
O
O
OH
O
OH
OH
OH
OH
OH
CH2OH
OH
CH2OH
OH
CH2OH
3
2
1
OCH3
O
OH
O
In the field of glycofuranoses, deoxygenated
D-arabinofuranose
NH
OH
O
O
O
derivatives were synthesized as biochemical probes to study sub-
O P
CH2
P O
O-
N
O
strate specificity of arabinosyltranferases of M. tuberculosis,37 and
O
O-
OH
OH
OH
CHROH
OH
C3H2OH
deoxygenated L-arabinofuranosides were prepared to investigate
their potential as acceptors in biocatalytic methods.38 An interest-
ing strategy to afford 2-deoxy furanosides starting from 2,3-anhy-
dro-furanosyl thioglycosides has been developed.39
OH OH
4
5*
R = H
4* R = 3H
As part of our project aimed to the characterization of galacto-
furanose related enzymes, we developed the synthesis of galacto-
furanosyl analogs deoxygenated at C-2, -3, or -6 (Fig. 3).28–32 We
found that they were resistant to the hydrolytic activity of the
Figure 1. Synthetic substrates for the characterization of b-D-Galf-processing
enzymes.
exob-D-galactofuranosidase, indicating that the hydroxyl groups
at these positions are essential for the enzyme recognition. Fre-
quently, glycopyranosidases have a more relaxed specificity. For
S
R
O
OH
O
OH
O
example, jack bean and almond
4-nitrophenyl -rhamnopyranoside at a rate approaching that
for the
-mannopyranoside.40
In order to further characterize the substrate specificity of this
enzyme we developed a strategy for deoxygenation at C-5 of glyco-
a-mannosidases both hydrolyzed
OH
OH
OH
CH2OH
OH
a-D
CH2OH
a-D
7 R = NO2
R = NH2
6
8
furanosides. We reported the synthesis of methyl 5-deoxy-b-
galactofuranoside (methyl 5-deoxy- -arabino-hexofuranoside,
14 ), by a photoinduced electron transfer (PET) deoxygenation of
a derivative of the methyl (methyl- -galactofuranosid)uronate
D-
S
S
a-L
a
N
NH
N
NH
O
OH
O
OH
D
OCH3
(11) as the key step. The advantage of the reported synthesis relies
on the easy access to the furanosic derivative 11, obtained in one
step from D-galacturonic acid (10), the high regioselectivity for
the introduction of the photoreductible 3-(trifluormethyl)benzoyl
group, and the effectiveness of the PET deoxygenation of 12, as-
sisted by the carboxymethyl ester group at the vicinal position
(Scheme 1).32
OH
OH
OH
CH2OH
OH
CH2OH
10
9
Figure 2. Inhibitors of the exo-b-D-galactofuranosidase from Penicillium fellutanum.
Having compound 14
erties as inhibitor and substrate for the exob-
of P. fellutanum. Nevertheless, compound 14
substrate, because no chromophore is released by action of the en-
zyme. Thus, considering the high affinity of the chromogenic deriv-
a
in hand, we have now studied its prop-
-galactofuranosidase
is not a convenient
D
OCH3
O
NO2
O
NO2
O
OH
O
O
OH
a
OH
OH
OH
CH2OH
OH
CH2OH
OH
CH3
ative 1, we developed the synthesis of its 5-deoxy analog 19
(Scheme 2). The synthesis and biological evaluation of thioglyco-
a
side 23b (Scheme 3) and its deoxygenated analog 20
a is also re-
ported (Scheme 2).
OH
OH
OH
OH
O
HO
O
O
2. Results and discussion
HO
O
O
O
OH
OH
OH
2.1. Synthesis and chemical characterization
OH
OH
OH
CH2OH
OH
CH3
For the synthesis of 19a we envisioned a route starting from
14, which has been prepared in our laboratory by a sequence suc-
cinctly shown in Scheme 1. However, in the present case and in
contrast to the original synthesis of 14a 32
,
it was not necessary
to separate the mixture of anomers of the methyl glycoside 14,
obtained from -galacturonic acid, since the anomeric configura-
Figure 3. Synthetic 2,3 and 6-deoxygenated analogs of substrates for exo-b-D-
galactofuranosidase.
D
tion is lost during the followings steps (Scheme 2). In fact, we
chose acetates as protective groups of 14 because during the ace-
tolysis of benzoylated derivatives, transesterification was ob-
served.22 Starting from 15, the conditions for the acetolysis
reaction were optimized. In order to avoid the drastic conditions
chromatographic system for the purification of galactofuranosidas-
es by immobilization of 8, using lactone 6 for the elution of the
enzyme.34 By this approach, galactofuranosidase activity was
detected in T. cruzi for the first time.35

A. Bordoni et al. / Bioorg. Med. Chem. 18 (2010) 5339–5345
5341
OH
COOH
OCH3
O
OCH3
O
OH
O
OH
O
Amberlite IR120-H
MeOH
(3-CF3)C4H6COCl
CH2Cl2/C5H5N
HO
OH
OH
OH
OH
CO2CH3
OH
O
10
CO2CH3
F3C
11
11
α
β (71%) +
12
OCH3
OCH3
O
O
NaBH4/I2
THF
OH
OH
hυ
MCZ, MgClO4
OH
OH
CH2OH
H
H
H
H
CO2CH3
13
14
α
Scheme 1. Synthetic route to methyl 5-deoxy-
a-
L-arabino-hexofuranoside (14a, Ref. 32).
O
OAc
O
OAc
5.5% H2SO4/Ac2O
in CH2Cl2 (1:50)
OAc
OCH3
Ac2O, CH2Cl2, Py
14α,β
OAc
OAc
H
H
H
H
CH2OAc
CH2OAc
16
SnCl4
4-nitrophenol
15
SnCl4
4-methylthiophenol
O
O
OR
OR
H
S
CH3
O
NO2
OR
OR
H
H
H
CH2OR
CH2OR
18
(α/β 10:9) R = OAc
α R = H
17 (α/β 5:1) R = OAc
19α R = H
NaOMe/MeOH
CH2Cl2
NaOMe/MeOH
CH2Cl2
20
Scheme 2. Synthesis of 4-nitrophenyl 5-deoxy-
a-
L
-arabino-hexofuranoside (19
a
) and 4-methylphenyl 5-deoxy-1-thio-
a-
L-arabino-hexofuranoside (20a).
S
CH3
O
OBz
O
OBz
O
OH
OBz
S
CH3
1) SnCl4, CH2Cl2
0 ºC,10 min
OBz
OBz
OH
NaOMe/MeOH
CH2Cl2
OBz
OBz
OH
2) 4-methylthiophenol
CH2OBz
CH2OBz
CH2OH
23β
21
22 (β/α 9:1)
Scheme 3. Synthesis of 4-methylphenyl 1-thio-b-
D
-galactofuranoside (23b).
of the conventional acetolysis the removal of the anomeric
O-methyl group has been attempted under milder conditions.
Thus, trityl tetrafluorborate (Ph3CBF4) has been described as a chemo-
selective reagent for the hydrolysis of several methyl glycosides,
including pentofuranosides.41 However, in our hands this method
was not effective, as even under longer reaction times or higher
concentrations of the reagent, the hydrolysis was incomplete.
Similarly, the use of SnCl4 for the substitution of a benzoyloxy
anomeric substituent by an acetyloxy group,27 was also unsuc-
cessful for 15, probably due to the poorer capacity of the methox-
ide compared to an acetyloxy as leaving group. Also, treatment of
15 with acetic anhydride and trifluoroacetic acid42 afforded 16 in
low yield.
nent. Integration of the area of the H-1 signal
6.10 ppm, J1,2 < 0.5 Hz; b-anomer: 6.32 ppm, J1,2 = 4.0 Hz) indicated
a 3:1
/b ratio. The signals corresponding to H-5 and 50 were over-
lapped with those of the methyl groups of the acetates. A similar
anomeric relationship was determined from the 13C NMR spec-
trum, which showed in the anomeric region the resonance of C-1
(a-anomer:
a
at 99.0 ppm (
33.7 ppm (b-anomer) and 32.0 ppm (
the deoxygenated C-5 of both anomers.
SnCl4 was efficiently used in this laboratory as glycosylation
promoter for the synthesis of -Galf derivatives, such as simple gly-
a-anomer) and 93.7 ppm (b-anomer), and signals at
a-anomer) corresponding to
D
cofuranosides,44 thioglycosides,24 disaccharides,44 thiodisaccha-
rides,27 and oligosaccharides.45 Thus, treatment of 16 with SnCl4
and subsequent addition of 4-nitrophenol or 4-methylthiophenol
led to 17 and 18, respectively. The 1H NMR spectrum of 17 showed
a 5:1 ratio of 1,2-trans and 1,2-cis anomeric signals at d 5.71
(J1,2 < 0.5 Hz) and 5.93 (J1,2 = 4.5 Hz). The 13C NMR spectrum
Finally, the acetolysis was satisfactorily performed using acetic
anhydride and H2SO4 diluted in a CH2Cl2 solution,22,43 affording
compound 16 in 80% yield. The 1H NMR spectrum of crude 16 indi-
cated that the 1,2-trans anomer (a-anomer) was the major compo-

5342
A. Bordoni et al. / Bioorg. Med. Chem. 18 (2010) 5339–5345
showed, in the anomeric region, resonances at 103.5 and 98.1 ppm,
shifted upfield in comparison to signals of O-alkylglycoside 14,32 as
expected for O-aryl glycosides.46 The 1H NMR spectrum of 18
showed the resonances of H-1 at 5.39 ppm (J1,2 = 2.0 Hz) and
5.53 ppm (J1,2 = 4.9 Hz), corresponding to the 1,2-trans and 1,2-cis
diastereomers, respectively, in similar amount. The 13C NMR spec-
(Fig. 4). The difference in the affinity of 14a, which is not hydro-
lyzed, and 19a, a substrate with low affinity, correlates with the
differences observed between 1 (Km 0.31 mM)18,34 and 4 (Km
2.60 mM).49
Inhibition of the galactofuranosidase activity also depends on
the structure of the aglycone. Thus far, the best inhibitor we have
tested was lactone 6. As above exemplified for thioglycosides 7 and
8, also for nucleoside a high sensitivity of the enzyme was ob-
served. Thus, compound 9 (IC50 0.10 mM) was ten times more ac-
tive than the analog 10 (IC50 1.05 mM), suggesting that the change
in the flexibility of the imidazolidine ring facilitates the interaction
with the active site of the enzyme.25
trum of 18 showed the C-1 signals at 90.8 ppm (a-anomer) and
89.5 ppm (b-anomer), in agreement with values observed for other
thiogalactofuranosides.24,26 The diastereocontrol of the SnCl4-pro-
moted procedure for the per-O-acetyl derivative 16 was less effec-
tive than for the benzoylated derivatives of D
-galactofuranose,24,44
as result of the less efficient anchimeric effect.
Compounds 17 and 18 were O-deacetylated with NaMeO/MeOH/
CH2Cl2 to afford good yields of 19 and 20, respectively. Recrystalliza-
tion of compound 19 from water afforded the 1,2-trans glycoside
For the evaluation of the new compounds as in vitro inhibitors
of the exob-D-galactofuranosidase from P. fellutanum, the protocol
previously developed was followed. 4-Nitrophenyl b-D-galactofur-
19a
as a single diastereoisomer, as observed in the 1H NMR spec-
anoside (1)18 was used as substrate and lactone 6 as the reference
inhibitor (IC50 0.02 mM).24 Releasing of 4-nitrophenol from 1 was
employed as a measurement of galactofuranosidase activity. Com-
trum, which showed the anomeric signal as a singlet at 5.64 ppm
(J1,2 < 1.0 Hz). The signals corresponding to the diastereotopic
hydrogens at C-5 appeared at high fields (1.87 and 1.96 ppm). The
13C NMR spectrum showed the resonance of C-1 and C-5 at 108.5
pounds 11b, 13, 14
matic reaction, in concentrations ranging from 0.2 to 2.5 mM.
Compounds 11b, 13, and 14 did not show inhibitory activity
a, 19a, 20a, and 23b were subjected to the enzy-
and 38.2 ppm, respectively. The pure anomer 20
a
was obtained by
a
recrystallization from hexane/EtOAc (3:2). The 1H NMR spectrum
showed the anomeric proton as a doublet at d 5.19 (J1,2 = 4.5 Hz)
and H-5a and H-5b as a dddd at 1.84 and 1.99 ppm, respectively.
The larger J1,2 for free 1,2-trans 1-thio-galactofuranosides, compared
with O-galactofuranosides, was previously observed, and it was
attributed to a conformational shift toward conformations with
the anomeric sulfur atom in a quasiequatorial position,24 reflecting
the weaker anomeric effect of this atom.47
With the aim of evaluating the influence of HO-5 on the inhib-
itory activity of thioglycosides,24,26 the oxygenated analog 23, was
synthesized. Following the described procedure for 1-thiogalacto-
furanosides,24 SnCl4-promoted glycosylation of perbenzoylated
derivative 2148 with 4-methylthiophenol afforded 22, and subse-
quent deprotection with NaMeO/MeOH in CH2Cl2, led to thioglyco-
side 23b in 80% overall yield. NMR spectra of 23b showed the
presence of the 1,2-trans thioglycoside as the only product, and
they were in agreement with those previously reported for galac-
tofuranosyl thiogalactofuranosides.24,26
(not shown). Thus, the presence of a carboxymethyl ester at C-6,
the absence of HO-5, or both modifications prevent the interaction
with the enzyme. The thioglycoside 23b showed a low inhibitory
activity, with 30% inhibition at 0.25 mM, in accordance with the
activity of thiogalactofuranosides with similar structures
(Fig. 5).24 The 5-deoxy analog 20
a, instead, was not active, and
no inhibition was observed at concentrations up to 2.5 mM. These
results indicate the importance of HO-5 in the interaction with the
protein, so its absence abolishes the inhibitory activity due to the
presence of –S observed for thioglycoside 23b.
3. Conclusion
The synthesis of derivatives of b-
starting from -galacturonic acid, was successfully accomplished.
The derivatives were evaluated as substrate and/or inhibitors of
the exob- -galactofuranosidase from P. fellutanum. When evaluated
D-Galf deoxygenated at C-5
D
D
as inhibitors, they showed no activity. Indeed, deoxygenation at C-
5 in an inhibitor suppressed its activity. As substrate, compound
2.2. Biological evaluation
14a showed no activity, indicating the importance of HO-5 for
the sugar–enzyme interaction as previously observed for HO-2,
-3, and -6.28–30 However, an increase in the interaction of the gly-
cone 5-deoxy Galf with the enzyme was observed by introducing
the 4-nitrophenyl group at the anomeric position, as previously ob-
served for the oxygenated analogs.24
Previous studies with simple b-
strates for the exob- -galactofuranosidase activity have shown a
high aglycone specificity. 4-Nitrophenyl b- -Galf (1) is a good sub-
strate (Km 0.31 mM) and proved to be useful for the measurement
D-galactofuranosides as sub-
D
D
of the enzymatic activity,18,34 whereas benzyl b-
D-Galf (2, Km
3.7 mM)20 is less effective. On the other hand, the 4-methylumbel-
liferyl analog 3, considerably bulkier than 1, is not hydrolyzed by
the enzyme. The methyl glycoside 4 (Km 2.60 mM) has also been
kinetically characterized.49 To assess the ability of the 5-deoxy
0.8
0.6
derivatives as substrates for the exo-b-
pound 14 was incubated with the enzyme under the conditions
employed for substrate 1. The reaction was monitored by HPAEC-
D-galactofuranosidase, com-
a
6
5
PAD and no release of 5-deoxy galactose (tR 24.12 min) from 14
a
0.4
4
(tR 13.68 min) was detected.
3
A colorimetric assay was also performed, using compound 19
a
2
as substrate and monitoring the reaction by measurement of the 4-
nitrophenol released. A control reaction under the test conditions
(NaOAc buffer, pH 5.5, 37 °C, 1.5 h),24 showed some chemical
0.2
K
m
= 1.29 mM
1
0
-1
0
1
2
3
hydrolysis of 19a, in contrast with the oxygenated analog 1, which
1 / [S]
0.0
0.0
is stable. This different behavior is not surprising considering the
lability of the glycosidic linkage in deoxy sugars compared to the
oxygenated analogs.50 On the other hand, enzymatic hydrolysis
0.5
1.0
1.5
[S] (mM)
2.0
2.5
3.0
of 19a was also observed. The kinetic analysis, considering the
Figure 4. Kinetic analysis of substrate 19
a. Each point is the mean obtained from
chemical hydrolysis at each point, gave a Km value of 1.29 mM
three replicate experiments. Inset: Lineweaver–Burk reciprocal plot.

A. Bordoni et al. / Bioorg. Med. Chem. 18 (2010) 5339–5345
5343
(1.7 mL, 18.0 mmol) were added. The mixture was stirred at room
temperature during 8 h and then was kept at 4 °C for 16 h. TLC
examination showed the conversion of the starting material into
a single product (Rf = 0.75, toluene/EtOAc 4:1). The mixture was
evaporated under reduced pressure and co-evaporated with tolu-
ene. Compound 15 (1.33 g, 97%) was used without further purifica-
tion. For the major 1,2-trans anomer 1H NMR (CDCl3, 500 MHz): d
5.01 (d, 1H, J = 1.5 Hz, H-2), 4.88 (dd, 1H, J = 5.8, 1.5 Hz, H-3), 4.84
(s, 1H, H-1), 4.27–4.22 (m, 1H, H-6a), 4.18–4.13 (m, 1H, H-6b), 4.10
(ddd, 1H, J = 8.5, 5.5, 1.5 Hz, H-4), 3.36 (s, 3H, OCH3), 2.15–2.10 (m,
1H, H-5a), 2.09 (s, 6H, 2 COCH3), 2.04 (s, 3H, COCH3), 2.03–1.96 (m,
1H, H-5b). 13C NMR (CDCl3, 125.8 MHz): d 170.9, 170.3, 169.8
(COCH3), 106.3 (C-1), 81.8 (C-2), 80.1 (C-3), 78.8 (C-4), 61.0 (C-6),
54.7 (OCH3), 32.1 (C-5), 20.9, 20.8, 20.7 (COCH3). HRMS (ESI+) calcd
for C13H20O8Na+ [M+Na]+ 327.10504, found 327.10526.
20α
100
80
60
40
20
0
23β
6
0.0
0.5
1.0
1.5
2.0
2.5
Compound (mM)
4.2.2. 1,2,3,6-Tetra-O-acetyl-5-deoxy-a,b-L-arabino-hexofura
nose (16)
Figure 5. Effect of the concentration of added compounds on the enzymatic
hydrolysis of 1 by the exo-b- -galactofuranosidase from Penicillium fellutanum. The
To a solution of crude compound 15 in dry CH2Cl2 (18 mL)
D
numbers indicate the compound added. Each point is the mean obtained from three
replicate experiments.
cooled to 0 °C, acetic anhydride (0.33 mL, 3.5 mmol) and H2SO4
(21 lL, 0.38 mmol) were added. The mixture was stirred at room
temperature during 8 h and then was kept at 4 °C for 16 h. TLC
monitoring showed consumption of the starting material and the
formation of a main product of Rf 0.39 (hexane/EtOAc 3:2). The
reaction was stopped by dilution with CH2Cl2 (75 mL) and addition
of cold water (5 mL). After stirring for 15 min, the organic layer
was washed with satd aq NaHCO3 (3ꢀ 50 mL) and brine. The or-
ganic solution was dried (Na2SO4), concentrated, and the residue
was purified by column chromatography (hexane/EtOAc 85:15)
These results reinforce the concept of the high selectivity of the
b-D-galactofuranosidase, which requires all the hydroxyls of the
galactofuranosyl residue in order to produce an efficient sugar–
protein interaction.
On the other hand, the presence of (1?5)-linked b-D-galactofur-
anosides is a common feature of biologically important glycoconju-
gates.1–7 In Penicillium and Aspergillius species side chains of
to give compound 16 (0.26 g, 80%) as
a/b mixture in a 6:1 ratio.
(1?5)-interlinked b-D
-Galf units are inmunodominant.13 Alternat-
For the major product (
a
-anomer): 1H NMR (CDCl3, 500 MHz) d
ing b-(1?5) and b-(1?6) galactofuranosyl residues construct the
arabinogalactan of mycobacteria and a bifunctional galactofurano-
syl transferases, GlfT2, thought to be responsible for the polymer-
ization.2a The deoxy galactofuranosides described in the present
paper, lacking the HO-5, could be also useful for studies on the
inhibition and identification of the galactofuranosyltransferase
activities.
6.14 (s, 1H, H-1), 5.18 (dd, 1H, J = 1.9, 0.5 Hz, H-2), 4.98 (ddd, 1H,
J = 2.4, 1.9, 0.5 Hz, H-3), 4.29–4.22 (m, 2H, H-4, H-6a), 4.16–4.11
(m, 1H, H-6b), 2.13–2.05 (m, 14H, H-5a, H-5b, 4 COCH3); 13C
NMR (CDCl3, 125.8 MHz): d 170.8, 169.3, 169.2 (COCH3), 99.0 (C-
1), 81.3 (C-4), 80.8 (C-2), 79.4 (C-3), 60.6 (C-6), 32.0 (C-5), 21.0,
20.8, 20.7, 20.6 (COCH3). For the b-anomer: 1H NMR (CDCl3,
500 MHz): d 6.36 (d, 1H, J = 4.0 Hz, H-1), 5.33–5.19 (m, 2H, H-2,
H-3), 4.29–4.22 (m, 1H, H-6), 4.16–4.11 (m, 2H, H-4, H-6), 2.13–
4. Experimental
2.05 (m, 14H, H-5a, H-5b,
4
COCH3); 13C NMR (CDCl3,
125.8 MHz): d 170.3, 169.9, 169.4 (COCH3), 93.8 (C-1), 79.2 (C-4),
77.8, 75.5 (C-2, C-3), 60.6 (C-6), 33.7 (C-5), 20.9, 20.8, 20.7, 20.5
(COCH3). HRMS (ESI+) calcd for C14H20O9Na+ [M+Na]+ 355.09995,
found 355.10041.
4.1. General methods
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 10% (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). Melting points were determined with a
Fisher–Johns apparatus and are uncorrected. Optical rotations
were measured with a Perkin-Elmer 343 digital polarimeter. For
anomeric mixtures, the optical rotations were not measured. Nu-
clear magnetic resonance (NMR) spectra were recorded with a Bru-
ker AMX 500 spectrometer. Assignments of 1H and 13C were
assisted by 2D 1H-COSY and HSQC experiments. High resolution
mass spectra (HRMS ESI+) were recorded in a Bruker micrOTOF-
Q II spectrometer.
4.2.3. General procedure for SnCl4-promoted glycosylation
The procedure previously described was followed.44 To a solu-
tion of 16 or 2148 (0.25 mmol) in anhydrous CH2Cl2 (2.0 mL) cooled
at 0 °C, SnCl4 (0.03 mL, 0.4 mmol) was added. After 10 min of stir-
ring, 4-nitrophenol or 4-methylthiophenol (1.2 equiv) was added
and the solution was stirred at room temperature for 1.5–3.5 h.
The reaction mixture was washed with satd aq NaHCO3 and then
with brine. The organic solution was dried (Na2SO4) and concen-
trated. The residue was purified by column chromatography with
the solvent specified in each individual case.
4.2.3.1. 4-Nitrophenyl 2,3,6-tri-O-acetyl-5-deoxy-a,b-L-arabino-
hexofuranoside (17). The syrup obtained by SnCl4-promoted
glycosylation of 16 with 4-nitrophenol was purified by column chro-
matography (hexane/EtOAc 7:3), and fractions of Rf = 0.63 (hexane/
EtOAc, 1:1) afforded an amorphous solid (0.09 g, 84%) characterized
4.2. Synthesis
4.2.1. Methyl 2,3,6-tri-O-acetyl-5-deoxy-
anoside (15)
a,b-
L-arabino-hexofur
as an a/b-anomeric mixture of 17 in a 5:1 ratio. For the a
-anomer: 1H
NMR (CDCl3, 500 MHz) d 8.18 (d, 2H, J = 9.2 Hz, H-aromatic), 7.11 (d,
2H, J = 9.2 Hz, H-aromatic), 5.71 (s, 1H, H-1), 5.34 (d, 1H, J = 1.8 Hz,
H-2), 5.03 (dd, 1H, J = 5.1, 1.8 Hz, H-3), 4.28–4.25 (m, 1H, H-4),
4.24–4.20 (m, 1H, H-6a), 4.15–4.10 (m, 1H, H-6b), 2.14–1.98 (m,
To a solution of methyl 5-deoxy-a,b-L-arabino-hexofuranoside
(14, 0.41 g, 2.30 mmol)32 in anhydrous CH2Cl2 (30.0 mL) cooled
to 0 °C, pyridine (1.6 mL, 19.8 mmol) and acetic anhydride

5344
A. Bordoni et al. / Bioorg. Med. Chem. 18 (2010) 5339–5345
11H, H-5a, H-5b, 3 COCH3); 13C NMR (CDCl3, 125.8 MHz): d 170.7,
170.07, 169.6 (COCH3); 160.9 (C-10), 142.6 (C-40), 125.7 (C-30),
116.5 (C-20), 103.9 (C-1), 81.4 (C-2), 80.9 (C-4), 78.5 (C-3), 60.6 (C-
6), 32.0 (C-5), 20.8, 20.7, 20.6 (COCH3). For the b-anomer: 1H NMR
(CDCl3, 500 MHz) d 8.18 (d, 2H, J = 9.2 Hz, H-30), 7.11 (d, 2H,
NaOMe/MeOH (1.0 mL) was added and after 30 min of stirring it
was diluted with methanol and concentrated under reduced pres-
sure to two thirds of the volume. The solution was deionized by
elution with MeOH through a column with Amberlite IR-120 plus
cation-exchange resin (H+) and purified as indicated in each case.
´
J = 9.2 Hz, H-2), 5.94 (d, 1H, J = 4.5 Hz, H-1), 5.45 (dd, 1H, J = 6.7,
5.4 Hz, H-3), 5.22 (dd, 1H, J = 4.5, 6,7 Hz, H-2), 4.15–4.10 (m, 1H, H-
4), 4.09–4.04 (m, 1H, H-6a), 4.01–3.96 (m, 1H, H-6b), 2.14–1.98
(m, 10H, H-5a, 3 COCH3), 1.94–1.86 (m, 1H, H-5b); 13C NMR (CDCl3,
125.8 MHz): d 170.3, 170.16, 170.02 (COCH3), 161.1 (C-10), 142.7 (C-
40), 125.8 (C-30), 116.6 (C-20), 98.1 (C-1), 79.3 (C-4), 78.1 (C-3), 76.65
(C-2), 60.5 (C-6), 34.1 (C-5), 20.8, 20.7, 20.6 (COCH3). HRMS (ESI+)
calcd for C18H21NNaO10 [M+Na]+ 434.10632, found: 434.10584.
4.2.4.1. 4-Nitrophenyl 5-deoxy-
a
-
L
-arabino-hexofuranoside (4-
nitrophenyl 5-deoxy-b- -galactofuranoside, 19
D
a
). Compound
17 (0.04 g, 0,098 mmol) was O-deacetylated with NaOMe (0.35 M,
1.0 mL) in CH2Cl2 (3 mL) as described above. Column chromatogra-
phy (toluene/EtOAc 95:5) afforded a compound 19 (19.2 mg, 67%),
Rf = 0.33(EtOAc), whichcrystallizedfromwatergavepure a-anomer
(89%), mp 90–92 °C, [
a
]
ꢁ105 (c 0.3, MeOH). 1H NMR (CD3OD,
D
500 MHz): d 8.21 (ddd, 2H, J = 10.4, 5.8, 3.3 Hz, 1H, H-30), 7.19
(ddd, 2H, J = 10.4, 5.8, 3.3 Hz, 1H, H-20), 5.64 (s, 1H, H-1), 4.58 (dd,
J = 4.3, 1,8 Hz, 1H, H-2), 4.08 (ddd, 1H, J = 8.1, 7.0, 4.7 Hz, 1H, H-4),
3.82 (dd, J = 7.0, 4.3 Hz, 1H, H-3), 3.71 (ddd, J = 10.8, 6.9, 5.2 Hz, 1H,
H-6a), 3.64 (ddd, J = 10.8, 7.9, 6.2 Hz, 1H, H-6b), 1.96 (dddd,
J = 14.0, 7.9, 6.9, 4.7 Hz, 1H, H-5a), 1,87 (dddd, J = 14.0, 8.1, 6.2,
4.2.3.2. 4-Methylphenyl 2,3,6-tri-O-acetyl-5-deoxy-1-thio-
a,b-
L
-arabino-hexofuranoside (18). Compound 18 was obtained from
16 according to the general procedure of SnCl4-promoted gly-
cosylation with 4-methylthiophenol. After purification by column
chromatography (hexane/EtOAc, 85:15) amorphous solid com-
pound 18 (0.16 g, 87%) was obtained as an inseparable anomeric
13
5.2 Hz, 1H, H-5b); C NMR (CD3OD, 125.8 MHz): d 164.4 (C-10),
mixture (a/b 10:9), Rf = 0.53 (hexane/EtOAc 6:4). For the
a
-anomer:
144.4 (C-40), 127. 5 (C-30), 118.5 (C-20), 108.5 (C-1), 84.9 (C-2), 83.6
(C-4), 83.5 (C-3), 60.5 (C-6), 38.2 (C-5). HRMS (ESI+) calcd for
1H NMR (CDCl3, 500 MHz) d 7.40 (d, 2H, J = 9.2 Hz, H-aromatic), 7.38
(d, 2H, J = 9.2 Hz, H-aromatic), 5.39(dd, 1H, 3J = 2.0 Hz, 4J = 0.6 Hz, H-
1), 5.22 (t, 1H, J = 2.2 Hz, H-2), 4.96 (ddd, 1H, J = 5.9; 2.4 Hz,
4J = 0.6 Hz, H-3), 4.38 (ddd, 1H, J = 8.5, 5.8, 4.6 Hz, H-4), 4.23–4.19
(m, 1H, H-6a), 4.17–4.14 (m, 1H, H-6b), 2.33 (s, 3H, CH3Ar), 2.17,
2.12, 2.04 (3s, 9H, COCH3), 2.19–2.00 (m, 2H, H-5a, H-5b); 13C NMR
(CDCl3, 125.8 MHz): d 170.9, 170.2, 169.7 (COCH3), 138.1, 132.8,
130.1, 129.9 (C-aromatic), 90.8 (C-1), 82.0 (C-2), 80.0 (C-3), 78.5
(C-4), 60.9 (C-6), 32.7 (C-5), 21.1, 21.0, 20.8, 20.7 (3 COCH3, CH3Ar).
For the b-anomer: 1H NMR (CDCl3, 500 MHz) d 7.40 (d, 2H,
J = 9.2 Hz, H-aromatic), 7.11 (d, 2H, J = 9.2 Hz, H-aromatic), 5.53 (d,
1H, J = 4.9 Hz, H-1), 5.46 (dd, 1H, J = 4.9, 3.0 Hz, H-2), 5.09 (dd, 1H,
J = 3.7, 3.0 Hz, H-3), 4.29–4.25 (m, 1H, H-6a), 4.23–4.19 (m, 1H, H-
6b), 3.99 (ddd, 1H, J = 8.0, 5.0, 3.9 Hz, H-4), 2.33 (s, 3H, COCH3),
C
12H15NO7Na+ [M+Na]+: 308.07407, found: 308.07459.
4.2.4.2. 4-Methylphenyl 5-deoxy-1-thio-
side (4-methylphenyl 5-deoxy-1-thio-b-
20 ). A solution of 18 (0.14 g, 0.35 mmol) in anhydrous CH2Cl2
a-L-arabino-hexofurano-
D
-galactofuranoside,
a
(10 mL) was O-deacetylated with 1.4 M NaOMe/MeOH (0.75 mL)
according to the general procedure. Column chromatography
(toluene/EtOAc 15:85) afforded compound 20
a (0.08 g, 85%),
Rf = 0.48 (EtOAc), which was recrystallized (92%) from hexane/
EtOAc (3:2), mp 89–91 °C, [
a
]
D ꢁ312 (c 1, MeOH). 1H NMR (CD3OD,
500 MHz): d 7.41 (d, 2H, J = 8.6 Hz, H-aromatic); 7.15 (d, 2H,
J = 8.6 Hz, H-aromatic), 5.19 (d, 1H, J = 4.5 Hz, H-1), 4.01 (ddd, 1H,
J = 12.5, 8.1, 5.4 Hz, H-4), 3.99 (dd, 1H, J = 5.2, 4.5 Hz, H-2), 3.76
(ddd, 1H, J = 12.2, 10.8, 4.3 Hz, H-6a), 3.72 (dd, 1H, J = 8.8, 5.4 Hz,
H-3), 3.72 (ddd, 1H, J = 10.8, 7.9, 6.2 Hz, H-6b), 2.34 (s, 3H, CH3Ar),
1.99 (dddd, 1H, J = 13.8, 12.2, 7.7, 4.5 Hz, H-5a), 1.84 (dddd, 1H,
J = 13.8, 8.2, 6.4, 5.2 Hz, H-5b); 13C NMR (CD3OD, 125.8 MHz): d
139.5, 134.2, 133.3, 131.4 (C-aromatic), 93.9 (C-1), 84.5 (C-2),
83.2 (C-3), 80.9 (C-4), 60.7 (C-6), 37.6 (C-5), 22.0 (CH3Ar). HRMS
2.09, 2.07, 2.03 (3s, 9H, COCH3), 2.10–2.00 (m, 2H, H-5a, H-5b). 13
C
NMR (CDCl3, 125.8 MHz): d 170.9, 170.2, 169.7 (COCH3), 138.1,
132.8, 130.1, 129.9 (C-aromatic), 89.5 (C-1), 80.5 (C-4), 80.0 (C-3),
77.6 (C-2), 61.1 (C-6), 31.4 (C-5), 21.1, 21.0, 20.84, 20.83 (3 COCH3,
CH3Ar). HRMS (ESI+) calcd for C19H24O7SNa+ [M+Na]+: 419.11409,
found: 419.11349.
(ESI+) calcd for
293.08180.
C
13H19O4SNa+ [M+Na]+: 293.08267; found:
4.2.3.3. 4-Methylphenyl 2,3,5,6-tetra-O-benzoyl-1-thio-a,b-D-
galactofuranoside (22). The SnCl4-promoted procedure was fol-
lowed, startingfrom 2148 and 4-methylthiophenol. Column chroma-
tography (toluene) afforded thioglycoside 22 (0.60 g, 83%), Rf = 0.72
4.2.4.3. 4-Methylphenyl 1-thio-b-D-galactofuranoside (23b). Depro-
tection of 22 (0.39 g, 0.55 mmol) in anhydrous CH2Cl2 (15 mL) with
1.4 M NaMeO (1.25 mL) according to the general procedure, led to
compound 23 (0.15 g, 98%) which recrystallized from hexane/
(toluene/EtOAc 9:1), as an inseparable anomeric mixture (b/a 9:1).
For the b-anomer: 1H NMR (CDCl3, 500 MHz) d 8.08–7.06 (m, 20H,
H-aromatic), 6.11–6.09 (m, 1H, H-5), 5.78 (s, 1H, H-1), 5.69 (d, 1H,
J = 5.0 Hz, H-3), 5.65 (s, 1H, H-2), 4.96 (t, 1H, J = 4.4 Hz, H-4), 4.76
(dd, 1H, J = 4.5, 11.8 Hz, H-6a), 4.71 (dd, 1H, J = 6.9, 11.8 Hz, H-6b),
2.35 (s, 3H, CH3Ar); 13C NMR (CDCl3, 125.8 MHz): d 166.0, 165.7,
165.3, 165.2 (COPh), 138.2–125.3 (C-aromatic), 91.6 (C-1), 82.3 (C-
2), 81.4 (C-4), 77.9 (C-3), 70.3 (C-5), 63.4 (C-6), 21.1 (CH3Ph). For
EtOAc (1:1) gave pure b-anomer, mp 124–126 °C, [
a
]
ꢁ253 (c
D
1.0, MeOH). 1H NMR (D2O, 500 MHz): d 7.46 (d, 2H, J = 8.8 Hz, H-
aromatic), 7.27 (d, 2H, J = 8.8 Hz, H-aromatic), 5.21 (d, 1H,
J = 5.9 Hz, H-1), 4.13 (dd, 1H, J = 7.8, 6.0 Hz, H-3), 4.02 (t, 1H,
J = 5.9 Hz, H-2), 3.88 (dd, 1H, J = 7.8, 3.3 Hz, H-4), 3.80 (ddd, 1H,
J = 7.6, 4.7, 3.3 Hz, H-5), 3.67 (dd, 1H, J = 11.7, 4.7 Hz, H-6a), 3.62
(dd, 1H, J = 11.7, 7.6 Hz, H-6b), 2.34 (s, 3H, CH3Ar); 13C NMR
(D2O, 125.8 MHz): d 140.0, 134.0, 130.7, 128.7 (C-aromatic), 90.9
(C-1), 82.0 (C-4), 80.3 (C-2), 76.1 (C-3), 70.9 (C-5), 63.4 (C-6),
20.9 (CH3Ar). Anal. Calcd for C13H18O5S: C, 54.53; H, 6.34. Found:
C, 54.47; H, 6.21.
the a
-anomer: 1H NMR (CDCl3, 500 MHz) d 8.08–7.06 (m, 20H, H-
aromatic), 6.04–5.99 (m, 1H, H-5), 5.93–5.92 (m, 2H, H-2, H-3),
5.78–5.77 (m, 1H, H-1), 4.86 (dd, 1H, J = 4.1, 12.0 Hz, H-6a), 4.78–
4.74 (m, 1H, H-6b), 4.54 (t, 1H, J = 4.8 Hz, H-4), 2.37 (s, 3H, CH3Ar).
13C NMR (CDCl3, 125.8 MHz): d 166.0, 165.5, 165.3, 165.2 (COPh),
138.2–125.3 (C-aromatic), 90.0 (C-1), 81.6 (C-4), 78.1 (C-2), 77.0
(C-3), 70.6 (C-5), 63.4 (C-6), 21.2 (CH3Ar). Anal. Calcd for
4.3. Enzymatic assays
C41H34O9S: C, 70.07; H, 4.88. Found: C, 69.80; H, 4.78.
The enzymatic activity was assayed using the filtered medium of
4.2.4. General procedure for O-deacylation
Compounds 17, 18, or 22 (0.1 mmol) were dissolved in anhy-
drous CH2Cl2 (3 mL) and cooled to 0 °C. Then, a solution 0.35 M
a stationary culture of P. fellutanum as source of b-
idase and 4-nitrophenyl b-
-galactofuranoside (1) as substrate18,24
The standard assay was conducted with 50 L of 66 mM NaOAc
D-galactofuranos-
D
l

A. Bordoni et al. / Bioorg. Med. Chem. 18 (2010) 5339–5345
5345
10. (a) Kremer, L.; Dover, L. G.; Morehouse, C.; Hitchin, P.; Everett, M.; Morris, H. R.;
buffer (pH 4.9), 15
tofuranoside, and 20
final volume of 250
11b, 13, 14 , 20 , and 23b were incorporated in the amounts re-
quired to obtain a final concentration of 0.2–2.5 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 spectrophotometrically at 410 nm.
l
L of a 5 mM solution of 4-nitrophenyl b-
L (4 g protein) of the enzyme medium, in a
L. For the evaluation as inhibitors, compounds
D-galac-
Dell, A.; Brennan, J.; McNeil, M. R.; Flaherty, C.; Duncan, K.; Besra, G. S. J. Biol.
l
l
ˇ
Chem. 2001, 276, 26430; (b) Belánová, M.; Dianišková, P.; Brennan, P. J.;
l
Completo, G. C.; Rose, N. L.; Lowary, T. L.; Mikušová, K. J. Bacteriol. 2008, 190,
1141; (c) Rose, N. L.; Completo, G. C.; Lin, S. J.; McNeil, M.; Palcic, M. M.; Lowary, T.
L. J. Am. Chem. Soc. 2006, 128, 6721; (d) Mikusová, K.; Yagi, T.; Stern, R.; McNeil, M.
R.; Besra, G. S.; Crick, D. C.; Brennan, P. J. J. Biol. Chem. 2000, 275, 33890.
11. Lederkremer, R. M.; Agusti, R. Adv. Carbohydr. Chem. Biochem. 2009, 62, 311.
12. Golgher, D. B.; Colli, W.; Souto-Padron, T.; Zingales, B. Mol. Biochem. Parasitol.
1993, 60, 249.
13. Cousin, M. A.; Notermans, S.; Hoogerhout, P.; Van Boom, J. H. J. Appl. Bacteriol.
1989, 66, 311.
14. Daley, L. S.; Ströbel, G. A. Plant Sci. Lett. 1983, 30, 145.
15. Rietschel-Berst, M.; Jentoft, N. H.; Rick, P. D.; Pletcher, C.; Fang, F.; Gander, J. E.
J. Biol. Chem. 1977, 252, 3219.
a
a
For the evaluation of 14
in replacement of 4-nitrophenyl b-
dard assay in the amount needed for a final concentration of 0.3–
2.7 mM.24 For compound 14
, release of the 5-deoxy galactose
was monitored by HPAEC-PAD analysis. For 19 , the 4-nitrophenol
a
and 19
a
as substrates, they were used
D
-galactofuranoside in the stan-
a
16. Gander, J. E.; Jentoft, N. H.; Drewes, L. H.; Rick, P. D. J. Biol. Chem. 1974, 249,
2063.
a
17. (a) Completo, G. C.; Lowary, T. L. J. Org. Chem. 2008, 73, 4513; (b) Gandolfi-
Donadío, L.; Gallo-Rodriguez, C.; Lederkremer, R. M. Carbohydr. Res. 2008, 343,
1870; (c) Riordan, J. M.; Reynolds, R. C. Bioorg. Med. Chem. 2007, 15, 5629; (d)
Pathak, A. K.; Pathak, V.; Seitz, L.; Gurcha, S. S.; Besra, G. S.; Riordan, J. M.;
Reynolds, R. C. Bioorg. Med. Chem. 2007, 15, 5629; (e) Bohn, M. L.; Colombo, M.
I.; Pisano, P. L.; Stortz, C. A.; Rúveda, E. A. Carbohydr. Res. 2007, 342, 2522; (f)
Euzen, R.; Ferrières, V.; Plusquellec, D. Carbohydr. Res. 2006, 341, 2759; (g) Lee,
Y. J.; Lee, B.-Y.; Jeon, H. B.; Kim, K. S. Org. Lett. 2006, 8, 3971; (h) Wen, X.; Crick,
D. C.; Brennan, P. J.; Hultin, P. G. Bioorg. Med. Chem. 2003, 11, 3579; (i) Gandolfi-
Donadio, L.; Gallo-Rodriguez, C.; Lederkremer, R. M. J. Org. Chem. 2002, 67,
4430; (j) Pathak, A. K.; Besra, G. S.; Crick, D.; Maddry, J. A.; Morehouse, C. B.;
Suling, W. J.; Reynolds, R. C. Bioorg. Med. Chem. 1999, 7, 2407; (k) McAuliffe, J.
C.; Hindsgaul, O. J. Org. Chem. 1997, 62, 1234.
18. Varela, O.; Marino, C.; Lederkremer, R. M. Carbohydr. Res. 1986, 155, 247.
19. Marino, C.; Cancio, M. J.; Varela, O.; Lederekremer, R. M. Carbohydr. Res. 1995,
276, 209.
20. Mariño, K.; Baldoni, L.; Marino, C. Carbohydr. Res. 2006, 341, 2886.
21. Mariño, K.; Marino, C.; Lederkremer, R. M. Anal. Biochem. 2002, 301, 325.
22. Mariño, K.; Marino, C.; Lima, C.; Baldoni, L.; Lederkremer, R. M. Eur. J. Org. Chem.
2005, 2958.
released was spectrophotometrically measured. One unit of activ-
ity was defined as the amount of 4-nitrophenol formed per hour
per microgram of protein at 37 °C. Km and Vm values were deter-
mined by Lineweaver–Burk plot. Controls without enzyme were
incubated in parallel.
4.4. HPAEC-PAD analysis
Analysis by HPAEC-PAD was performed with a Dionex ICS-3000
HPLC system with pulse amperometric detection (PAD), set at
30 nA and E1 = +0.05 V, E2 = +0.60 V, and E3 = ꢁ0.60 V. The column
used was a CarboPac MA-10 anion-exchange analytical column
(4 ꢀ 250 mm), equipped with a MA-10 guard column (5 ꢀ 50 mm).
The following program was used: 600 mM NaOH, isocratically, at a
flow rate of 0.4 mL/min: tR Gal = 20.2 min, tR 5-deoxy galact-
ose = 24.12 min, and tR 14a = 13.68 min.
23. Bordoni, A.; Lima, C.; Mariño, K.; Lederkremer, R. M.; Marino, C. Carbohydr. Res.
2008, 343, 1863.
24. Marino, C.; Mariño, K.; Miletti, L.; Manso Alves, M. J.; Colli, W.; Lederkremer, R.
M. Glycobiology 1998, 8, 901.
25. Marino, C.; Herczegh, P.; Lederkremer, R. M. Carbohydr. Res. 2001, 333, 123.
26. Mariño, K.; Marino, C. Arkivoc 2005, XII, 341.
27. Repetto, E.; Marino, C.; Uhrig, M. L.; Varela, O. Bioorg. Med. Chem. 2009, 17,
2703.
28. Marino, C.; Chiocconi, A.; Varela, O.; de Lederkremer, R. M. Carbohydr. Res.
1998, 311, 183.
29. Chiocconi, A.; Marino, C.; de Lederkremer, R. M. Carbohydr. Res. 2000, 323, 7.
30. Chiocconi, A.; Marino, C.; Otal, E.; de Lederkremer, R. M. Carbohydr. Res. 2002,
337, 2119.
31. Bordoni, A.; Lederkremer, R. M.; Marino, C. Carbohydr. Res. 2006, 341, 2286.
32. Bordoni, A.; Lederkremer, R. M.; Marino, C. Tetrahedron 2008, 64, 1703.
33. Baldoni, L.; Marino, C. J. Org. Chem. 2009, 74, 1994.
34. Miletti, L. C.; Marino, C.; Mariño, K.; Lederkremer, R. M.; Colli, W.; Alves, M. J.
M. Carbohydr. Res. 1999, 320, 176.
35. Miletti, L. C.; Mariño, K.; Marino, C.; Colli, W.; Alves, M. J. M.; Lederkremer, R.
M. Mol. Biochem. Parasitol. 2003, 127, 85.
Acknowledgments
The authors acknowledge financial support from the University
of Buenos Aires (Project X-130), the National Research Council of
Argentina (CONICET, Project PIP 1683), and the National Agency
for Promotion of Science and Technology (ANPCyT, Project 1107).
R.M.L. and C.M. are Research Members of CONICET, and A.B. was
supported by a fellowship from CONICET.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.05.038.
36. (a) Kirschning, A.; Jesberger, M.; Schöning, K. U. Synthesis 2001, 507; (b)
Lederkremer, R. M.; Marino, C. Adv. Carbohydr. Chem. Biochem. 2007, 61, 143;
(c) Hou, D.; Lowary, T. L. Carbohydr. Res. 2009, 344, 1911.
References and notes
37. Pathak, A. K.; Pathak, V.; Suling, W. J.; Riordan, J. R.; Gurcha, S. S.; Besra, G. S.;
Reynolds, R. C. Bioorg. Med. Chem. 2009, 17, 872.
38. Lopez, G.; Nugier-Chauvin, C.; Rémond, C.; O’Donohue, M. Carbohydr. Res. 2007,
342, 2202.
39. Lowary, T.; Hou, D. Org. Lett. 2007, 9, 4487.
40. Nishio, T.; Miyake, Y.; Tsujii, H.; Hakamata, W.; Kadokura, K.; Oku, T. Biosci.,
Biotechnol., Biochem. 1996, 60, 2038.
41. Kumar, A.; Doddi, V. R.; Vankar, Y. D. J. Org. Chem. 2008, 73, 5993.
42. Witczak, Z. J.; Chhabra, R.; Chen, H.; Xie, X.-Q. Carbohydr. Res. 1997, 301, 167.
43. Gelin, M.; Ferrières, V.; Plusquellec, D. Eur. J. Org. Chem. 2000, 1423.
44. (a) Lederkremer, R. M.; Marino, C.; Varela, O. Carbohydr. Res. 1990, 200, 227; (b)
Marino, C.; Varela, O.; Lederkremer, R. M. Carbohydr. Res. 1989, 190, 65.
45. (a) Gandolfi-Donadío, L.; Gallo-Rodriguez, C.; Lederkremer, R. M. J. Org. Chem.
2003, 68, 6928; (b) Mendoza, V. M.; Kashiwagi, G. A.; Lederkremer, R. M.;
Gallo-Rodriguez, C. Carbohydr. Res. 2010, 345, 385.
46. Bock, K.; Pedersen, C. Adv. Carbohydr. Chem. 1983, 41, 177.
47. Zunszain, P.; Varela, O. J. Chem. Res. 1995, (S) 486, (M) 2910.
48. Marino, C.; Gandolfi-Donadío, L.; Gallo-Rodriguez, C.; Bai, Y.; Lederkremer, R.
M. In Carbohydrate Chemistry: Proven Methods, Kovac, P., Ed.; CRC Press,
accepted for publication.
1. Crick, D. C.; Mahaprata, S.; Brennan, P. J. Glycobiology 2001, 11, 107.
2. (a) Besra, G. S.; Khoo, K. H.; McNeil, M. R.; Dell, A.; Morris, H. R.; Brennan, P. J.
Biochemistry 1995, 34, 4257; (b) Bhamidi, S.; Scherman, M. S.; Rithner, C. D.;
Prenni, J. E.; Chatterjee, D.; Khoo, K.-H.; McNeil, M. R. J. Biol. Chem. 2008, 283,
12992.
3. Lederkremer, R. M.; Colli, W. Glycobiology 1995, 5, 547.
4. McConville, M. J.; Collidge, T. A.; Ferguson, M. A.; Schneider, P. J. Biol. Chem.
1993, 268, 15595.
5. Turco, S. J.; Descoteaux, A. Annu. Rev. Microbiol. 1992, 46, 65.
6. Peltier, P.; Euzen, R.; Daniellou, R.; Nugier-Chauvin, C.; Ferrières, V. Carbohydr.
Res. 2008, 343, 1897.
7. (a) Lowary, T. L. Curr. Opin. Chem. Biol. 2003, 7, 749; (b) Richards, M. R.; Lowary,
T. L. ChemBioChem 2009, 10, 1.
8. Ahrazem, O.; Prieto, A.; San-Blas, G.; Leal, J. A.; Jiménez-Barbero, J.; Bernabé, M.
Glycobiology 2003, 13, 743.
9. (a) Sanders, D. A. R.; Staines, A. G.; MacMahon, S. A.; McNeil, M. R.; Whitfield,
C.; Naismith, J. H. Nat. Struct. Biol. 2001, 8, 858; (b) Beis, K.; Srikannathasan, V.;
Liu, H.; Fullerton, S.; Bamford, V. A.; Sanders, D. A. R.; Whitfield, C.; McNeil, M.
R.; Naismith, J. H. J. Mol. Biol. 2005, 348, 971; (c) Beverley, S. M.; Owens, K. L.;
Showalter, M.; Griffith, C. L.; Doering, T. L.; Jones, V. C.; McNeil, M. R. Eukaryot.
Cell 2005, 4, 1147; (d) Dykhuizen, E. C.; May, J. F.; Tongpenyai, A.; Kiessling, L. L.
J. Am. Chem. Soc. 2008, 130, 6706.
49. Tuekam, B. A.; Park, Y.-I.; Unkefer, C. J.; Gander, J. E. Appl. Environ. Microbiol.
2001, 67, 4648.
50. BeMiller, J. N. Adv. Carbohydr. Chem. 1967, 22, 25.