Synthesis of 4-nitrophenyl β-D-fucofuranoside and β-D-fucofuranosyl-(1 → 3)-D-mannopyranose: Modified substrates for studies on catalytic requirements of β-D-galactofuranosidase

Article abstract of DOI:10.1016/S0008-6215(99)00241-4

Syntheses of 4-nitrophenyl β-D-fucofuranoside (6) and β-D- fucofuranosyl-(1 → 3)-D-mannopyranose (10) are reported. These compounds, as analogues of galactofuranosides, were used for studying the influence of the hydroxyl group at C-6 in the interaction o

Full text of DOI:10.1016/S0008-6215(99)00241-4

www.elsevier.nl/locate/carres
Carbohydrate Research 323 (2000) 7–13
Synthesis of 4-nitrophenyl b-
b- -fucofuranosyl-(1“3)- -mannopyranose: modified
substrates for studies on catalytic requirements of
b- -galactofuranosidase
D-fucofuranoside and
D
D
D
Alejandro Chiocconi, Carla Marino, Rosa M. de Lederkremer *
CIHIDECAR, Departamento de Qu´ımica Orga´nica, Facultad de Ciencias Exactas y Naturales,
Uni6ersidad de Buenos Aires, Pabello´n II, Ciudad Uni6ersitaria, 1428 Buenos Aires, Argentina
Received 24 May 1999; accepted 20 August 1999
Abstract
Syntheses of 4-nitrophenyl b-
ported. These compounds, as analogues of galactofuranosides, were used for studying the influence of the hydroxyl
group at C-6 in the interaction of the substrate with b- -galactofuranosidase. For the synthesis of the fucofura-
nosides, 2,3,5-tri-O-benzoyl-6-bromo-6-deoxy- -galactono-1,4-lactone (1) was the key intermediate, which upon
reduction of the lactone group with diisoamylborane, acetylation of the anomeric hydroxyl group, and catalytic
hydrogenolysis of the bromine at C-6, led to 1-O-acetyl-2,3,5-tri-O-benzoyl-a,b- -fucofuranose (4), a convenient
derivative for the preparation of fucofuranosides. Compound 4 was glycosylated in the presence of SnCl4, either with
4-nitrophenol for the preparation of 6, or with 2,5,6-tri-O-benzoyl- -mannono-1,4-lactone (7), for the synthesis of
disaccharide 10, via the glycosyl-aldonolactone approach. The synthetic route developed for the b- -fucofuranosides
-galactofuranosidase from
D
-fucofuranoside (6) and b-
D
-fucofuranosyl-(1“3)-
D
-mannopyranose (10) are re-
D
D
D
D
D
is simple and efficient. Compound 6 was not hydrolyzed by incubation with the exo b-
D
Penicillium fellutanum, showing that HO-6 is essential for interaction of the substrate with the enzyme. © 2000
Elsevier Science Ltd. All rights reserved.
Keywords: Deoxy sugars; b-
D
-Fucofuranosides; exo b-D-Galactofuranosidase
1. Introduction
arabinofuranosides, some galactofuranosyl
disaccharides, and galactopyranosides [1]. A
natural substrate for the enzyme is the pepti-
dophosphogalactomannan from P. fellutanum
[1,2]. Variations in the content of galactofura-
nosyl units were attributed to the action of the
In connection with our studies on the sub-
strate specificity of the exo b- -galactofura-
nosidase from Penicillium fellutanum, we are
synthesizing deoxy analogues of galactofura-
nosides. A limited number of substrates have
been previously tested with the enzyme,
namely, ethyl and methyl galactofuranosides,
D
b- -galactofuranosidase. Galactofuranose is
D
also an important component of parasite gly-
coconjugates [3] present as terminal units in
the lipopeptidophosphoglycan of Try-
panosoma cruzi [4,5], or internally in the
Leishmania lipophosphoglycan [6]. The deoxy
analogues of the galactofuranosides could also
* Corresponding author. Fax: +54-11-457-63352.
E-mail address: lederk@qo.fcen.uba.ar (R.M. de Lederkre-
mer)
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(99)00241-4

8
A. Chiocconi et al. / Carbohydrate Research 323 (2000) 7–13
be useful for studies on the interaction with
the glycosyltransferase involved in the con-
struction of the lipophosphoglycan.
gel column chromatography. The same reac-
tion at high temperature gave higher propor-
tions of the pyranosides [14].
In this context, we have previously reported
We have previously obtained crystalline
the synthesis of b- -Galf-(1“3)-mannopyra-
D
per-O-benzoyl-a,b-
D
-galactofuranose by di-
nose and the 3-deoxy analogues, 3-deoxy-b-
-xylo-hexofuranosyl-(1“3)-mannopyranose
-xylo-hexofura-
rect benzoylation of
D
-galactose at high tem-
D
perature [15,16]. The mixture of the furanosic
benzoates was used for the preparation of
galactofuranosyl disaccharides [16,17]. The
simplicity of the procedure led us to try direct
and 4-nitrophenyl 3-deoxy-b-
D
nose, and have shown that whereas the en-
zyme hydrolyzes the former, the absence of
OH-3 prevents the interaction [7]. The aim of
the present work was the synthesis of 6-deoxy
benzoylation of the analogous
D-fucose for
the synthesis of furanosic benzoates. When
benzoylation was performed at 60 °C, the
pyranosic forms were the main products, ac-
cording to the relative signal intensities in the
1H NMR spectrum. The a,b-furanosic forms
were obtained in only 12% yield. Benzoylation
at room temperature increased the yield of the
a-pyranosic derivative, as usual, and the fura-
nosic forms were obtained in 22% yield. The
proportions of the furanose benzoates were
similar to those reported for the acetylation of
fucose with acetic anhydride–sodium acetate
[18]. Although the mixtures were resolved by
column chromatography, the procedure was
not convenient for the preparation of a start-
ing material. Signals for H-1 were assigned by
comparison with those for perbenzoyl deriva-
analogues of the substrate, that is the b- -fu-
D
cofuranosides, and the evaluation of the influ-
ence of the primary hydroxyl group in the
interaction with the enzyme.
L
-Fucose is the more abundant enantiomer
in nature, and has only been found in the
pyranose configuration. The less abundant
D-
fucose also occurs as the furanoside. The a
anomer is present in the cell-wall antigen from
Eubacterium saburreum L 452 [8], and in the
O-antigen from different strains of Pseu-
domonas syringae [9,10]. b- -Fucofuranose
D
was found as a sulfated steroidal glycoside in
the starfish Dermaterias imbricata [11].
The b-
D
- and b- -fucofuranosides of sec-
L
ondary and tertiary alcohols, prepared from
the peracetylated fucofuranoses, have been
used for the determination of the absolute
configuration of the alcohols by NMR tech-
niques [12,13]. The method we now describe is
a good alternative for the synthesis of the
fucofuranosides.
tives of
D-galactose [15], and were 6.89 ppm
(a-p, J1,2 3.6 Hz), 6.86 ppm (a-f, J1,2 4.8 Hz),
6.81 (b-f, J1,2B1 Hz), and 6.33 ppm (b-p, J1,2
8.3 Hz).
We previously showed the convenience of
aldono-1,4-lactones as precursors for the syn-
thesis of glycofuranosides [19], deoxy sugars
[7], and furanosic disaccharides [16,17]. The
lactone moiety can be selectively substituted
[17], or deoxygenated via b-elimination reac-
tions [20], with the anomeric center ‘protected’
as a lactone group. Following controlled re-
duction of this group by diisoamylborane
[21,22], the free HO-1 is obtained, which can
be differentially derivatized or activated for a
glycosylation reaction.
2. Results and discussion
During recent years, considerable progress
on the synthesis of
L-fucopyranosides
(oligosaccharides) has been made, but few re-
ports describe the preparation of fucofura-
nosides [12,13].
The synthesis of glycofuranosides implies
the availability of a precursor with the appro-
priate ring size. Gardiner and Percival [14]
2,3,5-Tri-O-benzoyl-6-bromo-6-deoxy- -
D
galactono-1,4-lactone (1) has been prepared in
our laboratory in 90% yield as an intermediate
described the reaction of -fucose with 0.8%
L
for the synthesis of abequose (3,6-dideoxy- -
D
methanolic hydrogen chloride, at room tem-
perature. This glycosylation led to an
anomeric mixture of the furanosic (64%) and
pyranosic forms, which were isolated by silica
xylo-hexose) and its glycofuranosides [23]. We
now used compound 1 as a convenient precur-
sor for the synthesis of furanose derivatives of

A. Chiocconi et al. / Carbohydrate Research 323 (2000) 7–13
9
Scheme 1. (i) DSBH, THF; (ii) Ac2O, Py; (iii) H2, PdꢀC, Et3N; (iv) SnCl4, CH2Cl2; (v) NaOMe, MeOH.
D
-fucose. Thus, reduction of 1 with diisoamyl-
borane [21,22] afforded an anomeric mixture
of 2,3,5-tri-O-benzoyl-6-bromo-6-deoxy-
[16,17,24]. Condensation of 4 with 4-nitrophe-
nol in the presence of the Lewis acid gave the
b-glycoside 5, as established by NMR spec-
troscopy. NMR data for 5 resemble those for
D
-
galactofuranoses (2) in 95% yield (Scheme 1).
The 13C NMR spectrum of 2 showed two
signals in the anomeric region at 100.0 and
95.6 ppm, assigned to the b and a anomers,
respectively (ratio b/a ꢀ2.2). The signals at l
29.6 and 29.2 were assigned to the C-6 of both
anomers, and the other signals (Table 1) re-
semble those of the galactofuranosides previ-
ously described [16,17].
the b- -galactofuranoside derivatives [16] with
D
1
the expected differences. H NMR spectrum
(Table 2) showed H-1 and H-2 signals as
broad singlets in accordance with the 1,2-trans
configuration, and signals for H-5 and H-6
upfield shielded because of the absence of
oxygen at C-6. The characteristic pattern of
b- -galactofuranosides was observed in the
D
13C NMR spectrum, with signals for C-1 at
104.0 ppm and for C-2 and C-4 over 80 ppm
(Table 1).
Compound 2 was acetylated with the pur-
pose of having an appropriate leaving group
for the glycosylation reactions. Thus, com-
pound 3 was obtained (95%) in a b/a ratio
ꢀ3.3, according to the relative signal intensi-
By debenzoylation of 5 with sodium
methoxide, glycoside 6 was obtained in 51%
13
ties from the H and 13C NMR spectra. The
1
yield from 1. The C NMR chemical shifts of
1
6 (Table 1) were in good agreement with those
anomeric region of the H NMR spectrum
reported by Kobayashi for other b- -fuco-
D
(Table 2) showed a broad singlet at l 6.51,
which was assigned to the H-1 of the b
anomer. A doublet at 6.64 ppm was attributed
to H-1 of the a anomer.
Catalytic hydrogenolysis of 3 in ethyl ac-
etate afforded the 6-deoxy derivative 4 in 95%
yield. The removal of the bromine at C-6 was
evidenced by the upfield shift of the C-6 signal
(15.7 ppm b, 16.1 ppm a) in the 13C NMR
spectrum of 4 (Table 1), as compared with the
same signal in 3 (29.6 ppm).
1
furanosides [12,13]. The H NMR spectrum of
4-nitrophenyl b- -fucofuranoside (6, Table 2)
D
was completely assigned.
For the preparation of disaccharide 10, we
used the glycosyl-aldonolactone approach pre-
viously developed in this laboratory [7]. Com-
pound 4 and the partially benzoylated
mannono-1,4-lactone 7 [7] were convenient
Table 1
13C NMR (50.3 MHz) chemical shifts of compounds 1–6
Thus, the acylated fucofuranoses could be
obtained in a total yield of 81% starting from
Compound C-1
C-2
C-3
C-4
C-5
C-6
D
-galactono-1,4-lactone. This starting material
compares very favorably in price with -fu-
cose, previously used for the preparation of
a
2b
100.0
99.3
99.3
104.0
106.2
51.8
81.8
81.7
82.4
82.1
77.6
77.7
77.0
77.0
77.9
82.9
83.6
85.7
85.5
89.5
72.1
71.8
69.2
69.3
68.1
29.2
29.2
15.7
16.3
19.0
3b a
4b a
5 a
D
D-fucofuranose derivatives [12,13].
6 b
Glycosylation of 4 was performed using
SnCl4 as catalyst, on the basis of our experi-
a The chemical shifts are expressed for a solution in CDCl3,
ence on the stereoselective synthesis of O-
relative to internal Me4Si.
and S-glycosides of b- -galactofuranose
D
b In D2O.

10
A. Chiocconi et al. / Carbohydrate Research 323 (2000) 7–13
Table 2
1H NMR (200.1 MHz) chemical shifts of compounds 1–6
l (ppm), J (Hz)
Compound
H-1 (J1,2
)
H-2 (J2,3
)
H-3 (J3,4
)
H-4 (J4,5
)
H-5 (J5,6
)
H-6,6% (J6,6%)
1 a
6.10 (6.0)
5.52
5.61
5.60 (B1)
5.76 (B1)
4.30 (2.9)
5.80
5.52
5.61 (3.2)
5.64 (4.6)
5.68 b
5.20 (6.0)
4.95 (4.3)
4.88 (4.6)
4.61 (4.5)
4.60 (4.8)
3.85 b
5.80 (2.5)
4.45 (6.2)
5.83
3.73
3.78 (8.4)
3.77
1.55
1.55
2b a
3b a
4b a
5 a
5.72 (B0.5)
6.51 (B0.5)
6.50 (B0.5)
6.05 (B0.5)
5.71 (1.4)
5.55 (6.5)
5.68 b
6 c
4.00 (5.2)
3.85 (6.2)
1.16
a The chemical shifts are expressed for a solution in CDCl3, relative to internal Me4Si.
b Center of a complex multiplet.
c For a solution in D2O, relative to HOD as internal reference.
Scheme 2. (i) SnCl4, CH2Cl2; (ii) DSBH, THF; (iii) NaOMe, MeOH.
at l 5.14 (J1%,2% 1.2 Hz) and 5.12 (J1%,2% 1.5 Hz).
The mannopyranose signals appeared at 5.18
and 4.87 ppm, in good agreement with those
precursors for disaccharide 10 (Scheme 2).
Thus, condensation of 4 with 7 promoted by
SnCl4 led to the glycosyl-lactone derivative 8
1
reported for the disaccharide b-
D
-Galf-(1“
as a crystalline product in 65% yield. H and
13C NMR spectra were fully assigned by com-
parison with the spectra of the corresponding
3)- -mannopyranose [7]. The a/b ratio was
D
ꢀ1.7 from the relative signal intensities. The
13C NMR spectrum of 10 showed signals at
94.9 and 94.7 ppm for C-1 of the a and b
anomers, respectively, in accordance with val-
ues for mannopyranose, and signals at 105.1
monosaccharides. The b- -configuration of
D
the glycosidic linkage was confirmed by the
small J1,2 value (B0.5 Hz) [25]. The 13C NMR
spectrum showed the signals of the galacto-
furanosyl unit (105.9, 85.5 and 81.5 ppm for
C-1%, 4% and 2%, respectively), similar to those
of compound 5, and the signals for the lactone
moiety similar to the signals of lactone 7.
Reduction of 8 with diisoamylborane led to
disaccharide 9 with HO-1 free, in the a/b ratio
ꢀ2. Deacylation of 9 with sodium methoxide
afforded disaccharide 10 in 49% overall yield
and 104.8 ppm for C-1 of the b- -fucofura-
D
nosyl unit. The absence of a substituent at
C-6% elicits a downfield shift of the C-4% signal,
whereas the resonance corresponding to C-5%
is shifted upfield. The same effect was ob-
served on C-6 deoxygenation of
ranose [26].
D
-galactopy-
The synthetic route developed for the syn-
-fucofuranosides is particularly
from
D-galactono-1,4-lactone. The tautomeric
thesis of b-
D
expansion of the reducing unit was evidenced
efficient, because no chromatographic separa-
tion of pyranosic and furanosic forms is re-
quired. The methodology could be extended
1
in the H NMR spectrum. The H-1% chemical
shift for the b- -Fucf unit was influenced by
D
the anomeric configuration of the mannopyra-
nose unit, and appeared as two close doublets
to the synthesis of the
L
enantiomeric glyco-
furanosides, and is likewise convenient, since

A. Chiocconi et al. / Carbohydrate Research 323 (2000) 7–13
11
the corresponding starting material,
L
-galac-
and successively washed with 5% HCl, satd aq
NaHCO3, and then dried (Na2SO4). The syrup
obtained after evaporation of the solvent
tono-1,4-lactone, is less expensive than
fucose.
L
-
1
showed, in the H NMR (CDCl3) anomeric
The exo b- -galactofuranosidase from P.
D
region: l 6.89 ppm (a-p, J1,2 3.6 Hz, 5%), 6.86
ppm (a-f, J1,2 4.8 Hz, 6%), 6.81 (b-f, J1,2 B1 Hz,
6%), 6.33 ppm (b-p, J1,2 8.3 Hz, 83%).
fellutanum was incubated with fucofuranoside
6, under the conditions previously described as
the optimum for the incubation of 4-nitro-
At room temperature (rt). To a soln of -fu-
D
phenyl b- -galactofuranoside [24]. No hydrol-
D
cose (0.5 g, 2.77 mmol) in pyridine (7.0 mL),
BzCl (2.0 mL) was added, and the mixture was
stirred for 2 h. The soln was poured into ice
ysis of 6 was observed, even after 24 h of
incubation. Some reports showed that ana-
logues of substrates of some enzymes could act
as inhibitors of the enzymes [27]. Thus, we
incubated the enzyme in the presence of both
the substrate and compound 6. We observed
that the presence of 6 did not modify the
activity of the enzyme, even if preincubation
with 6 was performed prior to addition of the
substrate.
1
water and processed as already described. H
NMR (CDCl3), anomeric region: l 6.89 ppm
(a-p, J1,2 3.6 Hz, 46%), 6.86 ppm (a-f, J1,2 4.8
Hz, 14%), 6.81 (b-f, J1,2 B1 Hz, 8%), 6.33 ppm
(b-p, J1,2 8.3 Hz, 32%).
2,3,5-Tri-O-benzoyl-6-bromo-6-deoxy- -
D
galactofuranose (2).—To a soln of freshly pre-
pared bis(3-methyl-2-butyl)borane (36.8 mmol)
in anhyd THF (2.0 mL) [21,22], compound 1
(2.55 g, 4.6 mmol) was added. The soln was
stirred for 16 h at rt and then processed as
already described [21], affording 2 (2.42 g,
95%) as an amorphous solid; [h]D −19° (c 1,
CHCl3). Anal. Calcd for C27H23BrO8: C, 58.39;
H, 4.17. Found: C, 58.83; H, 4.38.
The lack of hydrolytic activity on the deoxy
structure indicates a high specificity of the
b- -galactofuranosidase with respect to the
D
glycone moiety, with both HO-3 and HO-6 of
the substrates being essential for interaction
with the enzyme.
1-O-Acetyl-2,3,5-tri-O-benzoyl-6-bromo-6-
3. Experimental
deoxy- -galactofuranose (3).—Compound 2
D
(2.0 g, 3.61 mmol) was conventionally acety-
lated (1:1 Ac2O–pyridine, 7.0 mL) to afford
compound 3 (2.07 g, 95%), which upon crystal-
lization from EtOH had a mp of 135–136 °C;
[h]D −10° (c 1, CHCl3). Anal. Calcd for
C29H23BrO8: C, 58.30; H, 4.22. Found: C,
58.10; H, 4.21.
General methods.—Melting points were de-
termined with a Thomas–Hoover apparatus.
Optical rotations were measured with a
Perkin–Elmer 343 polarimeter. NMR spectra
were recorded with a Bruker AC-200 spectrom-
eter. Column chromatography was performed
on Silica Gel 60 (200–400 mesh, E. Merck).
1-O-Acetyl-2,3,5-tri-O-benzoyl-h,i- -fuco-
D
2,3,5-Tri-O-benzoyl-6-bromo-6-deoxy-
actono-1,4-lactone (1) [23] and 2,5,6-tri-O-ben-
zoyl-
D
-gal-
furanose (4).—A soln of compound 3 (1.0 g,
1.68 mmol) in EtOAc (10 mL) containing Et3N
(1 mL) was hydrogenated with PdꢀC (0.15 g)
at 45 psi (3 atm) for 3 h. The catalyst was
filtered and the filtrate was successively washed
with 5% HCl, satd aq NaHCO3, and then dried
(Na2SO4). After evaporation of the solvent, the
syrup obtained (0.83 g, 95%) was recrystallized
from EtOH and had a mp of 99–100 °C; [h]D
−19° (c 1, CHCl3). Anal. Calcd for C29H26O19:
C, 67.18; H, 5.05. Found: C, 67.06; H, 5.14.
D-mannono-1,4-lactone (7) [7] were pre-
pared as previously described. Enzymatic
assays were performed as reported [24].
Benzoylation of
D
-fucose
At high temperature. The procedure previ-
ously described for the preparation of penta-
O-benzoyl-a,b-
D
-galactofuranose [15] was
-fucose (0.5
adapted for -fucose. A soln of
D
D
g, 2.77 mmol) in anhyd pyridine (7.0 mL) was
stirred for 2 h at 100 °C. The temperature was
lowered to 60 °C, and BzCl was added drop-
wise. The reaction was performed for 1.5 h at
60 °C. The mixture was then poured into ice
water, and the syrup was dissolved in CH2Cl2
4-Nitrophenyl 2,3,5-tri-O-benzoyl-i- -fuco-
D
furanoside (5).—To a soln of 4 (1.0 g, 2.0
mmol) in dry CH2Cl2 (10.0 mL), SnCl4 (0.25
mL, 2.0 mmol) was added, and the soln was
stirred for 10 min at 0 °C. Then, 4-nitrophenol

12
A. Chiocconi et al. / Carbohydrate Research 323 (2000) 7–13
(0.30 g, 2.4 mmol) was added and the mixture
was stirred for 3 h at rt. The mixture was poured
into satd aq NaHCO3, extracted with CH2Cl2
and the organic extract was washed with water,
dried (MgSO4), filtered and evaporated. The
residue was purified by column chromatogra-
phy (19:1 toluene–EtOAc) to obtain com-
pound 5 (0.70 g, 60%) as a syrup; [h]D −97° (c
1, CHCl3). Anal. Calcd for C33H27O11: C, 66.33;
H, 4.55. Found: C, 66.52; H, 4.70.
pound 1. The syrup obtained (8, 0.57 g, 95%)
was purified by chromatography on a short
column (19:1 toluene–EtOAc); [h]D −75° (c 1,
13
CHCl3); C NMR (CDCl3) inter alia: l 106.0
(C-1%); 99.9, 95.5 (C-1, both anomers), 15.9,
15.4 (C-6% both anomers). Anal. Calcd for
C54H46O16: C, 68.20; H, 4.82. Found: C, 68.41;
H, 4.99.
3-O-i-
D
-Fucofuranosyl- -mannose (10).—
D
Compound 9 (0.30 g, 0.32 mmol) was sus-
pended in a 0.5 M soln of NaOMe in MeOH
(10.0 mL) and stirred until complete dissolution
occurred (2 h). The soln was made neutral with
Dowex 50W (H+), and concd. Methyl benzoate
was eliminated by repeated evaporation with
water. Compound 10 (0.10 g, 97%) was ob-
4-Nitrophenyl i- -fucofuranoside (6).—
D
Compound 5 (0.35 g, 0.6 mmol) was suspended
in a 0.5 M soln of NaOMe in MeOH (10.0 mL),
and stirred for 2 h. The soln was neutralized
with Dowex 50W (H+) resin, concd and water
was evaporated from the residue several times,
affording compound 6 (0.16 g, 94%); [h]D
−145° (c 1.4, CH3OH). Anal. Calcd for
C12H15NO7: C, 50.53; H, 5.30. Found: C, 50.33;
H, 5.47.
1
tained as a syrup; [h]D −13° (c 0.9, H2O); H
NMR (D2O) inter alia: l 5.18 (d, H-1 a anomer,
J1,21.8 Hz), 5.14 (d, H-1% b anomer, J1%,2% 1.2 Hz),
5.12 (d, H-1% b anomer, J1%,2% 1.5 Hz), 4.87 (d,
H-1 b anomer, J1,2 1.0 Hz), 1.22 (d, H-6% a
anomer, J5%,6% 6.1 Hz), 1.21 (d, H-6% b anomer, J
2,5,6-Tri-O-benzoyl-3-O-(2,3,5-tri-O-ben-
zoyl-i-
D
-fucofuranosyl)- -mannono-1,4-lac-
D
6.6 Hz);13C NMR (D2O) l: 105.1, 104.8
tone (8).—To a soln of 4 (0.57 g, 1.1 mmol) in
dry CH2Cl2 (10 mL), SnCl4 (0.14 mL, 1.21
mmol) was added, and the mixture was stirred
for 10 min at 0 °C. Compound 7 (0.58 g, 1.2
mmol) was added, and stirring was continued
for 3 h at rt. The soln was processed as
described for 5. The glycosyl-lactone obtained
(8, 0.68 g, 65%) was crystallized from EtOH,
and after recrystallization from the same sol-
vent, had a mp of 182–183 °C; [h]D −47° (c 1,
5%,6%
(C-1%), 94.9, 94.7 (C-1), 88.1 (C-4%), 82.4 (C-2%),
78.3 (C-3%), 75.9–66.0 (C-2,3,4,5), 68.3 (C-5%),
61.6 (C-6), 19.1, 16.4 (C-6%). Anal. Calcd for
C12H22O10: C, 44.17; H, 6.80. Found: C, 44.41;
H, 7.04.
Assays for specificity of exo i-
nosidase from P. fellutanum.—The enzyme was
incubated with 4-nitrophenyl b- -galactofura-
noside as a control reaction, with 4-nitrophenyl
b- -fucofuranoside (6) for studying the specifi-
D
-galactofura-
D
1
CHCl3); H NMR (CDCl3): l 8.09–7.13 (H-
D
aromatic), 6.05 (d, H-2, J2,3 3.8 Hz), 5.85 (m,
H-5%), 5.72 (d, H-2%, J2%,3% 0.8 Hz), 5.53 (bs, H-1%,
J1%,2%B0.5 Hz), 5.45 (dd, H-3%, J3%,4% 3.3 Hz), 5.31
(m, H-5), 5.12–5.01 (H-3,4,6a), 4.65 (dd, H-6b,
city of the enzyme, or with both, in order to
determine if compound 6 inhibited the enzyme.
The assays were done by incubating 100 mL of
the culture medium containing the enzyme (20
mg of protein), with 60 mL of a 5 mM soln of the
substrate in 100 mL of 66 mM NaOAc buffer
(pH 4.0), in a final volume of 500 mL. The
enzymatic reaction was started by the addition
of the enzyme, and after 1.5 h at 37 °C it was
stopped with 1 mL of 0.1 M Na2CO3 buffer (pH
9.0). The released 4-nitrophenol was measured
spectrophotometrically at 410 nm.
J5,6b 3.0 Hz, J
12.6 Hz), 4.39 (d, H-4%, J4%,5%
6a,6b
4.5 Hz), 1.13 (d, 3 H-6%, J 5%,6% 6.4 Hz); 13C NMR
(CDCl3): l 168.7 (C-1), 165.7–164.9 (CꢁO
benzoates), 133.9–125.6 (C-aromatics), 105.9
(C-1%), 85.5 (C-4%), 81.5 (C-2%), 77.0, 76.1 (C-
4,3%), 71.8 (C-3), 70.6 (C-5%), 69.2 (C-2), 67.9
(C-5), 61.8 (C-6), 15.5 (C-6%). Anal. Calcd for
C54 H44O16: C, 68.35; H, 4.67. Found: C, 68.64;
H, 4.45.
2,5,6-Tri-O-benzoyl-3-O-(2,3,5-tri-O-ben-
Acknowledgements
zoyl-i-
D
-fucofuranosyl)- -mannofuranose (9).
D
—Compound 8 (0.60 g, 0.63 mmol) was re-
duced with bis(3-methyl-2-butyl)borane (2.6
mmol) in THF (5 mL), as described for com-
We thank CONICET (Consejo Nacional de
Investigaciones Cient´ıficas y Te´cnicas) and the

A. Chiocconi et al. / Carbohydrate Research 323 (2000) 7–13
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