The glycosyl-aldonolactone approach for the synthesis of β-d-Galf-(1→3)-d-Manp and 3-deoxy-β-d-xylo-hexofuranosyl-(1→3)-d-Manp

Article abstract of DOI:10.1016/S0008-6215(98)00214-6

A convenient synthesis of free β-d-Galf-(1→3)-d-Manp (8a) is reported. The disaccharide is present as external unit in the lipopeptidophosphoglycan (LPPG) of Trypanosoma cruzi and internally in the lipophosphoglycan (LPG) of Leishmania. Condensation of 2,

Full text of DOI:10.1016/S0008-6215(98)00214-6

Carbohydrate Research 311 (1998) 183±189
The glycosyl-aldonolactone approach for the
synthesis of ꢀ-d-Galf-(1!3)-d-Manp and
3-deoxy-ꢀ-d-xylo-hexofuranosyl-(1!3)-d-Manp
Carla Marino, Alejandro Chiocconi, Oscar Varela,
Rosa M. de Lederkremer *
Â
CIHIDECAR, Departamento de Quõmica Organica, Facultad de Ciencias Exactas y Naturales,
Â
Universidad de Buenos Aires, Pabellon II, Ciudad Universitaria, 1428 Buenos Aires, Argentina
Â
Received 24 April 1998; accepted 28 July 1998
Abstract
A convenient synthesis of free ꢀ-d-Galf-(1!3)-d-Manp (8a) is reported. The disaccharide is
present as external unit in the lipopeptidophosphoglycan (LPPG) of Trypanosoma cruzi and
internally in the lipophosphoglycan (LPG) of Leishmania. Condensation of 2,5,6-tri-O-benzoyl-d-
mannono-1,4-lactone (1) with 1,2,3,5,6-penta-O-benzoyl-d-galactofuranose, promoted by SnCl4,
led to the ꢀ-glycosyl-lactone, a key intermediate for disaccharide 8a, readily obtained by successive
reduction of the lactone with diisoamylborane and debenzoylation. As in the LPG of Leishmania
the HO-3 group of the galactofuranose is glycosylated by ꢁ-d-Galp, we also synthesized 3-deoxy-
ꢀ-d-xylo-hexofuranosyl-(1!3)-d-Manp (8b) and p-nitrophenyl 3-deoxy-ꢀ-d-xylo-hexofuranoside
for studying the in¯uence of HO-3 in the interaction with speci®c glycosidases. The disaccharide
8a, and its corresponding alditol, were good substrates for the ꢀ-d-galactofuranosidase from
Penicillium fellutanum, whereas the 3-deoxyglycosides were not hydrolyzed by the enzyme. # 1998
Elsevier Science Ltd. All rights reserved
Keywords: ꢀ-d-Galactofuranose disaccharides; Glycosyl-lactone; Deoxy sugars; ꢀ-d-Galactofuranosidase;
Trypanosoma cruzi; Leishmania
1. Introduction
present ꢀ-(1!3)-linked to ꢁ-mannose, either as a
terminal non-reducing unit, as in the lipopeptido-
phosphoglycan (LPPG) of T. cruzi [2,3], or inter-
nal, as in the Leishmania lipophosphoglycan (LPG)
[4]. As expected, since galactofuranose is absent in
mammalian glycoconjugates, it is antigenic [5].
Mutant cells of L. donovani, which were defective
in the biosynthetic step introducing the internal
galactofuranose, were destroyed by macrophages,
con®rming the role of galactofuranose for intra-
cellular survival of the parasite [6].
In recent years we have been interested in the
glycobiology of galactofuranose, particularly in
regard to its presence in Trypanosomatids [1].
Parasites of this family, such as Trypanosoma cruzi
and Leishmania species, are infectious to humans.
It is interesting that in the glycoinositolphos-
pholipid anchor-like structures, galactofuranose is
* Corresponding author. E-mail: varela@qo.fcen.uba.ar
0008-6215/98/$Ðsee front matter # 1998 Elsevier Science Ltd. All rights reserved
PII S0008-6215(98)00214-6

184
C. Marino et al./Carbohydrate Research 311 (1998) 183±189
Derivatives of ꢀ-d-Galf-(1!3)-ꢁ-d-Manp have
We have recently reported studies on the synth-
esis of inhibitors of the ꢀ-d-galactofuranosidase
from Penicillium fellutanum [15]. It was interesting
to explore the substrate speci®city of this enzyme
towards disaccharides 8a and 8b. As already
described, deoxy sugar derivatives are very useful
in de®ning the importance of each speci®c hydro-
xyl group for the interaction between carbohy-
drates and proteins [16].
been synthesized [7±10]. However, deprotection to
the free disaccharide was not reported in any of
these instances.
Glycosyl-lactones were previously used in our
laboratory as precursors of galactofuranose-con-
taining disaccharides [11,12]. Selectively sub-
stituted aldonolactones, used as glycosylating
agents for monosaccharides, are readily obtained
from the corresponding aldonolactones [13,14].
The lactone can be chemoselectively reduced to the
furanoid sugar. Complete deprotection results in
isomerization of the reducing end to the more
stable pyranose. Thus, in the present work, the
disaccharide ꢀ-d-Galf-(1!3)-d-Manp (8a) was
prepared in few steps.
Taking in consideration that the Galf is linked
through its HO-3 to ꢁ-d-Galp in the LPG of
Leishmania [4], we also synthesized the 3-deoxy
analogue 8b which, lacking the HO-3, could be
useful for studies on inhibition of the particular
glycosyltransferase involved in the construction of
the LPG glycan.
2. Results and discussion
In previous work, we showed that benzoylation
of d-mannono-1,4-lactone with 3.3 molar equiva-
lents of benzoyl chloride in pyridine a€orded tri-
benzoate 1 as the main product (60%), isolated by
column chromatography [13]. We have now facili-
tated the isolation and improved the yield of 1
(70%) by crystallization from benzene of the com-
pound from the crude benzoylation mixture.
Partially benzoylated aldonolactone 1 was a
useful precursor for the target disaccharides 8a and
Scheme 1. (i) SnCl4, Cl2CH2; (ii) NaBH4/MeOH, NaOMe/MeOH; (iii) DSB/THF; (iv) NaOMe/MeOH.

C. Marino et al./Carbohydrate Research 311 (1998) 183±189
185
8b, as the free HO-3 could be readily glycosylated
by using a Lewis acid catalyst, as previously
described for the preparation of other glycosyl-
lactones [11,12]. Thus, condensation of 1 with 2a in
the presence of SnCl4 gave the glycosyl-lactone 3a
in 85% yield. The anomeric con®guration of the
glycosidic linkage was established as ꢀ by means of
per-O-benzoylated derivatives of d-lyxofuranose,
the analogous pentose [20,21].
O-Debenzoylation of 5a with NaOMe gave the
pure disaccharide 8a in 70% overall yield from 2a.
1
The H NMR spectrum showed in the anomeric
region broad singlets for H-10 (5.15), H-1ꢁ (5.10),
and H-1ꢀ (4.86 ppm). This disaccharide constitutes
the terminal unit of the glycosidic chains in the
LPPG isolated from epimastigote forms of Trypa-
nosoma cruzi [2,3]. The unit for Galf-ꢀ(1!3)-Manp
is also present in the core oligosaccharide of LPG
of Leishmania, but in this case it is located in the
middle of the chain [4]. Although some derivatives
have been previously prepared, this is the ®rst
synthesis of free disaccharide 8a. The earlier
reports described the syntheses of the methyl gly-
coside [7], and the 8-methoxycarbonyloctyl [8]
derivatives of 8a, using the orthoester method.
McAuli€e and Hindsgaul [9] described the synthesis
of methyl 3-O-(2,3,5,6-tetra-O-acetyl-ꢀ-d-galacto-
furanosyl)-2-O-benzyl-4,6-O-benzylidene-ꢁ-d-man-
nopyranoside, starting from the peracetylated
dithioacetal of d-galactose. The same protected
disaccharide has also been prepared, using phenyl
1-selenogalactofuranoside as the glycosyl donor
[10].
the H NMR spectrum of 3a, which showed H-10
1
and H-20 signals as broad singlets, in accordance
with the 1,2-trans con®guration. The 13C NMR
spectrum showed signals at ꢂ 106.7 (C-10), 83.9 (C-40),
and 81.3 (C-20) corresponding to the ꢀ-galactofur-
anose unit, and the signals of the lactone carbons
were clearly assigned by comparison with those in
compound 1 [13]. In particular, the resonance of
C-3 appeared shifted down®eld (4.6 ppm), as
expected because of the glycosylation of HO-3.
Reduction of 3a with sodium borohydride, fol-
lowed by O-deacylation with sodium methoxide,
a€orded crystalline 3-O-ꢀ-d-galactofuranosyl-d-
1
mannitol (4). The H NMR spectrum of 4 showed
a broad singlet for H-10 (ꢂ 5.12, J1 ,2 < 1 Hz), and
in its 13C NMR the anomeric signal (109.1 ppm),
low-®eld signals for C-40 and C-20 (83.7 and
82.1 ppm), and three resonances for the CH2OH
groups, at 63.9, 63.6, and 63.4 ppm, were observed.
The disaccharide alditol 4 has been previously iso-
lated from the lichen Peltigera horizontalis by
methanolic extraction [17], and this is the ®rst
synthesis and spectroscopic characterization of the
compound.
0
0
The same approach was used to synthesize the
analogous 3-deoxy disaccharide 8b. In this case,
lactone 1 was condensed with 1-O-acetyl-3-deoxy-
2,5,6-tri-O-benzoyl-ꢀ-d-xylo-hexofuranoside (2b)
[22], in the presence of SnCl4. The glycosyl-lactone
3b was isolated from the reaction mixture by crys-
tallization from ethanol (65% yield). The 13C
NMR spectrum of 3b showed the distinctive reso-
nance for the deoxy carbon (C-3, 31.7 ppm) and
some up®eld displacement for C-20 and C-40 with
respect to the spectrum of 3a.
Reduction of the lactone group of 3b with di-
isoamylborane gave compound 5b in 90% yield.
Treatment of 5b with NaOMe a€orded the
30-deoxy disaccharide 8b in 92% yield. The spec-
troscopic behavior of compounds 5b and 8b was
very similar to that of 5a and 8a, respectively.
Thus, they showed similar chemical shifts for their
anomeric signals and also the anomeric composi-
tions were alike.
Reduction of the lactone group of 3a with di-
isoamylborane [18,19] gave 5a in 88% yield. The
anomeric region of the 13C NMR spectrum of 5a
showed the resonances for C-1 Galf (106.6 and
106.4 ppm) and C-1 Manf at ꢂ 99.9 (ꢀ anomer)
and 95.6 (ꢁ anomer). The ꢁ:ꢀ ratio was estab-
lished as 1.4:1 from the relative signal intensities.
Standard benzoylation of the anomeric HO of 5a
gave a mixture of the diastereoisomeric per-O-
benzoyl derivatives having the ꢁ (6) and the ꢀ (7)
con®gurations, which were separated by column
chromatography and individually characterized.
1
Thus, the anomeric proton signal in the H NMR
spectrum of 6, centered at 6.77 ppm with J1,2
2.4 Hz indicates the ꢁ con®guration for this com-
1
pound. The H NMR of 7 showed a similar che-
We have previously reported the synthesis of 4-
nitrophenyl ꢀ-d-galactofuranoside (10a) [23] as a
useful substrate for the detection of ꢀ-d-galacto-
furanosidase activity of Penicillium fellutanum [15].
Now, compound 10b, the 3-deoxy analogue of 10a,
was prepared with the purpose of investigating the
mical shift (6.82 ppm), but the J1,2 value of 4.8 Hz
indicates the ꢀ con®guration at C-1. Furthermore,
these data and also the chemical shifts observed for
C-1 in the 13C NMR spectra of 6 and 7, are in
good agreement with the values observed for the

186
C. Marino et al./Carbohydrate Research 311 (1998) 183±189
speci®city of the enzyme. Compound 2b was con-
densed with 4-nitrophenol in the presence of SnCl4,
to give compound 9b stereoselectively in 86%
a convenient synthon for the synthesis of higher
oligosaccharides of the natural glycoconjugates.
We also observed that HO-3 plays an important
role in the interaction between the substrate and
the ꢀ-d-galactofuranosidase.
1
yield. The H NMR spectrum of 9b showed a
broad singlet at 6.02 ppm for the anomeric proton
and indicates the 1,2-trans con®guration. The other
signals were in good agreement with those reported
for the 3-deoxy-d-xylo-hexofuranosides [22].
Standard debenzoylation of 9b a€orded com-
pound 10b, in 95% yield. Its 13C NMR spectrum
showed the signal of the anomeric carbon with a
similar chemical shift as that observed in com-
pound 10a, and the signals corresponding to the
other carbons of the ring shifted up®eld because of
the absence of OH-3.
Incubation of the enzyme with the analogue 10b
under the same conditions used with substrate 10a,
showed that the galactofuranosidase can not
hydrolyze the glycosidic linkage within the pH
range of 4±9 and incubation times of 1.5±5 h.
Moreover, hydrolysis of substrate 10a was not
modi®ed by the presence of 10b, showing that the
3-deoxy-galactofuranoside did not interact with the
enzyme.
The substrate speci®city of ꢀ-d-galactofur-
anosidase could be monitored by high-pH anion-
exchange chromatography with pulsed ampero-
metric detection (HPAEC-PAD). Compounds 4,
8a, and 8b were incubated with the enzyme under
the same conditions that compound 10a, and the
digests were analyzed by HPAEC-PAD after con-
venient dilution with water. Disaccharide 8a
(tr=23.17 min), was hydrolyzed to Gal (tr=
3.62 min) and Man (tr=4.02). The alditol disac-
charide 4 (tr=9.45 min) was also a good substrate
for the enzyme, and gave galactose and mannitol.
Incubation of 4 and 8a during 18 h resulted in com-
plete hydrolysis of the disaccharides.
In accord with the results obtained with gly-
coside 10b, the 3-deoxy disaccharide 8b
(tr=10.12 min) was also found resistant to hydroly-
sis by ꢀ-d-galactofuranosidase. No new peaks
appeared in the `monosaccharide region' of the
chromatogram, con®rming that the enzyme does
not hydrolyze the glycosidic linkage in absence of
the OH-3 group in the nonreducing unit.
3. Experimental
General methods.ÐMelting points were deter-
mined with a Thomas±Hoover apparatus. Optical
rotations were measured with a Perkin±Elmer 343
polarimeter. NMR Spectra were recorded with a
Bruker AC 200 spectrometer. Column chromato-
graphy was performed on Silica Gel 60 (200±400
mesh, Merck). Thin-layer chromatography (TLC)
was carried out on precoated aluminium plates of
Silica Gel 60F254 (Merck), using the following sol-
vents: (a) 9:1 toluene±EtOAc, (b) 7:1:2 1-PrOH±
NH3±H2O. The spots were visualized by exposure
to UV light and by spraying the plates with 10%
(v/v) H2SO4 in EtOH, followed by heating.
1,2,3,5,6-Penta-O-benzoyl-d-galactofuranose (2a)
was prepared by benzoylation of d-galactose at
100 ꢀC [11]. 1-O-Acetyl-2,5,6-tri-O-benzoyl-3-de-
oxy-ꢀ-d-xylo-hexofuranose (2b) was prepared as
previously described [22].
2,5,6-Tri-O-benzoyl-d-mannono-1,4-lactone (1).Ð
To a stirred solution of d-mannono-1,4-lactone
(1.0 g, 5.62 mmol) in pyridine (7.5 mL), cooled in
an ice-water bath, BzCl (2.0 mL, 17.04 mmol) was
slowly added. The mixture was stirred for 1.5 h at
ꢀ
0 C, and then poured into ice-water (200 mL).
After 2 h the syrup was separated and dissolved in
CH2Cl2. The organic solution was washed with aq
NaHCO3 (100 mL) and aq NaCl (2Â100 mL),
dried (MgSO4) and the solvent evaporated to
a€ord an amorphous solid. Addition of benzene
(80 mL) to the solid led to crystalline compound 1
(1.93 g, 70%) which upon recrystallization from
ethanol had mp 135±136 ꢀC, lit: 136±138 ꢀC [13].
2,5,6-Tri-O-benzoyl-3-O-(2,3,5,6-tetra-O-benzoyl-
b-d-galactofuranosyl)-d-mannono-1,4-lactone (3a).Ð
To a solution of 1,2,3,5,6-penta-O-benzoyl-d-
galactofuranose (2a, 0.35 g, 0.50 mmol) in dry
CH2Cl2 (10 mL) was added, SnCl4 (0.08 mL,
0.66 mmol) and the solution was stirred for 10 min
at 0 ꢀC. Compound 1 (0.25 g, 0.50 mmol) was
added, and the mixture was stirred overnight at
room temperature. The solution was poured into
aq NaHCO3, extracted with CH2Cl2 and the
organic extract was washed with water, dried
In summary, with this simple approach we e-
ciently synthesized ꢀ-d-Galf-(1!3)-d-Manp (8a)
and 3-deoxy-ꢀ-d-xylo-hexofuranosyl-(1!3)-d-
Manp (8b). These disaccharides could be useful for
biosynthetic studies of the Leishmania LPG.
Moreover 8a, which could be readily acylated, is

C. Marino et al./Carbohydrate Research 311 (1998) 183±189
187
(MgSO4), ®ltered and evaporated. The residue was
puri®ed by column chromatography (19:1 toluene±
EtOAc), and the fractions containing the product
having Rf 0.62 (solvent a) were pooled and con-
centrated. The resulting crystalline mass was
recrystallized from EtOH a€ording compound 3a
(0.45 g, 85%), mp 92±93 ꢀC; [ꢁ]d 42ꢀ (c 1,
(C-40 ꢁ,ꢀ), 82.2, 81.7 (C-20 ꢁ,ꢀ), 77.7 (C-30 ꢁ,ꢀ),
75.1±69.8 (C-2,3,4,5, and 50 for both anomers), and
63.7±63.4 (C-6,60 for both anomers). Anal. Calcd
for C61H50O18: C, 68.40; H, 4.70. Found: C, 68.25;
H, 4.48.
1,2,5,6-Tetra-O-benzoyl-3-O-(2,3,5,6-tetra-O-
benzoyl-b-d-galactofuranosyl)-a-d-mannofuranose
(6) and 1,2,5,6-tetra-O-benzoyl-3-O-(2,3,5,6-tetra-
O-benzoyl-b-d-galactofuranosyl)-b-d-mannofuranose
(7).ÐCompound 5a (0.2 g, 0.19 mmol) was con-
ventionally benzoylated with BzCl (1.0 mL) in pyr-
idine (2.0 mL). After 2 h, TLC examination of the
solution (solvent a) showed two main products
which were separated by column chromatography
(49:1 toluene±EtOAc) after conventional work up
of the reaction mixture. From fractions having Rf
0.50 compound ꢀ6 (0.07 g, 37%) was isolated as a
1
CHCl3); H NMR (CDCl3): ꢂ 8.25±7.05 (H-aro-
matic), 6.02 (d, J2,3 3.8 Hz, H-2), 5.86 (m, 2 H, H-
5,50), 5.74 (s, J1 ,2 <1 Hz, H-20), 5.54 (d, J2 ,3
0
0
0
0
3.6 Hz, H-30), 5.51 (s, H-10), and 5.08±4.23 (m, 7 H,
H-3,4,6a,6b,40,60a,60b); 13C NMR (CDCl3) ꢂ 169.0
(C-1), 165.6±164.6 (C=O benzoate), 133.9±128.3
(C-aromatic), 106.9 (C-10), 83.9 (C-40), 81.3 (C-20),
77.6, 76.4 (C-4,30), 73.1 (C-3), 70.9, 70.6 (C-2,50),
68.2 (C-5), 63.6, 63.1 (C-6,60). Anal. Calcd for
C61H48O18: C, 68.54; H, 4.53. Found: C, 68.76; H,
4.75.
1
syrup, [ꢁ]d +12 (c 1, CHCl3); H NMR (CDCl3)
b-d-Galactofuranosyl-(1!3)-d-mannitol (4).Ð
Compound 3a (0.32 g, 0.3 mmol) was suspended in
MeOH (10 mL) and NaBH4 (0.20 g, 5.3 mmol) was
added. After stirring overnight, 0.5 M NaOMe in
MeOH (5 mL) was added. The stirring was con-
tinued for 3 h, when TLC monitoring showed a
single spot (Rf 0.28, RGal 1, solvent b). The solution
was deionized with Dowex 50W (H+) resin and
then co-evaporated with water several times. The
syrup was crystallized from iPrOH±EtOH a€ord-
anomeric region ꢂ: 6.77 (J1,2 2.4 Hz, H-1); 13C
NMR (CDCl3) anomeric region ꢂ: 106.4 (C-10),
99.4 (C-1). Anal. Calcd for C68H54O19: C, 69.50; H,
4.63. Found: C, 69.79 ; H, 4.61.
Evaporation of the solvent from slower-moving
fractions (Rf 0.43) a€orded syrupy 7 (0.09 g, 41%)
which had [ꢁ]d 59ꢀ (c 1, CHCl3); 1H NMR
(CDCl3) anomeric region ꢂ: 6.82 (J1,2 4.8 Hz, H-1);
13C NMR (CDCl3) anomeric region ꢂ: 106.6 (C-10),
93.2 (C-1). Anal. Calcd for C68H54O19: C, 69.50; H,
4.63. Found: C, 69.31; H, 4.48.
ꢀ
ing 4 (0.089 g, 89%), which had mp 150±151 C,
[ꢁ]d 55ꢀ (c 1, H2O), lit: [ꢁ]d 61ꢀ (c 2, H2O) [17];
3-O-b-d-Galactofuranosyl-d-mannose (8a).Ð
Compound 5a (0.40 g, 0.37 mmol) was suspended
in a 0.5 M solution of NaOMe in MeOH (10.0 mL),
and stirred until complete dissolution (1 h), when
TLC showed a single spot (Rf 0.32, solvent b). The
solution was made neutral with Dowex 50W (H+)
resin and concentrated to a€ord compound 8a
1H NMR (D2O), anomeric region, ꢂ 5.120 (H-10,
J1 ,2 1.5 Hz); 13C NMR (D2O) ꢂ 109.1 (C-1 ), 83.7
(C-40), 82.1 (C-20),77.8, 76.9 (C-3,30) 71.6, 71.5,
71.4, 70.3 (C-2,4,5,50), 63.9, 63.6, 63.4 (C-1,6,60).
Anal. Calcd for C12H24O11: C, 41.86, H, 7.03.
Found: 41.69, H, 6.87.
0
0
(0.12 g, 94%), which had [ꢁ]d 47ꢀ (c 1, H2O); H
1
2,5,6-Tri-O-benzoyl-3-O-(2,3,5,6-tetra-O-benzoyl-
b-d-galactofuranosyl)-d-mannofuranose (5a).ÐTo
a solution of freshly prepared bis(3-methyl-2-
butyl)borane (1.9 mmol) in anhydrous tetra-
hydrofuran (2.0 mL) a solution of compound 3a
(0.50 g, 0.47 mmol) in tetrahydrofuran (3 mL) was
added. The solution was stirred for 16 h at room
temperature and then processed as already descri-
bed [18]. Boric acid was eliminated by co-evapora-
tion with MeOH, and the syrup was puri®ed by a
short column chromatography (19:1 toluene±
EtOAc). Fractions having Rf 0.22 (solvent a) were
pooled and evaporated to give syrupy 5a (0.44 g,
88%), [ꢁ]d 35ꢀ (c 1, CHCl3); 13C NMR (CDCl3)
ꢂ 106.6 (C-10ꢀ of the ꢁ anomer), 106.4 (C-10ꢀ of
the ꢀ anomer), 99.9 (C-1ꢀ), 95.6 (C-1ꢁ), 83.1, 82.2
NMR (D2O) anomeric region ꢂ: 5.16 (bs, H-1 ꢁ-
Manp), 5.10 (bs, H-1 ꢀ-Galf), 4.83 (bs, H-1 ꢀ-
Manp); 13C NMR (D2O) ꢂ: 105.7, 105.5 (C-1 Galf),
94.8, 94.7 (C-1 Manp), 84.0, 83.9 (C-40), 82.3 (C-
20), 77.9 (C-30), 76.9±66.2 (C-2,3,4,5,50 of both
anomers) and 63.8, 61.8 (C-6,60). Anal. Calcd for
C12H22O11: C, 42.11; H, 6.48. Found: C, 42.36; H,
6.61.
2,5,6-Tri-O-benzoyl-3-O-(2,5,6-tri-O-benzoyl-3-
deoxy-b-d-xylo-hexofuranosyl)-d-mannono-1,4-
lactone (3b).ÐTo a solution of 1-O-acetyl-2,5,6-tri-
O-benzoyl-3-deoxy-ꢀ-d-xylo-hexofuranose (2b,
0.57 g, 1.1 mmol) in dry CH2Cl2 (5 mL) cooled at
ꢀ
0 C, was added SnCl4 (0.16 mL, 1.3 mmol). After
15 min of stirring, a solution of lactone 1 (0.59 g,

188
C. Marino et al./Carbohydrate Research 311 (1998) 183±189
1.2 mmol) in CH2Cl2 (2 mL) was added, and the
mixture was stirred overnight at room temperature.
The mixture was treated as described for 3a. The
glycosyl-lactone 3b was crystallized from EtOH
from the crude syrup (0.66 g, 65%). After recrys-
tallization from the same solvent, compound 3b
furanose (2b, 0.57ꢀg, 1.1 mmol) in dry CH2Cl2
(5 mL) cooled at 0 C, SnCl4 (0.16 mL, 1.3 mmol)
was added. After 15 min, 4-nitrophenol (0.17 g,
1.2 mmol) was added, and the mixture was stirred
overnight at room temperature. The mixture was
processed as described for 3a. From the crude
syrup, compound 9b (0.54 g, 86%) crystallized
upon addition of EtOH. After recrystallization
ꢀ
ꢀ
1
had mp 152±153 C; [ꢁ]d 71 (c 1, CHCl3); H
NMR (CDCl3): ꢂ 8.09±7.13 (H-aromatic), 5.99 (d,
ꢀ
0
0
J2,3 4.2 Hz, H-2), 5.85 (m, H-5), 5.52 (d, J2 ,3 a
6.9 Hz, H-20), 5.50 (bs, H-10), 5.42 (m, H-50), 5.13
(dd, J5,6a 2.4 Hz, J6a,6b 12.6 Hz, H-6a), 5.03 (m, 2
H, H-3,4), 4.65 (dd, J5,6b 4.6 Hz, H-6b), 4.45 (m,
from the same solvent, it had mp 57±59 C, [ꢁ]d
80ꢀ (c 1, CHCl3); H NMR (CDCl3): ꢂ 8.19±7.10
1
(H-aromatic), 6.02 (J1,2 <0.5, H-1), 5.78 (m, H-5),
0
5.67 (d, J2,3 7.0, J2,3 2.0 Hz, H-2), 4.78 (J3,4 8.2,
H-40), 4.26 (m, 2 H, H-6a,6b), 2.76 (m, J3 a,4
J3 ,4 6.2 Hz, H-4), 4.71 (J6,6 12.0 Hz, H-6), 4.66 (H-
0
0
0
0
0
8.2 Hz, J3 a,3 b 14.6 Hz0, H-30a), and 2.08 (dd, J3 b,4
60), 2.90 (J3,3 14.5 Hz, H-3), 2.29 (H-3 ); 13C NMR
0
0
0
0
0
5.1 Hz, H-30b); 13C NMR (CDCl3) ꢂ 169.3 (C-1),
165.7±164.9 (C=O benzoates), 133.8±127.7 (C-
aromatics), 106.5 (C-10), 78.4, 77.8 (C-40,20), 76.4
(C-4), 72.0 (C-3), 71.7 (C-50), 70.9 (C-2), 68.1 (C-5),
63.7, 62.4 (C-6,60), 31.7 (C-30). Anal. Calcd for
C54H44O16: C, 68.34; H, 4.68. Found: C, 68.45; H,
4.89.
(CDCl3) ꢂ: 166.1±165.7 (C=O benzoate), 161.0
(C-1, p-nitrophenyl), 142.5 (C-4, p-nitrophenyl),
133.5±128.4 (C-aromatic), 125.7 (C-2,6 p-nitrophe-
nyl), 116.5 (C-3,5 p-nitrophenyl), 104.4 (C-1), 78.0
(C-4,2), 71.7 (C-5), 63.4 (C-6), 32.8 (C-3). Anal.
Calcd for C33H27NO10: C, 66.33; H, 4.55. Found:
C, 66.06; H, 4.32.
2,5,6-Tri-O-benzoyl-3-O-(2,5,6-tri-O-benzoyl-3-
deoxy-b-d-xylo-hexofuranosyl)-d-mannofuranose
(5b).ÐCompound 3b (1.0 g, 1.05 mmol) was
reduced with diisoamylborane (4.2 mmol) as just
described for the preparation of 5a. Syrupy com-
pound 5b (0.90 g, 90%) crystallized from EtOH;
mp 148±149 ꢀC, [ꢁ]d 59ꢀ (c 0.8, CHCl3); 13C
NMR (CDCl3) ꢂ 106.6, 106.1 (C-10 ꢀ-Galf for ꢁ
and ꢀ anomers), 99.8 (C-1 ꢀ-Manf), 95.4 (C-1 ꢁ-
Manf), 79.1, 78.2, 78.0, 77.5 (C-20,40), 74.2±69.7 (C-
2,3,4,5 and 50 for both anomers), 63.7 (C-6,60) and
32.0, 31.8 (C-30 for both anomers). Anal Calcd.
for C54H46O16: C, 68.20; H, 4.88. Found: C, 68.48;
H, 5.00.
3-O-(3-Deoxy-b-d-xylo-hexofuranosyl)-d-mannose
(8b).ÐCompound 5b (0.50 g, 0.52 mmol) was stir-
red with a 0.5 M solution of NaMeO in MeOH
(10.0 mL), until complete dissolution occurred. The
solution was made neutral with Dowex 50W (H+)
resin and concentrated to a€ord compound 8b
(0.15 g, 89%), which had [ꢁ]d 130ꢀ (c 1, H2O); 1H
NMR (D2O) anomeric region ꢂ: 5.10 (bs, H-1 ꢀ-
Galf and H-1 ꢁ-Manp), 4.81 (bs, H-1 ꢀ-Manp); 13C
NMR (D2O) ꢂ: 105.9, 105.6 (C-1 ꢀ-Galf), 94.9,
94.7 (C-1ꢁ,ꢀ-Manp), 79.3 (C-40), 75.4±66.0 (C-
2,20,3,4,5,50), 63.8, 61.8 (C-6,60) and 34.8 (C-30).
4-Nitrophenyl-3-deoxy-b-d-xylo-hexofuranoside
(10b).ÐCompound 9b (0.60 g, 0.85 mmol) was
stirred with a 0.5 M solution of NaOMe±MeOH,
during 2 h. The mixture was made neutral by
addition of Dowex 50W (H+) resin, ®ltered, and
the ®ltrate concentrated to a€ord syrupy com-
pound 10b (0.23 g, 95%); [ꢁ]d 170ꢀ (c 1, H2O); 1H
1
NMR (D2O) ꢂ: 5.81 (bs, H-1); H NMR (Me2SO-
d6) ꢂ: 8.20 (J2,3 9.9 Hz, H-3 of p-nitrophenyl), 7.20
(H-2 of p-nitrophenyl), 5.70 (J1,2<0.5 Hz, H-1),
4.30 (H-2,4), 3.45 (H-5,6,60), 2.38 (m, H-3), 1.80
(m, J2,3 2.6, J3 ,4 5.4, J3,3 13.2 Hz, H-30); 13C NMR
(Me2SO-d6) ꢂ: 161.7 (C-1 of p-nitrophenyl), 141.3
(C-4 of p-nitrophenyl), 125.6 (C-2,6 of p-nitro-
phenyl), 116.5 (C-3,5 of p-nitrophenyl), 106.5 (C-1),
79.7 (C-4), 74.1 (C-2), 72.5 (C-5), 62.5 (C-6), 34.0
(C-3). Anal. Calcd for C12H15NO7: C, 50.53; H,
5.30. Found: C, 50.62; H, 5.23.
0
0
0
Assays of b-d-galactofuranosidase activity.ÐThe
®ltered medium of a stationary culture (20 days) of
Penicillium fellutanum (previously P. charlesii G
Smith NRRL 1987) was used as the enzyme source
[24]. Protein was determined using bovine serum
albumin as standard [25]. The enzymatic assays
were made with 0.31 ꢃmol of the tested substrates,
100 ꢃL of 66 mM NaOAc bu€er (pH 4) and
100 ꢃL of the enzyme medium (20 ꢃg protein), in a
®nal volume of 500 ꢃL. The enzymatic reactions
.
Anal. Calcd for C12H22O10 2H2O: C, 39.78; H,
7.23. Found: C, 40.50; H, 7.18.
4-Nitrophenyl 2,5,6-tri-O-benzoyl-3-deoxy-b-d-
xylo-hexofuranoside (9b).ÐTo a solution of 1-O-
acetyl-2,5,6-tri-O-benzoyl-3-deoxy-ꢀ-d-xylo-hexo-
ꢀ
were incubated for 1.5 h or as indicated, at 37 C
and stopped in two di€erent ways. In the case of
compounds 10a and 10b, they were stopped with

C. Marino et al./Carbohydrate Research 311 (1998) 183±189
189
1 mL of sodium carbonate bu€er (pH 9.0), and the
4-nitrophenol released was measured spectro-
photometrically at 410 nm.
In the case of compounds 4, 8a and 8b, 30 ꢃL of
theꢀ incubation mixture was heated for 2 min at
80 C and conveniently diluted. Aliquots of 30 ng
were analyzed by HPAEC.
HPAEC analysis of enzymatic digests.ÐSamples
were analyzed using HPAEC-PAD, with a Carbo-
Pac PA1 column (4Â25 mm). Eluants were 200 mM
NaOH, water, and 250 mM NaOAc±150 mM
NaOH, respectively. The column was equilibrated
in 20 mM NaOH. After 10 min, a linear gradient of
NaOAc, to a limit concentration of 125 mM over
30 min, was used. The pulsed amperometric detector
sensitivity was set at 100 mA (attenuation full scale)
and the pulse potentials were as follows: E1=0.05
V, t1=480 ms (Range 2, position 5); E2=0.6 V,
t2=120 ms (Position 2); E3= 0.6 V, t3=60 ms
(Position 1). The time constant was set to 3 s.
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Acknowledgements
We thank CONICET (Consejo Nacional de
Investigaciones Cientõ®cas y Tecnicas) and the
University of Buenos Aires for ®nancial support.
R.M. de Lederkremer and O. Varela are research
members of CONICET.
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