Facile synthesis of methyl α- and β-d-[6-3H]galactofuranosides from d-galacturonic acid. Substrates for the detection of galactofuranosidases

Article abstract of DOI:10.1016/j.carres.2008.02.019

Galactofuranose metabolism is a good target for the development of novel chemotherapeutic agents for the treatment of some microbial infections. A simple procedure for the synthesis of methyl (methyl α,β-d-galactopyranosid)uronate followed by NaB³H₄ reduction gave a straightforward access to radiolabeled substrates for galactofuranosidases.

Full text of DOI:10.1016/j.carres.2008.02.019

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 1863–1869
Note
Facile synthesis of methyl a- and b-D-[6-3H]galactofuranosides
from D-galacturonic acid. Substrates for the detection
of galactofuranosidases
Andrea Bordoni, Carlos Lima, Karina Marino, Rosa M. de Lederkremer
˜
and Carla Marino*
CIHIDECAR (CONICET), Departamento de Quı´mica Orga´nica, Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, Pabello´n II, Ciudad Universitaria, 1428 Buenos Aires, Argentina
Received 15 January 2008; received in revised form 19 February 2008; accepted 23 February 2008
Available online 29 February 2008
Abstract—Galactofuranose metabolism is a good target for the development of novel chemotherapeutic agents for the treatment of
some microbial infections. A simple procedure for the synthesis of methyl (methyl a,b-D-galactopyranosid)uronate followed by
NaB3H4 reduction gave a straightforward access to radiolabeled substrates for galactofuranosidases.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: D-Galacturonic acid; Galactofuranosides; Galactofuranosidases; Penicillium varians; Methyl glycofuranosides
Galactofuranosyl (Galf) residues are present in glyco-
conjugates of many pathogenic bacteria, fungi, and pro-
tozoan parasites, and are highly immunogenic.1–3 The
fact that Galf is essential for the survival or virulence
of various pathogenic bacteria3,4 but is absent in higher
eukaryotes has generated great interest in its biosyn-
thetic pathways. Labeled substrates are important as
they are sensitive tools for the characterization of
enzymes. Although the metabolism of b-D-galacto-
furanosides has been well characterized,3–9 the a-D-Galf
biosynthetic and metabolic machinery still remain
unclear. The description of the enzymes involved in its
metabolism is important considering that glycoconju-
gates containing a-D-Galf residues have been described
in several pathogenic microorganisms including bacte-
ria, (Escherichia coli,10 Streptococcus pneumoniae,11
and Hafnia alvei12) and fungi (Paracoccidioides brasilien-
sis, causative agent of paracoccidioidomycosis.13) Peni-
cillium varians, a nonpathogenic fungus synthesizes
varianose, an extracellular (cell wall) polysaccharide
with the repeating unit I.14,15
→
6)-β-D-Galf-(1→5)-β-D-Galf-(1
2
1
α-D-Glcp-(1→2)-α-D-Galf
I
The role of varianose during fungal growth is still
uncertain but in Penicillium fellutanum, another filamen-
tous fungus, the extracellular glycopeptide peptidophos-
phogalactomannan (pPGM) has been described as a
reserve source of phosphate, choline, and/or carbo-
hydrates.16 These nutrients are released by enzyme-cata-
lyzed depolymerization and, among other hydrolases, a
b-D-galactofuranosidase is involved in this degrada-
tion.17 By analogy with the physiological role of pPGM
in P. fellutanum, the presence of enzymes with the specific
role of hydrolyzing varianose could be proposed in P. var-
ians. b-D-Galactofuranosidase activity has been detected
in Penicillium funiculosum,18 then called P. varians.19
Degradation of varianose would require the presence of
an a-D-galactofuranosidase and an a-D-glucosidase.
The synthesis of methyl a-D-[6-3H]galactofuranoside
(5a*) reported in the present study affords a sensitive sub-
strate for the detection of a-D-galactofuranosidases.
*
Corresponding author. Tel./fax: +54 11 4576 3352; e-mail:
cmarino@qo.fcen.uba.ar
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.02.019

1864
A. Bordoni et al. / Carbohydrate Research 343 (2008) 1863–1869
As part of our ongoing project on the development of
1). The pyranose glycosides were not detected by
NMR spectroscopy.
synthetic tools for the characterization of the enzymes
involved in the galactofuranosyl metabolism,20–24 we
have previously developed a synthetic sequence for the
introduction of a tritium label at the 6-position of
galactofuranose derivatives. The key step was the oxida-
tion of the C-6 hydroxyl group of a conveniently pre-
pared derivative with pyridinium chlorochromate,
followed by the reduction with NaB3H4. Using this
sequence, methyl b-D-[6-3H]galactofuranoside (5b*)21
and UDP-b-D-[6-3H]galactofuranoside (UDP-Galf*)23
were obtained. We have shown the usefulness of 5b*
for the detection of the known exo-b-D-galactofuranosi-
dase from P. fellutanum21 and combined with other
synthetic tools, the compound also allowed us to detect,
for the first time, exo-b-D-galactofuranosidase activity in
Trypanosoma cruzi. In the latter case, the availability of
the radioactive material was essential for the detection
because the colored biological material, in addition to
the low level of enzyme activity, precluded the use of
colorimetric assays.22
We envisioned using D-galacturonic acid (1) as a con-
venient starting material for the preparation of 5a*. It
was previously described that by heating 1 with
methanol under reflux in the presence of a cation
exchange resin (H+ form), methyl (methyl a-D-galacto-
pyranosid)uronate (4) was obtained. However, the same
reaction at room temperature for 8 h afforded methyl
D-galacturonate 225 (Scheme 1).
In our case, the treatment of 1 with methanol in the
presence of Amberlite IR-120H for 8 h at 35 °C in an
orbital shaker afforded the furanosidic isomer 3 as an
a:b mixture in a 3:1 ratio, but a significant proportion
of the nonglycosylated product, 2, was still present.
After keeping the reaction for 48 h, the NMR spectra
showed that the furanosidic derivative 3 was the only
product, in a 2.6:1 b:a ratio as deduced by the integra-
tion of the well-resolved H-5 doublets (Table 1, entry
These results suggest that esterification of the carb-
oxylic group, which proceeds faster than glycosylation,26
favors the formation of the furanosidic ring. For com-
parison, we performed the same reaction with D-galact-
ose. We observed by NMR spectroscopy that the crude
product was composed mainly of the methyl b-D-galac-
tofuranoside (5b), and minor amounts of the a-furanos-
idic and the a,b-pyranosidic isomers (Table 1, entry 2).
Using column chromatography, compound 5b was iso-
lated in 60% yield. The preference of galactose to afford
the furanosidic form by acid catalysis in methanol is well
known. By the traditional Fisher glycosylation, 53% of
methyl b-D-Galf was obtained after chromatographic
purification.27 By comparison, the procedure using resin
described here has the advantage of an easier work-up.
As methyl b-D-Galf is widely used as a starting material
in the synthesis of galactofuranose containing mole-
cules,28 we consider this to be a valuable method.
By treating glucose with methanol in the presence of
Amberlite IR-120H, a mixture composed by the four
glycosides was obtained, but the reaction was not
Table 1. Methyl glycosides formed by the [H+]-resin catalyzed
procedure
Entry
Saccharide
Product distribution (%)
b-f
a-f
b-p
a-p
1
2
D-Galacturonic acid
D-Galactose
71a
62b
68a
60b
27a
11a
29a
28b
17a
—
—
6a
9a
3
4
D-Glucosec
D-Mannose
29a,d
10a
66a
3a
20a
a Determined by NMR spectroscopy.
b Isolated pure products after column chromatography.
c 34% of unreacted glucose was detected.
d Corresponds to the integration of the signals of the a-f and b-p
glycosides, which were not resolved.
OCH3
O
OH
O
OH
HO
OH
COCH
3O
CH2OH
Amberlite IR120 (H+)
MeOH
O
+
OCH3
OH
HO
OH
HO
OH
OH
OH
OH
OH
CO2CH3
OH
CO2CH3
1
2
3β
3α
NaB3H4, MeOH
OCH3
HO
O
OH
O
OH
COCH
3O
+
OCH3
OH
HO
OH
OCH3
OH
OH
OH
C3H2OH
C3H2OH
4
5β∗
5α∗
Scheme 1.

A. Bordoni et al. / Carbohydrate Research 343 (2008) 1863–1869
1865
complete after 48 h (Table 1, entry 3). As expected,
mannose led to methyl a-D-Manp as the major product
(Table 1, entry 4). For the synthesis of D-glycofurano-
siduronic acids and D-glycofuranosides of long chain
alcohols, a stereospecific procedure involving the
complexation of the C-5 and C-6 hydroxyl groups with
alkaline-earth cations was described previously.29 Our
results confirm that the configuration at C-4 of galactose
5α
5β
Gal
A
3000
2500
2000
1500
1000
500
0
0
5
10
15
20
25
30
Time (minutes)
5β
5α
Gal
B
C
150 0
10 0 0
500
0
0
5
10
15
20
25
30
Time (minutes)
5β
5α
Gal
2000
1500
1000
500
0
0
5
10
15
20
25
30
Time (minutes)
5β
5α
Gal
D
2000
1500
1000
500
0
0
5
10
15
20
25
30
Time (minutes)
Figure 1. HPAEC-PAD analysis of [6-3H]methyl-b-D-galactofuranoside (5b*) and [6-3H]methyl-a-D-galactofuranoside (5a*) and enzymatic assays.
(A) 5b*, (B) 5a*, (C) enzymatic assay on P. varians medium cultures of 30 days, using 5a* as a substrate, (D) enzymatic assay on P. varians medium
cultures of 30 days, using 5b* as a substrate. A carboPac MA-1 column with the conditions indicated in Section 1 was used. Authentic samples of
methyl b-D-Galf (5b), methyl a-D-Galf (5b) and galactose were used.

1866
A. Bordoni et al. / Carbohydrate Research 343 (2008) 1863–1869
is responsible for the preference for the furanosidic
forms, and that no complexation of the C-5/C-6 diol is
necessary, at least in methanol.
In summary, conditions to afford furanosidic deriva-
tives of D-galacturonic acid and D-galactose in high yield
by the reaction of the free monosaccharides with meth-
anol in the presence of Amberlite IR-120H have been
described. This is an easy methodology, which combined
with the reduction of the carboxylate ester group of 3
with NaB3H4 constitutes a direct strategy for accessing
to [3H]-labeled galactofuranosides.
The simplicity of the procedure for the preparation of
3, followed by the reduction of the ester group consti-
tutes a direct strategy for accessing [3H]-labeled galacto-
furanosides for the studies of galactofuranosidases. It is
well known that the reduction of carboxylic esters with
NaBH4 is usually slow, but that the presence of an
heteroatom at the vicinal position favors the reaction
as it has been observed in aldonolactones.30 In the case
of 3, we observed that complete reduction occurred by
the use of an excess of NaBH4 (3 equiv) in methanol
at room temperature for 3 h. The influence of the hydro-
xyl group at the a-position is evidenced by the fact that
the 5-deoxy analogue of 3b could not be reduced under
similar conditions, and the reactivity of the NaBH4 had
to be enhanced by the addition of I2, as we have recently
reported.24 Compounds 5a and 5b were analyzed by
high performance anion-exchange chromatography with
pulse amperometric detection (HPAEC-PAD), and con-
ditions for good resolution were established (Fig. 1, see
Section 1).
1. Experimental
1.1. General methods
Thin-layer chromatography (TLC) was performed on
0.2 mm Silica Gel 60 F254 (Merck) aluminum supported
plates. Detection was effected by exposure to UV light
and by spraying with 10% (v/v) H2SO4 in EtOH and
charring. Column chromatography was performed on
Silica Gel 60 (200–400 mesh, Merck). NMR spectra
were recorded with a Bruker AC 200 spectrometer at
200 MHz (1H) and 50 MHz (13C) or with a Bruker
AM 500 spectrometer at 500 MHz (1H) and 125 MHz
(13C). Melting points were determined with a Fisher-
Johns apparatus and are uncorrected. Optical rotations
were measured with a Perkin–Elmer 343 polarimeter,
with a path length of 1 dm.
Reduction of 3b by using NaB3H4, afforded 5b* in a
significantly shorter way (Fig. 1, panel A). Although
the stereoselectivity in favor of 3a is poor, the efficiency
of the chromatographic separation from 3b and the
simplicity of the route for introducing the tritium label
favor its use. Next, compound 3a was reduced in a
methanolic solution of NaB3H4, affording 5a*, which
showed the same chromatographic properties as nonra-
dioactive 5a (Fig. 1, panel B).
1.2. General procedure for methylation promoted by
cation exchange resin
A suspension of the substrate (5.55 mmol) in MeOH
(20.0 mL) with 1.0 g of Amberlite IR-120H was stirred
at 35 °C in an orbital shaker at 120 rpm for 48 h.
After filtration, the solvent was removed under dimin-
ished pressure. The following products were thus
obtained:
With compound 5a* in hand as a new substrate for
a-D-galactofuranosidase detection, and using other
substrates previously developed,20,31,32 we investigated
enzyme activities in P. varians cultures. The culture med-
ium of different ages was filtered, dialyzed, lyophilized,
and used for the assays. The presence of b-D-galactof-
uranosidase activity was evaluated using 4-nitrophenyl
b-D-galactofuranoside as a substrate.20,31 The activity
detected was in accordance with the results previously
reported.18 Using 4-nitrophenyl a-D-glucopyranoside
as a substrate,33 no a-glucosidase activity was detected.
The radiolabeled substrates 5b* and 5a* were used under
the conditions described for the detection of b-D-galacto-
furanosidase activity in P. fellutanum21 and the activity
was followed by HPAEC-PAD. No a-D-galactofurano-
sidase activity was detected under the conditions used
in this study (Fig. 1, panel C). However, the effectiveness
of the radiolabeled substrates was demonstrated by the
detection of b-galactofuranosidase in a culture medium,
by the release of radioactive galactose from 5b (Fig. 1,
panel D). It cannot be ruled out that other conditions
(pH, temperature) are required for the activity of an
a-galactofuranosidase or if an endo-enzyme is acting in
P. varians.
1.2.1. Methyl (methyl b-D-galactofuranosid)uronate
(3b). TLC examination of the crude product from D-
galacturonic acid (1.00 g) showed a main component
with Rf = 0.41 (EtOAc, twice developed) and a lower
moving product (Rf = 0.36). NMR analysis showed
the presence of 3 in a b/a ratio of 2.6:1 as indicated by
the integration of the H-5 signals. The syrup (1.11 g)
was purified by column chromatography (49:1 EtOAc–
toluene). Fractions of Rf = 0.48 afforded syrupy 3b
(0.71 g, 62%), [a]D À125 (c 1, MeOH); lit.34 [a]D À112
1
(c 1.38, MeOH); H NMR (500 MHz, D2O): d 4.89 (d,
J = 1.9 Hz, 1H, H-1), 4.50 (d, J = 2.7 Hz, 1H, H-5),
4.29 (dd, J = 2.7, 6.5 Hz, 1 H, H-4), 4.18 (dd, J = 3.9,
6.5 Hz, 1H, H-3), 4.03 (dd, J = 1.9, 3.9 Hz, 1H, H-2),
3.81 (s, 3 H, OCH3), 3.38 (s, 3H, CO2CH3); 13C NMR
(50.3 MHz, D2O): d 174.2 (C-6), 109.0 (C-1), 84.1, 81.0
(C-2,C-4), 76.3 (C-3), 70.0 (C-5), 55.6 (COOCH3), 53.5
(OCH3).

A. Bordoni et al. / Carbohydrate Research 343 (2008) 1863–1869
1867
1.2.2. Methyl (methyl a-D-galactofuranosid)uronate
(3a). Fractions of Rf = 0.36 afforded methyl (methyl
a-D-galactofuranosid)uronate (3a, 0.31 g, 28%). Recrys-
tallized from 1:1 hexane–EtOAc it gave mp 63–64 °C,
[a]D +95 (c 1, MeOH); lit.34 mp 64–65 °C, [a]D +93 (c
3 h, TLC analysis showed complete consumption of
the starting material (14:1:1 nPrOH–NH3–H2O). The
solution was deionized through a column of Amberlite
IR-120H resin by elution with MeOH. Evaporation of
the solution under reduced pressure and several co-evap-
orations with MeOH afforded a syrup, which was puri-
fied by column chromatography (100:7 EtOAc–MeOH).
The following compounds were thus obtained:
Methyl b-D-galactofuranoside (5b), 0.66 g, 82%, with
the same chromatographic and spectroscopic properties
as an authentic sample of 5b.35,36
1
1.17, MeOH); H NMR (500 MHz, D2O): d 4.85 (d,
J = 4.8 Hz, 1H, H-1), 4.41 (d, J = 3.8 Hz, 1H, H-5),
4.18 (apparent t, J = 9.5 Hz, 1H, H-3), 4.14 (dd,
J = 4.7, 8.4 Hz, 1H, H-2), 4.12 (dd, J = 3.8, 7.5 Hz,
1H, H-4), 3.80 (s, 3H, OCH3), 3.41 (s, 3H, CO2CH3);
13C NMR (125.8 MHz, D2O): d 174.4 (C-6), 102.6
(C-1), 82.3 (C-4), 76.5 (C-2), 73.8 (C-3), 70.9 (C-5),
56.1 (COOCH3), 53.6 (OCH3).
Methyl a-D-galactofuranoside (5a), 0.63 g, 78%, with
the same chromatographic and spectroscopic properties
as those reported.27,36
1.2.3. Other glycosylation reactions.
1.2.3.1. D-Galactose. TLC analysis of the crude
product showed four spots, which were identified as
methyl b-D-Galf, methyl a-D-Galf, and methyl a,b-D-
Galp. The mixture was purified by column chromato-
graphy (100:7?100:12, EtOAc–MeOH). Fractions of
Rf = 0.59 (14:1:1 nPrOH–NH3–H2O) afforded methyl
b-D-galactofuranoside (0.64 g, 60%), [a]D +110 (c 1,
water); in agreement with data from the literature.27,36
Fractions of Rf = 0.55 corresponded to methyl a-D-
Galf,35 which could not be well separated from the pyr-
anose forms.
1.4. Reduction methyl (methyl D-galactofuranosid)uro-
nates (3) with NaB3H4
The substrate (2.2 mg, 9.9 lmol) was reduced in MeOH
(0.3 mL) with 10 mCi of NaB3H4 in 0.1 M KOH. The
mixture was left overnight at room temperature, and
then solid NaBH4 (2 mg, 0.05 mmol) was added. After
4 h, the solution was decationized as described in Sec-
tion 1.3 and concentrated under reduced pressure. The
material was further deionized through a IWT TMD-8
(mixed form) resin column, and the solvent was
evaporated.
1.2.3.2. D-Glucose and D-mannose. The crude syrups
were analyzed by NMR spectroscopy36 (Table 1, entries
3 and 4).
1.5. HPAEC-PAD analysis
Analysis by HPAEC-PAD was performed with a Dio-
nex ICS-3000 HPLC system with pulse amperometric
detection (PAD), set at 30 nA and E1 = + 0.05 V,
E2 = +0.60 V, and E3 = À0.60 V. The column used
was a CarboPac MA-10 anion-exchange analytical
column (4 Â 250 mm), equipped with a MA-10 guard
column (5 Â 50 mm). The following program was used:
1.3. Reduction of methyl (methyl D-galactofuran-
osid)uronates (3) with NaBH4
To a suspension of the substrate (0.92 g, 4.17 mmol) in
MeOH (15 mL), NaBH4 (0.41 g, 12.69 mmol) was added
in 4 portions (4 Â 0.12 g) every 0.5 h. After stirring for
600
400
200
0
1,2
1
0.8
0.6
0.4
0.2
0
0
5
10
15
20
25
30
Time (culture days)
Figure 2. Enzymatic activity in filtered and lyophilized culture media from Penicillium varians.
b-D-galactofuranosidase: determined using 4-
–
nitrophenyl b-D-galactofuranoside as a substrate.
a-D-glucosidase: monitored using 4-nitrophenyl a-D-glucopyranoside.
a-D-
–
–
galactofuranosidase: monitored using [6-3H]methyl-a-D-galactofuranoside (5a*) as a substrate. Each point is the mean obtained from three replicate
experiments.

1868
A. Bordoni et al. / Carbohydrate Research 343 (2008) 1863–1869
6. Sanders, D. A. R.; Staines, A. G.; MacMahon, S. A.;
MacNeil, M. R.; Whitfield, C.; Naismith, J. H. Nat.
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Biochemistry 2003, 42, 2104–2109.
600 mM NaOH, isocratically, at a flow rate of 0.4 mL/
min. Radioactivity in the fractions was determined in
a Rack-beta Wallack liquid scintillation counter using
a scintillation cocktail (Optiphase ‘Hisafe’ 3, LKB).
t
R 5a = 9.9 min,
tR 5b = 13.6 min, tR Man = 16.5 min,
8. Rose, N. L.; Completo, G. C.; Lin, S.-J.; McNeil, M.;
Palcic, M. M.; Lowary, T. L. J. Am. Chem. Soc. 2006, 128,
6721–6729.
tR Gal = 20.2 min, tR Glc = 18.2 min.
´ ˇ
´
ˇ
´
9. Belanova, M.; Dianiskova, P.; Brennan, P. J.; Completo,
1.6. Culture of P. varians
´
G. C.; Rose, N. L.; Lowary, T. L.; Mikusˇova, K. J.
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An inoculum of P. varians (CBS 386.48) was grown
under the conditions previously described14 with glucose
(5 g/L) as a carbon source. The filtered medium of
different ages was dialyzed (membrane tubing MWCO
8000), lyophilized, and used for the enzymatic assays
(Fig. 2).
´
´
Jimenez-Barbero, J.; Bernabe, M. Glycobiology 2003, 13,
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1.7. Enzyme assays
14. Haworth, W. N.; Raistrick, H.; Stacey, M. Biochem. J.
1935, 29, 2668–2678.
Exo-b-D-galactofuranosidase activity was measured by
15. Jansson, P.-E.; Lindberg, B. Carbohydr. Res. 1980, 82, 97–
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16. Gander, J. E.; Jentoft, N. H.; Drewes, L. H.; Rick, P. D. J.
Biol. Chem. 1974, 249, 2063–2072.
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19. Species fungorum. Global Species database. http://
www.speciesfungorum.org.
using
4-nitrophenyl
b-D-galactofuranoside31
or
[6-3H]methyl b-D-galactofuranoside,21 and a-D-glucosi-
dase activity was monitored by using 4-nitrophenyl a-
D-glucoside as a substrate.33 a-D-Galactofuranosidase
activity was tested by incubating the culture medium
(20 lL, 50 mg total protein) with 5a* (40,000 cpm) as
a substrate, 12 lL of 66 mM sodium acetate buffer
(pH 5) in a final volume of 15 lL, and incubated
overnight at 37 °C. The sample was centrifuged for
40 min at 10,000g through an Ultrafree-MC centrifu-
gal filter (MW 5000). The filtrate was analyzed by
HPAEC-PAD.
20. Miletti, L.; Marino, C.; Marino, K.; Lederkremer, R. M.;
˜
Colli, W.; Manso Alves, J. M. Carbohydr. Res. 1999, 320,
176–182.
21. Marino, K.; Marino, C.; Lederkremer, R. M. Anal.
˜
Biochem. 2002, 301, 325–328.
22. Miletti, L.; Marino, K.; Marino, C.; Colli, W.; Manso
˜
Alves, J. M.; Lederkremer, R. M. Mol. Biochem. Parasitol.
2003, 127, 85–88.
23. Marino, K.; Marino, C.; Lima, C.; Baldoni, L.; Leder-
˜
Acknowledgments
kremer, R. M. Eur. J. Org. Chem. 2005, 2958–
2964.
24. Bordoni, A.; Lederkremer, R. M.; Marino, C. Tetrahedron
2008, 64, 1703–1710 and references cited therein.
25. Cadotte, J. E.; Smith, F.; Spriestersbach, D. J. Am. Chem.
Soc. 1952, 74, 1501–1504.
We thank the Universidad de Buenos Aires, ANPCyT,
and CONICET for financial support. R.M.L. and
C.M. are research members of CONICET, and A.B.
was supported by a fellowship from CONICET.
26. (a) Jansen, E. F.; Jang, R. J. Am. Chem. Soc. 1946, 68,
1475–1477; (b) Larsson, K.; Petersson, G. Carbohydr. Res.
1974, 34, 323–329; (c) Vlahov, J.; Snatzke, G. Liebigs Ann.
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