Synthesis of ethoxy-linked pseudo-disaccharides incorporating a crown ether macrocycle and lectin recognition

Article abstract of DOI:10.1016/S0008-6215(99)00194-9

Six ethoxy-linked pseudo-disaccharides incorporating a non-reducing d- galactopyranosyl residue and a glucose bearing an 18-crown-6-ether were synthesized by the trichloracetimidate method. Deprotection left four novel structures from which two were tested against the galactose-specific lectin Kluyveromyces bulgaricus. Their minimum inhibitory concentration (MIC) was in the same order of magnitude as compared to previously prepared oligosaccharides without spacer.

Full text of DOI:10.1016/S0008-6215(99)00194-9

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Carbohydrate Research 321 (1999) 214–227
Synthesis of ethoxy-linked pseudo-disaccharides
incorporating a crown ether macrocycle and lectin
recognitionꢀ
Be´atrice Dumont-Hornebeck a, Jean-Pierre Joly a,*, Joe¨l Coulon b, Yves Chapleur a
a Groupe Sucres, CNRS UMR 7565, Uni6ersite´ Henri Poincare´-Nancy I, Case Officielle 79, BP 239,
F-54506 Nancy-Vandœu6re, France
b Laboratoire de Biochimie Microbienne, Faculte´ des Sciences Pharmaceutiques et Biologiques,
Uni6ersite´ Henri Poincare´-Nancy I, 5 rue A. Lebrun, BP 403, F-54501 Nancy, France
Received 17 October 1998; revised 5 July 1999; accepted 26 July 1999
Abstract
Six ethoxy-linked pseudo-disaccharides incorporating a non-reducing
D
-galactopyranosyl residue and a glucose
bearing an 18-crown-6-ether were synthesized by the trichloracetimidate method. Deprotection left four novel
structures from which two were tested against the galactose-specific lectin Kluy6eromyces bulgaricus. Their minimum
inhibitory concentration (MIC) was in the same order of magnitude as compared to previously prepared oligosaccha-
rides without spacer. © 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Sugar crown ethers; Pseudo-disaccharides; Trichloracetimidates; Ozonolysis; Galactosylation; Kluy6eromyces bulgari-
cus; Lectin recognition
two-carbon tether between the two sugar
residues, by a similar method. Two poss-
ibilities have been explored according to the
position of the ethylene glycol spacer: (i) at
C-1 of the glycose unit bearing the crown
ether (Scheme 1(A)); (ii) at C-4 of this unit
(Scheme 1(B)).
1. Introduction
In Part 1 of this series [1], we reported the
synthesis of di- and trisaccharides bearing an
18-crown-6-ether
trichloracetimidate as donor and secondary
alcohol groups as acceptors. Assuming that an
increase in the distance between the galactose
residue and the macrocycle would be
beneficial to the recognition of the galactose
unit by a specific lectin, we now report the
synthesis of pseudo-disaccharides, having a
macrocycle
using
a
2. Results and discussion
The allyl group, easily introduced by
alkylation and cleaved by ozone [2], was used
throughout this study as the common
precursor of the two-carbon spacer. The
ꢀ Lectin-targeted crown ethers, Part 2. For Part 1, see Ref.
[1]. Part of the University Thesis of B. Dumont-Hornebeck
(Nancy, 1997), presented in part at the 18th International
Carbohydrate Symposium, Milan, 21–26 July, 1996.
* Corresponding author. Fax: +33-3-8391-2479.
E-mail address: jean-pierre.joly@meseb.uhp-nancy.fr (J.-P.
Joly)
synthesis of allyl
D-glucopyranoside crown
ethers is summarised in Scheme 2.
Protected crown ethers 3 and 4 were
obtained by usual methods [3] from allyl
0008-6215/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(99)00194-9

B. Dumont-Hornebeck et al. / Carbohydrate Research 321 (1999) 214–227
215
Scheme 1.
D
-glycosides 2 and 1 in 89 and 64% yield,
−10 °C in the same solvents on allyl glycoside
6 to afford 17 almost quantitatively (Scheme 4).
The synthesis of 18 and 19 has been reported
[1]. The preferred strategy to synthesize
6-amino-crown ethers was to introduce the
amine-functionality after the glycosylation step
of the 6-bromo or 6-azido precursors. The
6-azido-6-deoxy sugar 21 could be obtained in
two steps from 18 after quantitative
4-O-alkylation in a two-phase system (allyl
bromide, 50% aqueous NaOH) at room
temperature (Scheme 5). In this case, the crown
ether could play both the role of substrate and
phase-transfer catalyst. Nucleophilic displace-
ment of the bromide in 20 by sodium azide in
N,N-dimethylformamide around 130 °C also
afforded 21 in excellent yield, but a more polar
by-product appeared if the reaction time
respectively in two steps.
Two methyl groups were easily introduced in
5 under phase-transfer catalysis conditions
(Me2SO4, 50% aqueous NaOH, room
temperature) to afford 6 in 96% yield (Scheme
3). Curiously, mono-alkylation at C-4 via a
classical protection/O-alkylation/deprotection
sequence failed in our hands to give the target
alcohol 9 in good yield; a major degradation
product (not further investigated) was obtained
at the deprotection step and (p-TsOH, MeOH)
in addition to 9 as the minor product (ꢀ35%).
Nevertheless, the same sequence applied in the
b-series gave the monomethyl ether 12 from
which the 6-azido-6-deoxy sugar 14 could be
prepared by tosylation and subsequent
nucleophilic displacement by sodium azide
(38% overall yield from 3). Finally, the allyl
1
exceeded 1 h. The H NMR spectrum of this
group
underwent
ozonolysis
at
low
by-product showed the absence of allylic signals
between 5.1 and 5.9 ppm and its EIMS+
spectrum (70 eV) displayed a molecular peak at
temperature and the intermediate ozonide was
reduced in situ with sodium borohydride to give
the acceptor 15 in 69% yield. Optimisation of
the ozonolysis step was achieved within 1 h at
m/z 509 [M] + and another intense peak at m/z
’
Scheme 2. (i) (ClCH2CH2)2O, 50% aqueous NaOH, Bu4NHSO4; (ii) catechol, K2CO3, n-BuOH.

216
B. Dumont-Hornebeck et al. / Carbohydrate Research 321 (1999) 214–227
Scheme 3.
+
’
481 [M–28] . As a similar reaction has been
reported by Lamberth and Bednarski on allyl
glycosides bearing an azide at C-2 [4], we
postulated that an intramolecular cycloaddi-
tion took place when the azido-sugar was
heated over too long a period and that, at least,
a nitrogen molecule was likely to be extruded
to produce an aziridine. As for 21, the allyl
ether 23 was isolated by the phase-transfer
reaction of 22 with allyl bromide. Monosubsti-
tuted glycol ethers 24, 25 and 26 were isolated
after an in situ ozonolysis/reduction sequence
of the corresponding 4-O-allyl precursors 23,
20 and 21, respectively. Alternatively, the 6-de-
oxysugar 24 could be obtained by quantitative
reduction of 25 with lithium aluminium hy-
dride. The primary alcohol 15 was glycosylated
hol as acceptor. Classical Zemple´n deacetyla-
tion afforded the deprotected carriers 32, 33, 34
and 38 quantitatively from their respective
precursors 28, 31, 30 and 35. For unclear
reasons, the reduction of the azide 37 failed to
yield the corresponding amine (Scheme 7, R=
Ac, R1=NH2) even in a Paar reactor under 5
MPa hydrogen pressure in the presence of 10%
palladium on charcoal.
The in vitro flocculation inhibition capabili-
ties of oligosaccharides A, B, C and D prepared
in Part 1 of this series [1] toward the yeast
Kluy6eromyces bulgaricus (Kb CWL1) lectin
[5,6] are compared with those of the presently
synthesized galactosides 34 and 38 in Table 1.
with
D
-galactose trichloracetimidates to give
the ethoxy-linked (1“1)-disaccharide 27 in
55% yield (Scheme 6).
Boron trifluoride diethyl etherate was pre-
ferred to trimethylsilyl triflate (Me3SiOTf) as
catalyst since better yield and selectivity had
previously been observed with this Lewis acid
used in a two-fold excess with crown ethers as
acceptors [1]. In the same manner, glycols 16,
17, 24, 25 and 26 were reacted as donors to yield
pseudo-disaccharides 28, 29, 30, 35, 36 and 37
(Scheme 7).
However, the cleavage of the benzylidene
acetal in 28 to yield mainly 29 after only 2 h of
reaction at room temperature was unexpected.
Judging from the 1H NMR coupling constants,
only a b-glycosidic linkage could be ascertained
in all these galactosides in opposition to a
preceding result obtained with a primary alco-

B. Dumont-Hornebeck et al. / Carbohydrate Research 321 (1999) 214–227
217
Scheme 4. (i) O3, THF–MeOH, −75 °C, then NaBH4, −75 °C“rt; (ii) O3, THF–MeOH, −10 °C, then NaBH4, −10 °C“rt.
3. Experimental
These biological results altogether gave con-
spicuous proof of the in vitro activity of the
non-reducing
D
-galactose moiety after the gly-
Synthesis of crown ethers and general meth-
ods and materials.—The syntheses of
cosylation step with acceptors bearing a crown
ether (compounds A, B, C, D [7,8]) or with a
functionalised monoether of ethylene glycol
(34 and 38). More precisely, the incorporation
of an intermediate sugar as hydrophilic spacer
in compound D does not really change the
recognition by the lectin. The presence of a
free amine group at C-6 of the acceptor
slightly increased the minimum inhibitory
concentration (MIC) of the disaccharide B.
However, one must admit that the primary
amine group is mainly protonated in the ac-
etate buffer (pHꢀ4.5) used in all tests, and
that the MIC of B may be different at a
physiological pH (ꢀ7.2). The best results
were measured with compound C incorporat-
ing two galactose units without spacer and
with compound 34. Yet these differences are
small and cannot be considered as significant
improvements.
D
-galac-
tose trichloracetimidate and crown ethers A,
B, C and D have been described in Part 1 of
this series [1]. Due to shortage of products,
elemental analysis has not been performed.
Allyl [2,3 - b](11,12 - benzo - 1,4,7,10,13,16-
hexaoxacyclooctadeca-11-ene)-4,6-O-benzyl-
idene-2,3-dideoxy-i-
To a stirred dispersion of allyl 4,6-O-benzyli-
dene- -glucopyranosides [9] (31.5 g, 102
D
-glucopyranoside (3).—
D
mmol) and n-Bu4NHSO4 (69.3 g, 2.0 equiv) in
bis(2-chloroethyl)ether (300 mL) were added
50% aq NaOH (300 mL) below 10 °C. The
two-phase system was vigorously stirred below
15 °C for 6 h, the reaction being monitored by
TLC (EtOAc). The reaction mixture was then
partitioned between chilled water (800 mL)
and CH2Cl2 (500 mL). The aq phase was
extracted with CH2Cl2 (3×200 mL), the or-
ganic phases were combined, washed with wa-
ter (2×100 mL), dried over MgSO4,
concentrated under reduced pressure, and the
excess of reagent finally removed in vacuo.
Chromatography with 9:1 n-hexane–EtOAc
successively yielded allyl 4,6-O-benzylidene-
In summary, four simple models (com-
pounds 32, 33, 34 and 38) incorporating
a two-carbon spacer between two hexopyra-
nosides, have been synthesised in good yields
by a convergent approach using the tri-
chloroacetimidate method from
-galactose. A unique b-glycosidic linkage
D
-glucose and
D
was observed in the main isolated reaction
products. Deprotected compounds 34 and
38 gave similar results to oligosaccharides A,
B, C and D [1] when tested in vitro towards
a galactose-specific lectin. Taking all our re-
sults into account, it is strongly suggested that
the lectin isolated from K. bulgaricus recog-
nises the non-reducing
D
-galactose residue of
these six compounds in a very similar
manner.
Scheme 5.

218
B. Dumont-Hornebeck et al. / Carbohydrate Research 321 (1999) 214–227
Table 1
Comparative minimum inhibitory concentrations (MICs) of
-glucose, -galactopyranoside, lac-
D
D
-galactose, methyl b-
D
tose, pseudo-disaccharides 34, 38 and disaccharide derivatives
A–D towards Kb CWL1 a
b
Carbohydrate
MIC (mM)
D
D
-Glucose
Negative
3.50
1.75
1.25
2.65
3.75
2.10
2.65
2.00
-Galactose
Methyl b-
Lactose
A [1]
B [1]
C [1]
D [1]
34
D
-galactopyranoside
Scheme 6. (i)
D-Galactose trichloracetimidates (2 equiv),
,
crushed activated 4 A MS, CH2Cl2, rt, 30 min, then Et2O·BF3
(2 equiv), rt, 20 h.
2,3-O-bis[2-(2-chloroethoxy)ethyl]-b- -gluco-
D
pyranoside (6.52 g, 12.5%) and allyl 4,6 - O -
benzylidene - 2,3 - O - bis[2 - (2 - chloroethoxy)-
38
2.50
a The lectin activity was 3.4 AU/mg of protein (1 UA“in-
hibition capacity of a 0.5 mg/mL lectin solution towards a
given yeast cell suspension).
ethyl]-a- -glucopyranoside (19.0 g, 36.4%)
D
as colourless gums pure enough for the next
step. From condensation of allyl 4,6-O-ben-
zylidene-2,3-O-bis[2-(2-chloroethoxy)ethyl]-
b The MIC is the lowest concentration that totally inhibits
flocculation (mean value of three measurements).
b- -glucopyranoside (4.00 g, 7.67 mmol) with
D
pyrocatechol (1.69 g, 2 equiv) according to
Ref. [10], after 24 h of reflux in n-BuOH,
usual work-up and chromatography with 1:1
EtOAc–n-hexane, the crown ether 3 was ob-
tained (3.856 g, 90%) as a white solid: mp
Allyl [2,3 - b](11,12 - benzo - 1,4,7,10,13,16-
hexaoxacyclooctadeca-11-ene)-4,6-O-benzyl-
idene-2,3-dideoxy-h-
Cyclisation of allyl 4,6-O-benzylidene-2,3-O-
bis[2-(2-chloroethoxy)ethyl]-a- -glucopyrano-
D
-glucopyranoside (4).—
D
1
123–125 °C; [h]D −42.0° (c 1, CHCl3); H
side (6.0 g, 11.5 mmol) with pyrocatechol
(2.53 g, 2 equiv) afforded, after 24 h of reflux
in n-BuOH, usual work-up and chromatogra-
phy with 1:1 EtOAc–n-hexane, the crown-
ether 4 (4.495 g, 70%) as a white solid: mp
NMR (CDCl3): l 7.42 (m, 2 H, H-2, -6, Ar),
7.4–7.3 (m, 3 H, H-3,-4, -5, Ar), 6.9 (bs, 4 H,
catechol), 5.92 (dddd, 1 H, OCH2ꢀCHꢁCH2),
5.52 (s, 1 H, Hb), 5.33 (dq, 1 H, Jtrans 17.0 Hz,
OCH2ꢀCHꢁCHH), 5.21 (dq, 1 H, Jcis 10.5 Hz,
Jgem 51.5 Hz, OCH2ꢀCHꢁCHH), 4.48 (d,
1 H, H-1), 4.42–3.62 (m, 20 H, 9×OCH2,
H-3, -6%), 3.55 (bt, 1 H, J3–4 8.0 Hz, J4–5 9.0
Hz, H-4), 3.53 (dd, 1 H, J5–6 9.0 Hz, J6–6% 14.0
Hz, H-6), 3.35 (m, 1 H, H-5), 3.27 (t, 1 H, J1–2
1
153–155 °C; [h]D +38.6° (c 1, CHCl3); H
NMR (CDCl3): l 7.47 (m, 2 H, Ar), 7.35 (m,
2 H, Ar), 7.23 (d, 1 H, Ar), 6.88 (bs, 4 H,
catechol), 5.92 (dddd, 1 H, OCH2ꢀCHꢁCH2),
5.52 (s, 1 H, Hb), 5.33 (dq, 1 H, Jtrans 17.0 Hz,
OCH2ꢀCHꢁCHH), 5.22 (dq, 1 H, Jcis 10.5 Hz,
Jgem51.5 Hz, OCH2ꢀCHꢁCHH), 4.98 (d, 1
H, H-1), 4.28–3.75 (m, 21 H, 9×OCH2, H-3,
7.8 Hz, J2–3 8 Hz, H-2); EIMS: m/z 558
+
’
[M] .
Scheme 7.

B. Dumont-Hornebeck et al. / Carbohydrate Research 321 (1999) 214–227
219
-5, -6%), 3.7 (dd, 1 H, J5–6 3.5 Hz, J6–6% 10.0 Hz,
H-6), 3.55 (t, 1 H, J3–4 ꢀJ4–5 9 Hz, H-4), 3.48
H, H-1), 4.23–3.7 (m, 20 H, 9×OCH2, H-6,
-6%), 3.63 (m, 2 H, H-3, -5), 3.54 (s, 3 H, OCH3
on C-4), 3.39 (s, 3 H, OCH3 on C-6), 3.38 (d,
1 H, J1–2 3.7 Hz, J2–3 9.5 Hz, H-2), 3.25 (t, 1
(dd, 1 H, J1–2 3.7 Hz, J2–3 9.5 Hz, H-2);
+
’
EIMS: m/z 558 [M] .
Allyl [2,3 - b](11,12 - benzo - 1,4,7,10,13,16-
hexaoxacyclooctadeca - 11 - ene) - 2,3 - dideoxy -
H, J3–4 ꢀJ4–5 9.5 Hz, H-4); EIMS: m/z 498
+
’
[M] .
Allyl [2,3 - b](11,12 - benzo - 1,4,7,10,13,16-
hexaoxacyclooctadeca - 11 - ene) - 2,3 - dideoxy -
6-O-trityl-h- -glucopyranoside (7).—To
h-D
-glucopyranoside (5).—Crown ether
4
(2.00 g, 3.58 mmol) was dissolved in AcOH
(60 mL) at 90 °C and distilled water (40 mL)
was then dropped onto the soln at the same
temperature. The reaction was monitored by
TLC (EtOAc) and was completed after 3 h.
The solvents were evaporated under reduced
pressure and the remaining gum dissolved in
CH2Cl2 (100 mL), washed by aq satd
NaHCO3 (15 mL) and twice by water (10
mL). The isolated organic phase was then
dried over MgSO4 and concentrated under
reduced pressure to yield 5 (1.63 g, \99%) as
a colourless homogeneous gum pure enough
for the next step: [h]D +71.6° (c 1, CHCl3);
1H NMR (CDCl3–D2O): l 6.88 (bs, 4 H,
catechol), 5.9 (dddd, 1 H, OCH2ꢀCHꢁCH2),
5.31 (dq, 1 H, Jtrans 17.0 Hz, OCH2ꢀ
D
a
stirred solution of diol 5 (1.63 g, 3.47 mmol)
in toluene (10 mL) and pyridine (1.0 mL) was
added trityl chloride (1.45 g, 1.5 equiv) and
the mixture heated to a gentle reflux. The
reaction was monitored by TLC (EtOAc) and
stopped after 10 h, allowed to cool, concen-
trated to a gum, which was dissolved in
CH2Cl2 (50 mL) to give, after usual work-up
and chromatography on deactivated silica gel
(1% NEt3) with 1:2 EtOAc–n-hexane, the pro-
tected alcohol 7 (1.53 g, 60%) as a white foam:
1
mp 63–65 °C; [h]D +37.0° (c 1, CHCl3); H
NMR (CDCl3): l 7.47 (d, 6 H, Ar), 7.34–7.18
(m, 9 H, Ar), 6.88 (bs, 4 H, catechol), 5.96
(dddd, 1 H, OCH2ꢀCHꢁCH2), 5.33 (dq, 1 H,
Jtrans 17.0 Hz, OCH2ꢀCHꢁCHH), Jgem 51.5
Hz, OCH2ꢀCHꢁCHH), 5.22 (dq, 1 H, Jcis 10.0
Hz, 5.01 (d, 1 H, H-1), 4.29–3.7 (m, 19 H,
8.5×OCH2, H-4, -5), 3.6 (t, 1 H, J3–4 ꢀ9.0
Hz, H-3), 3.57 (d, 1 H, OCHH), 3.45 (dd, 1
H, J1–2 3.5 Hz, J2–3 9.0 Hz, H-2), 3.37 (dd, 1
H, J5–6% 3.5 Hz, H-6%), 3.3 (dd, 1 H, J5–6 5.0
CHꢁCHH), 5.2 (dq, 1 H, Jcis 10.0 Hz, Jgem
5
1.5 Hz, OCH2ꢀCHꢁCHH), 4.94 (d, 1 H, H-1),
4.37–3.61 (m, 20 H, 9×OCH2, H-6%,-5), 3.57
(t, 1 H, J4–5 8.5 Hz, H-4), 3.45 (t, 1 H,
J3–4 ꢀ8.0 Hz, H-3), 3.44 (dd, 1 H, J5–6 9.0 Hz,
J6–6% 14.0 Hz, H-6), 3.4 (dd, 1 H, J1–2 3.8 Hz,
+
’
J2–3 8.5 Hz, H-2); EIMS: m/z 470 [M] .
Allyl [2,3 - b](11,12 - benzo - 1,4,7,10,13,16-
hexaoxacyclooctadeca - 11 - ene) - 2,3 - dideoxy -
Hz, J6–6% 9.5 Hz, H-6), 2.8 (bs, 1 H, OH);
+
’
EIMS: m/z 712 [M] .
4,6-di-O-methyl-h-
D
-glucopyranoside (6).—To
Allyl [2,3 - b](11,12 - benzo - 1,4,7,10,13,16-
hexaoxacyclooctadeca - 11 - ene) - 2,3 - dideoxy -
a soln of 5 (0.174 g, 0.369 mmol) in toluene
(10 mL) were added 10 mL of 50% aq NaOH
and Me2SO4 (1 mL) under the fume board.
The resulting mixture was magnetically stirred
for 20 h at room temperature (rt) and then
quenched with chilled water (40 mL). The
isolated aq phase was extracted with CH2Cl2
(3×50 mL) and the combined organic phases
washed with water (2×10 mL), dried over
MgSO4, and concentrated under reduced pres-
sure to yield the crown ether 6 (0.176 g, 96%)
as a white amorphous solid: mp 81–83 °C;
4-O-methyl-6-O-trityl-h- -glucopyranoside
D
(8).—A soln of Me2SO4 (1 mL, 8.5 equiv) in
toluene (5 mL) was vigorously stirred for 5
min at rt with 50% aq NaOH (10 mL) under
the fume board. Then 7 (0.89 g, 1.24 mmol)
was added and the mixture stirred again for
20 h. Chilled water (100 mL) was added and
the organic phase was extracted with CH2Cl2
(3×50 mL). The organic extracts were com-
bined and washed with water (3×20 mL),
dried (MgSO4), concentrated in vacuo, and
purified by elution with 1:1 EtOAc–n-hexane
through basic alumina to yield 8 (0.85 g, 95%)
as a pale yellow gum: [h]D +40.3° (c 1,
1
[h]D +68.1° (c 1, CHCl3); H NMR (CDCl3):
l 6.88 (bs, 4 H, catechol), 5.9 (dddd, 1 H,
OCH2ꢀCHꢁCH2), 5.3 (dq, 1 H, Jtrans 17.0 Hz,
OCH2ꢀCHꢁCHH), 5.18 (dq, 1 H, Jcis 10.0 Hz,
Jgem 51.5 Hz, OCH2ꢀCHꢁCHH), 4.96 (d, 1
1
CHCl3); H NMR (CDCl3): l 7.46 (d, 6 H,
Ar), 7.33–7.2 (m, 9 H, Ar), 6.87 (bs, 4 H,

220
B. Dumont-Hornebeck et al. / Carbohydrate Research 321 (1999) 214–227
5.33 (dq, 1 H, Jtrans 17.2 Hz, OCH2ꢀ
CHꢁCHH), 5.2 (dq, 1 H, Jcis 10.0 Hz, Jgem
catechol), 5.9 (dddd, 1 H, OCH2ꢀCHꢁCH2),
5.19 (dq, 1 H, Jcis 10.0 Hz, Jgem 51.5 Hz,
OCH2ꢀCHꢁCHH), 4.92 (d, 1 H, H-1), 4.24–
4.02 (m, 7 H, 3.5×OCH2), 4.0–3.7 (m, 13 H,
5.5×OCH2, H-5, -6%), 3.66 (t, 1 H, H-3), 3.57
(s, 3 H, OCH3), 3.38 (dd, 1 H, J5–6 3.0 Hz,
J6–6% 11.5 Hz, H-6), 3.36 (dd, 1 H, J1–2 3.5 Hz,
5
1.5 Hz, OCH2ꢀCHꢁCHH), 4.37 (d, 1 H, H-1),
4.25–3.6 (m, 20 H, 9×OCH2, H-4, -5), 3.57
(t, 1 H, J3–4 9.0 Hz, H-3), 3.4–3.3 (m, 2 H,
H-6, -6%), 3.24 (dd, 1 H, J1–2 7.0 Hz, J2–3 ꢀ8.5
Hz, H-2), 2.9 (bs, 1 H, OH); EIMS: m/z 712
+
’
[M] .
J2–3 9.5 Hz, H-2), 3.22 (t, 1 H, J3–4 ꢀJ4–5 9.0
+
’
Hz, H-4); EIMS: m/z 726 [M] .
Allyl [2,3-b](11,12 - benzo - 1,4,7,10,13,16-
hexaoxacyclooctadeca - 11 - ene) - 2,3 - dideoxy -
4-O-methyl-6-O-trityl-i- -glucopyranoside
Allyl [2,3 - b](11,12 - benzo - 1,4,7,10,13,16-
hexaoxacyclooctadeca - 11 - ene) - 2,3 - dideoxy -
D
4-O-methyl-h-
D
-glucopyranoside (9).—A soln
(11).—The same procedure was used as de-
scribed for 8 except for the reaction time,
which was 14 h. From the alcohol 10 (0.94 g,
1.3 mmol) was obtained, after usual work-up
and chromatography on deactivated silica gel
(1:1 EtOAc–n-hexane), the crown ether 11
(0.79 g, 83%) as a pale yellow gum: [h]D
of 8 (0.65 g, 0.89 mmol) and p-TsOH (17 mg,
0.1 equiv) in MeOH (80 mL) was magnetically
stirred at rt. The reaction, monitored by TLC
(EtOAc), was stopped after 24 h by addition
of NaHCO3 (0.15 g, 2.0 equiv). The mixture
was stirred again for 15 min, concentrated
under reduced pressure to a gum, which was
dissolved in CH2Cl2 (50 mL), washed twice
with water (15 mL), dried (MgSO4), concen-
trated in vacuo and purified by chromatogra-
phy (2:1 EtOAc–n-hexane) on silica gel to
yield 9 (0.16 g, 35%) as a homogeneous gum:
1
−11.5° (c 1, CHCl3); H NMR (CDCl3): l
7.55–7.4 (m, 6 H, Ar), 7.33–7.2 (m, 9 H, Ar),
6.9 (s, 4 H, catechol), 5.99 (dddd, 1 H,
OCH2ꢀCHꢁCH2), 5.35 (dq, 1 H, Jtrans 17.0 Hz,
OCH2ꢀCHꢁCHH), 5.22 (dq, 1 H, Jcis 10.0 Hz,
Jgem51.5 Hz, OCH2ꢀCHꢁCHH), 4.43 (ddd, 1
H, OCHH), 4.36 (d, 1 H, H-1), 4.3–3.7 (m, 18
H, H-5, 8.5×OCH2), 3.62 (bt, 1 H, J3–4 ꢀ9.0
Hz, H-3), 3.5–3.13 (m, 6 H, OCH3, H-4, -6,
1
[h]D +78.3° (c 1, CHCl3); H NMR (CDCl3):
l 6.88 (bs, 4 H, catechol), 5.89 (dddd, 1 H,
OCH2ꢀCHꢁCH2), 5.3 (dq, 1 H, Jtrans 17.0 Hz,
OCH2ꢀCHꢁCHH), 5.19 (dq, 1 H, Jcis 10.0 Hz,
Jgem 51.5 Hz, OCH2ꢀCHꢁCHH), 4.93 (d, 1
H, H-1), 4.23–3.65 (m, 21 H, 9×OCH2, H-5,
-6, -6%), 3.64 (t, 1 H, H-3), 3.55 (s, 3 H,
OCH3), 3.34 (dd, 1 H, J1–2 3.6 Hz, J2-3 9.0 Hz,
-6%), 3.07 (dd, 1 H, J1–2 7.0 Hz, J2–3 9.0 Hz,
+
’
H-2); EIMS: m/z 726 [M] .
Allyl [2,3 - b](11,12 - benzo - 1,4,7,10,13,16-
hexaoxacyclooctadeca - 11 - ene) - 2,3 - dideoxy -
4-O-methyl-i- -glucopyranoside (12).—The
D
H-2), 3.21 (t, 1 H, J3–4 ꢀJ4–5 9.0 Hz, H-4), 1.9
same procedure was used as described for 9
except for the reaction time, which was 14 h.
From the trityl ether 11 (0.74 g, 1.02 mmol)
the crown ether 12 (0.254 g, 50%) was ob-
tained after usual work-up and chromatogra-
phy on silica gel (1:2 EtOAc–n-hexane) as a
pale yellow gum: [h]D −20.8° (c 1, CHCl3);
1H NMR (CDCl3): l 6.89 (bs, 4 H, catechol),
5.91 (dddd, 1 H, OCH2ꢀCHꢁCH2), 5.3 (dq, 1
H, Jtrans 17.0 Hz, OCH2ꢀCHꢁCHH), 5.18 (dq,
1 H, Jcis 10.0 Hz, Jgem 51.5 Hz, OCH2ꢀ
CHꢁCHH), 4.43 (ddd, 1 H, OCHH), 4.37 (d,
1 H, J1–2 7.3 Hz, H-1), 4.3–3.73 (m, 19 H,
8.5×OCH2, H-6, -6%), 3.69 (ddd, 1 H, J4–5
10.0 Hz, J5–6 4.5 Hz, J5–6% 8.0 Hz, H-5),
3.55 (s, 3 H, OCH3), 3.33 (t, 1 H, J3–4 9.0 Hz,
H-3), 3.23–3.12 (m, 2 H, J2–3 ꢀ8 Hz,
+
’
(bs, 1 H, OH); EIMS: m/z 484 [M] .
Allyl [2,3 - b](11,12 - benzo - 1,4,7,10,13,16-
hexaoxacyclooctadeca - 11 - ene) - 2,3 - dideoxy -
6-O-trityl-i-
D
-glucopyranoside
(10).—The
same procedure was used as described for 5
except for the reaction time, which was only 2
h. From crown ether 3 (0.844 g, 1.51 mmol)
was obtained the corresponding diol (0.75 g,
99%) as a homogeneous gum pure enough for
the next step. The same procedure of trityla-
tion and purification was used as described for
7 except for reaction time, which was 14 h.
From 0.71 g of the starting material was ob-
tained 0.97 g (92%) of trityl ether 10 as a pale
yellow foam: mp B50 °C; [h]D −13.6° (c 1,
1
CHCl3); H NMR (CDCl3): l 7.46 (d, 6 H,
Ar), 7.33–7.2 (m, 9 H, Ar), 6.88 (bs, 4 H,
H-2, -4), 1.7 (bs, 1 H, OH); EIMS: m/z 484
+
’
[M] .
catechol), 5.96 (dddd, 1 H, OCH2ꢀCHꢁCH2),

B. Dumont-Hornebeck et al. / Carbohydrate Research 321 (1999) 214–227
221
CHꢁCHH), 4.35 (ddd, 1 H, OCHH), 4.34 (d,
1 H, H-1), 4.25–3.7 (m, 18 H, 8.5×OCH2,
H-6%), 3.52 (s, 3 H, OCH3), 3.39 (dd, 1 H,
H-6), 3.34 (m, 1 H, J5–6 4.0 Hz, J5–6% 6.5 Hz,
H-5), 3.31 (t, 1 H, H-3), 3.19 (dd, 1 H, J1–2 7.5
Allyl [2,3 - b](11,12 - benzo - 1,4,7,10,13,16-
hexaoxacyclooctadeca - 11 - ene) - 2,3 - dideoxy -
4-O-methyl-6-O-tosyl-i- -glucopyranoside
D
(13).—To a stirred soln of the alcohol 12
(0.23 g, 0.464 mmol) in abs pyridine (10 mL)
was added p-TsCl (0.27 g, 3 equiv) and the
mixture was heated to 100 °C under argon.
The reaction was monitored by TLC (EtOAc)
and cooled after 13 h, concentrated to a gum,
which was dissolved in CH2Cl2 (50 mL) and
washed with HCl (0.1 N, 20 mL), satd
NaHCO3 (10 mL), and water (10 mL) to
afford, after elution through neutral alumina
(EtOAc), the crown ether 13 (0.30 g, 99%) as
a colourless gum: [h]D −15.3° (c 1, CHCl3);
1H NMR (CDCl3): l 7.78 (d, 2 H, Ar), 7.32
(d, 2 H, Ar), 6.89 (bs, 4 H, catechol), 5.85
(dddd, 1 H, OCH2ꢀCHꢁCH2), 5.27 (dq, 1 H,
Jtrans 17.0 Hz, OCH2ꢀCHꢁCHH), 5.16 (dq, 1
H, Jcis 10.0 Hz, Jgem51.5 Hz, OCH2ꢀ
CHꢁCHH), 4.25 (d, 1 H, H-1), 4.3–3.65 (m,
20 H, 9×OCH2, H-6, -6%), 3.48 (s, 3 H,
OCH3), 3.31 (m, 1 H, J4–5 10, J5-6 2.0 Hz, J5–6%
5.5 Hz, H-5), 3.28 (t, 1 H, H-3), 3.1 (bt, 1 H,
J1–2 7.5 Hz, J2–3 9.0 Hz, H-2), 3.07 (t, 1 H,
Hz, J2–3 9.0 Hz, H-2), 3.07 (t, 1 H, J3–4 ꢀJ4–5
+
’
9 Hz, H-4); EIMS: m/z 509 [M] .
(2-Hydroxy-ethyl) 6 - azido - [2,3 - b](11,12-
benzo-1,4,7,10,13,16-hexaoxacyclooctadeca-11-
ene)-4-O-methyl-2,3,6-trideoxy-i- -gluco-
D
pyranoside (15).—A 1:9 O3–O2 mixture was
bubbled through a stirred soln of crown ether
14 (0.20 g, 0.39 mmol) in 1:1 THF–MeOH
(20 mL) cooled between −70 and −78 °C.
The ozonolysis was monitored by TLC
(EtOAc) and stopped by streaming pure O2
into the solution after 3 h. Two equiv of
NaBH4 (30 mg) were then added and the
resulting stirred solution allowed to warm to
rt. The reduction was also monitored by TLC
(EtOAc) and stopped by evaporation of the
solvents after 24 h. The residue was dissolved
in CH2Cl2 (50 mL), washed with water (2×10
mL), dried over MgSO4, and chro-
matographed on a silica gel column with
EtOAc to yield 15 (0.140 g, 69%) as a colour-
less gum: [h]D −31.0° (c 1, CHCl3); IR
J3–4 ꢀJ4–5 9 Hz, H-4), 2.45 (s, 3 H, ArCH3);
+
’
EIMS: m/z 638 [M] .
1
(NaCl) w 2100 cm−1 (N3); H NMR (CDCl3):
Allyl 6-azido-[2,3-b](11,12-benzo-1,4,7,10,-
13,16 - hexaoxacyclooctadeca - 11 - ene) - 4 - O -
l 6.95–6.83 (m, 4 H, catechol), 4.35 (d, 1 H,
H-1), 4.25–3.7 (m, 20 H, 10×OCH2), 3.53 (s,
3 H, OCH3), 3.5–3.4 (m, 2 H, J6–6% 13.0 Hz,
H-6, -6%), 3.35 (m, 1 H, J5–6 5.5 Hz, J5–6% 3.0
Hz, H-5), 3.34 (t, 1 H, H-3), 3.26 (t, 1 H, J1–2
methyl - 2,3,6 - trideoxy - i -
D
- glucopyranoside
(14).—To a stirred soln of the tosylate 13
(0.28 g, 0.432 mmol) in DMF (10 mL) was
added NaN3 (56 mg, 2 equiv) and the result-
ing mixture immediately heated to 150 °C.
The reaction was monitored by TLC (EtOAc)
— the azido-sugar producing a typical brown-
ish colour after being sprayed with dil H2SO4
and heated to around 250 °C — and was
allowed to cool after 7 h. The solvent was
evaporated in vacuo and the residue dissolved
in CH2Cl2 (50 mL) and washed with water
(3×10 mL) to afford after rapid chromatog-
raphy on neutral alumina (CH2Cl2“EtOAc)
the crown ether 14 (0.22 g, 99%) as a pale
yellow wax: mp B50 °C; [h]D −40.5° (c 1,
7.5 Hz, J2–3 8.0 Hz, H-2), 3.08 (t, 1 H, J3–4
ꢀ
J4–5 8.0 Hz, H-4), 1.65 (bs, 1 H, OH); EIMS:
+
’
m/z 527 [M] .
(2-Hydroxy-ethyl) [2,3-b](11,12-benzo-1,4,-
7,10,13,16 - hexaoxacyclooctadeca - 11 - ene)-
4,6-O-benzylidene-2,3-dideoxy-h- -glucopyr-
D
anoside (16).—The same procedure was used
as for the synthesis of 15 except for the reac-
tion time, which was 8 h for the ozonolysis and
12 h for the reduction steps. From 3 (0.15 g,
0.27 mmol) the alcohol 16 (79 mg, 53%) was
isolated as a colourless wax, after chromatog-
raphy on silica gel with 1:1 EtOAc–n-hexane
and finally with 4:1 EtOAc–EtOH. MpB
1
CHCl3); IR (NaCl) w 2099 cm−1 (N3); H
1
NMR (CDCl3): l 6.88 (bs, 4 H, catechol), 5.9
(dddd, 1 H, OCH2ꢀCHꢁCH2), 5.3 (dq, 1 H,
Jtrans 17.0 Hz, OCH2ꢀCHꢁCHH), 5.17 (dq, 1
H, Jcis 10.0 Hz, Jgem51.5 Hz, OCH2ꢀ
50 °C; [h]D +39.4° (c 1, CHCl3) H NMR
(CDCl3): l 7.5–7.41 (m, 2 H, Ar), 7.4–7.3 (m,
3 H, Ar), 6.89 (bs, 4 H, catechol), 5.52 (s, 1 H,
Hb), 4.99 (d, 1 H, H-1), 4.3–3.67 (m, 23 H,

222
B. Dumont-Hornebeck et al. / Carbohydrate Research 321 (1999) 214–227
79
+
’
Hz, H-4); EIMS: m/z 546 [ BrM] and 548
10×OCH2, H-3, -6, -6%), 3.61 (m, 1 H, J5–6
81
+
’
[ BrM] .
2.5 Hz, J5–6% 7.5 Hz, H-5), 3.53 (t, 1 H, J3–4
ꢀ
Methyl 4 - O - allyl - 6 - azido - [2,3 - b](11,12-
benzo-1,4,7,10,13,16-hexaoxacyclooctadeca-11-
ene)-2,3,6-trideoxy-h- -glucopyranoside (21)
J4–5 9.5 Hz, H-4), 3.46 (dd, 1 H, J1–2 3.5 Hz,
J2–3 9.0 Hz, H-2), 1.9 (bs, 1 H, OH); EIMS:
+
’
m/z 562 [M] .
D
(2-Hydroxy-ethyl) [2,3-b](11,12-benzo-1,4,-
7,10,13,16 - hexaoxacyclooctadeca - 11 - ene) -
Method A. The same procedure and
reagents were used as for the synthesis of 20.
From the crown ether 19 [1] (0.558 g, 1.25
mmol) the azide 21 (0.60 g, 98%) was obtained
as a colourless wax, after usual work-up and
chromatography on silica gel (1:1“9:1
EtOAc–n-hexane): [h]D +73.5° (c 1, CHCl3);
2,3-dideoxy-4,6-di-O-methyl-h- -glucopyra-
D
noside (17).—The same procedure was used as
for the synthesis of 16 except for the tempera-
ture, which was only −10 °C and for the
reaction time, which was respectively, 1 h for
the ozonolysis and 5 h for the reduction.
From 7 (0.155 g, 0.31 mmol), the crude alco-
hol 17 (0.16 g, \ 99%) was obtained as a
homogeneous colourless gum, following usual
work-up (but without chromatography): [h]D
IR (NaCl): w 2100 cm−1 (N3); H NMR
1
(CDCl3): l 6.87 (bs, 4 H, catechol), 5.87
(dddd, 1 H, OCH2ꢀCHꢁCH2), 5.23 (dq, 1 H,
Jtrans 17.0 Hz, OCH2ꢀCHꢁCHH), 5.13 (dq, 1
H, Jcis 10.5 Hz, Jgem 51.5 Hz, OCH2ꢀCHꢁ
CHH), 4.77 (d, 1 H, H-1), 4.36–3.77 (m, 17
H, 8.5×OCH2), 4.325 (dd, 1 H, J 5.5 Hz, Jgem
12.5 Hz, OCHH), 3.62 (t, 1 H, H-3), 3.73 (m,
1 H, H-5), 3.5 (dd, 1 H, J5–6% 2.0 Hz, H-6%),
3.42 (s, 3 H, OCH3), 3.39 (dd, 1 H, J1–2 3.5
Hz, J2–3 9.0 Hz, H-2), 3.37 (dd, 1 H, J5–6 6.0
Hz, J6–6% 13.5 Hz, H-6), 3.27 (t, 1 H, J3–4 ꢀJ4–
1
+58° (c 1, CHCl3); H NMR (CDCl3): l 6.88
(bs, 4 H, catechol), 4.92 (d, 1 H, H-1), 4.2–
3.72 (m, 22 H, 10×OCH2, H-5, -6%), 3.7–3.6
(m, 2 H, H-3, -6), 3.51 (s, 3 H, OCH3), 3.38 (s,
3 H, OCH3), 3.36 (dd, 1 H, J1–2 3.5 Hz, J2–3
9.5 Hz, H-2), 3.18 (t, 1 H, J3–4 ꢀJ4–5 9.5 Hz,
H-4), 1.23 (bs, 1 H, OH); EIMS: m/z 502
+
’
+
’
[M] .
5 9 Hz, H-4); EIMS: m/z 509 [M] , 481
+
’
Methyl 4-O-allyl-[2,3-b](11,12-benzo-1,4,-
7,10,13,16-hexaoxacyclooctadeca-11-ene)-6-
[M–N2] .
Method B. To a stirred soln of 20 (1.0 g,
1.826 mmol) in DMF (80 mL) was added
NaN3 (0.18 g, 1.5 equiv) and the solution
heated to 130 °C. The reaction was monitored
by TLC (1:1 EtOAc–n-hexane), and was com-
plete after 1 h. The solvent was evaporated,
the residue dissolved in CH2Cl2 (100 mL),
washed twice with water (20 mL), dried over
MgSO4, and concentrated under reduced pres-
sure. Chromatography of the residue (3:2
EtOAc–n-hexane) yielded the azide 21 (0.92
g, \99%) as a colourless wax identical in all
aspects to the previous compound obtained
from 19.
bromo - 2,3,6 - trideoxy - h -
D
- glucopyranoside
(20).—A soln of the crown ether 18 (0.55 g,
1.08 mmol) in toluene (10 mL) and allyl bro-
mide (1 mL, ꢀ10 equiv) was vigorously
stirred for 14 h at rt with 50% aq NaOH (10
mL) under an efficient fume board. Chilled
water (80 mL) was added and the organic
phase extracted with CH2Cl2 (3×50 mL). The
organic extracts were combined and washed
with water (3×20 mL), dried (MgSO4), con-
centrated in vacuo, and purified by chro-
matography on silica gel to yield 20 (0.58 g,
\99%) as a colourless gum: [h]D +71.6° (c 1,
1
CHCl3); H NMR (CDCl3): l 6.87 (bs, 4 H,
Methyl 4 - O - allyl - [2,3 - b](11,12 - benzo-
1,4,7,10,13,16-hexaoxacyclooctadeca-11-ene)-
catechol), 5.90 (dddd, 1 H, OCH2ꢀCHꢁCH2),
5.24 (dq, 1 H, Jtrans 17.0 Hz, OCH2ꢀCHꢁ
CHH), 5.14 (dq, 1 H, Jcis 10.5 Hz, Jgem51.5
Hz, OCH2ꢀCHꢁCHH), 4.37 (dd, 1 H, J 5.5
Hz, Jgem 12.5 Hz, OCHH), 4.80 (d, 1 H, H-1),
4.22–3.78 (m, 18 H, 7.5×OCH2, H-6%, -7,
-7%), 3.73 (m, 1 H, H-5), 3.65 (t, 1 H, H-3),
3.57 (dd, 1 H, J5–6 6 Hz, J6–6% 11.5 Hz, H-6),
3.4 (s, 3 H, OCH3), 3.38 (dd, 1 H, J1–2 3.5 Hz,
J2–3 9.0 Hz, H-2), 3.31 (t, 1 H, J3–4 ꢀJ4–5 9.0
2,3,6-trideoxy-h-
D
-glucopyranoside
(23).—
The same procedure and reagents were used as
for the synthesis of 21. From the crown ether
22 (0.75 g, 1.75 mmol) after 24 h of reaction,
following the usual work-up and chromatog-
raphy on silica gel (2:1 EtOAc–n-hexane) the
crown ether 23 (0.74 g, 90%) was obtained as
a colourless gum: [h]D +62.3° (c 1, CHCl3);
1H NMR (CDCl3): l 6.89 (4 H, catechol), 5.92

B. Dumont-Hornebeck et al. / Carbohydrate Research 321 (1999) 214–227
223
Methyl [2,3-b](11,12-benzo-1,4,7,10,13,16-
hexaoxacyclooctadeca-11-ene)-6-bromo-4-O-
(2 - hydroxy - ethane - 1 - yl) - 2,3,6 - trideoxy - h -
(dddd, 1 H, OCH2ꢀCHꢁCH2), 5.26 (dq, 1 H,
Jtrans 17.0 Hz, OCH2ꢀCHꢁCHH), 5.14 (dq, 1
H, Jcis 10.5 Hz, Jgem51.5 Hz, OCH2ꢀCHꢁ
CHH), 4.72 (d, 1 H, H-1), 4.33 (dd, 1 H, J 5.5
Hz, Jgem 12.5 Hz, OCHH), 4.23–3.63 (m, 18
H, 8.5×OCH2, H-5), 3.59 (t, 1 H, H-3), 2.37
(s, 3 H, OCH3), 2.85 (t, 1 H, J3–4 ꢀJ4–5 9.0
Hz, H-4), 2.36 (dd, 1 H, J1–2 3.5 Hz, J2–3 9.0
D
-glucopyranoside (25).—The same procedure
was used as for the synthesis of 17 except for
the reaction time, which was 3 h for the
ozonolysis and 4 h for the reduction (TLC
with 2:1 EtOAc–n-hexane). From the allyl
ether 20 (0.64 g, 1.16 mmol), following usual
work-up (but without chromatography), the
crude glycol 25 (0.65 g, \99%) was obtained
as a colourless gum: [h]D +59.7° (c 1,
Hz, H-2), 1.24 (d, 3 H, J5–6 6.0 Hz, H-6);
+
’
EIMS: m/z 509 [M] .
Methyl [2,3-b](11,12-benzo-1,4,7,10,13,16-
hexaoxacyclooctadeca - 11 - ene) - 4 - O - (2 - hy -
1
droxy-ethane-1-yl)-2,3,6-trideoxy-h-
pyranoside (24)
D
-gluco-
CHCl3); H NMR (CDCl3): l 6.9 (bs, 4 H,
catechol), 4.82 (d, 1 H, H-1), 4.27–3.65 (m, 22
H, 10×OCH2, H-5, -6), 3.6 (dd, 1 H, J5–6 5.0
Hz, J6–6% 11.5 Hz, H-6%), 3.43 (s, 3 H, OCH3),
3.42 (dd, 1 H, J1–2 3.5 Hz, J2–3 9 Hz, H-2),
3.36 (t, 1 H, J3–4 8.5 Hz, H-3), 3.29 (t, 1 H,
J4–5 6 Hz, H-4), 1.65 (s, 1 H, OH); EIMS: m/z
Method A. The same procedure and
reagents were used as for the synthesis of 17
except for the reaction time, which was respec-
tively 2 h for the ozonolysis and 14 h for the
reduction. From 23 (0.56 g, 1.19 mmol) the
crown ether 24 (0.56 g, 99%) was obtained as
colourless crystals after the usual work-up but
without chromatography: mp 57–59 °C
79
+
81
+
’
’
550 [ BrM] and 552 [ BrM] .
Methyl 6-azido-[2,3-b](11,12-benzo-1,4,-
7,10,13,16-hexaoxacyclooctadeca-11-ene)-4-
O-(2-hydroxy-ethane-1-yl)-2,3,6-trideoxy-h-
1
(EtOAc); [h]D +55.0° (c 1, CHCl3); H NMR
(CDCl3): l 6.85 (bs, 4 H, catechol), 4.69 (d, 1
H, H-1), 4.25–3.65 (m, 21 H, 10×OCH2,
OH), 3.62 (m, H-5), 3.58 (t, H-3), 3.39 (dd, 1
H, J1–2 3.5 Hz, J2–3 9.0 Hz, H-2), 3.36 (s, 3 H,
OCH3), 3.0 (bt, 1 H, J3–4 ꢀ9.0 Hz, J4–5 9.5
D-glucopyranoside (26).—The same procedure
was used as for the synthesis of 15 except for
the reaction time, which was 14 h (TLC with
2:1 EtOAc–n-hexane). From the allyl ether 21
(0.40 g, 0.785 mmol) the glycol 26 (0.25 g,
61%) was obtained, following usual work-up
and chromatography (2:3 EtOAc–n-hexane),
as a colourless gum: [h]D +64.2° (c 1,
Hz, H-4), 1.25 (d, 3 H, J5–6 6.0 Hz, H-6);
+
’
EIMS: m/z 472 [M] .
Method B. A soln of the crown ether
25 (0.30 g, 0.544 mmol) (vide infra) in THF (5
mL) was dropped over 10 min onto a stirred
suspension of LiAlH4 (83 mg, 4 equiv) in THF
(5 mL) at 0 °C under argon. After addition,
the temperature was raised up to rt under
stirring. The reaction was monitored by TLC
(EtOAc) and stopped after 45 min at rt
by slow addition of EtOAc (1 mL). Hydrolysis
of the hydride excess was completed with satd
aq Na2SO4 (0.5 mL), the suspension fil-
tered through a sintered glass and the re-
maining salts rinsed twice with THF (25 mL).
The solvents were eliminated under reduced
pressure and the residue dissolved in CH2Cl2
(50 mL), washed twice with water (10 mL),
dried over MgSO4, and concentrated to a
colourless gum, which crystallised slowly on
standing. Yield: 0.247 g (99%) of white crys-
tals identical in all aspects to the above glycol
24.
1
CHCl3); IR (NaCl): w 2098 cm−1 (N3); H
NMR (CDCl3): l 6.89 (bs, 4 H, catechol), 4.79
(d, 1 H, H-1), 4.25–3.65 (m, 22 H, 10×
OCH2, H-5, -6%), 3.61 (t, 1 H, H-3), 3.51 (dd,
1 H, J5–6 3 Hz, J6–6%, 13 Hz, H-6), 3.4 (s, 3 H,
OCH3), 3.39 (dd, 1 H, J1–2 3.5 Hz, J2–3 9 Hz,
H-2), 3.31 (t, 1 H, J3–4 ꢀJ4–5 9.5 Hz, H-4),
+
’
2.05 (s, 1 H, OH); EIMS: m/z 513 [M] .
Ethyl-2-(2,3,4,6-tetra-O-acetyl-i- -galac-
D
topyranosyloxy) 6-azido-[2,3-b](11,12-benzo-
1,4,7,10,13,16-hexaoxacyclooctadeca-11-ene)-
4-O-methyl-2,3,6-trideoxy-i-
oside (27).—To a soln of the acceptor 15 (0.10
g, 0.194 mmol) and -galactose trichlorace-
D
-glucopyran-
D
timidate (0.19 g, 2 equiv) in CH2Cl2 (10 mL)
were added crushed molecular sieves (ꢀ1 g,
activated at 105 °C and stored in vacuo over
P2O5) and the suspension magnetically stirred
under Ar at rt for 30 min. BF3·OEt2 (50 mL, 2
equiv) in CH2Cl2 (10 mL) was then dropped

224
B. Dumont-Hornebeck et al. / Carbohydrate Research 321 (1999) 214–227
1
CHCl3); H NMR (CDCl3): l 6.9 (bs, 4 H,
catechol), 5.36 (d, 1 H, J4–5 3.0 Hz, H-4 Gal),
5.15 (t, 1 H, H-2 Gal), 5.0 (dd, 1 H, J2–3 10.5
Hz, J3–4 3.5 Hz, H-3 Gal), 4.9 (d, 1 H, J1–2
3.75 Hz, H-1 Glc), 4.5 (d, 1 H, J1–2 8.0 Hz,
H-1 Gal), 4.3–3.55 (m, 27 H, 10×OCH2,
H-3, -4, -6, -6% Glc, H-5, -6, -6% Gal), 3.53 (dd,
1 H, J1–2 3.0 Hz, J2–3 7.5 Hz, H-2 Glc), 3.4
(m, 1 H, J5–6 2.5 Hz, J4–5 9.0 Hz, J5–6% 6.0 Hz,
H-5 Glc), 2.65 (bs, 2 H, OH), 2.2–1.9 (4 s,
each 3 H, 4×OAc); LSIHRMS: calcd for
C36H52O20Na 827.2950, found m/z 827.2956.
over a period of 15 min. The reaction was
monitored by TLC (1:1 EtOAc–n-hexane)
and stopped after 20 h by addition of
NaHCO3 (90 mg, 4 equiv). The suspension
was filtered and the filtrate washed with water
(2×20 mL), dried with MgSO4, and concen-
trated under reduced pressure. Chromatogra-
phy of the residue on a silica gel column (1:1
EtOAc–n-hexane) yielded the target com-
pound 27 (0.116 g, 55%) as a colourless gum:
[h]D +7.7° (c 0.5, CHCl3); IR (NaCl): w 2099
1
cm−1 (N3); H NMR (CDCl3): l 6.9 (bs, 4 H,
catechol), 5.36 (d, 1 H, H-4 Gal), 5.17 (t, 1 H,
H-2 Gal), 4.99 (dd, 1 H, J2–3 10.5 Hz, J3–4 3.5
Hz, H-3 Gal), 4.52 (d, 1 H, J1–2 8.0 Hz, H-1
Gal), 4.31 (d, 1 H, H-1 Glc), 4.27–3.65 (m, 24
H, 10×OCH2, H-6% Glc, H-5, -6, -6% Gal),
3.52 (s, 3 H, OCH3), 3.45–3.3 (m, 2 H, H-5, -6
Glc), 3.29 (t, 1 H, H-3 Glc), 3.12 (t, 1 H, J1–2
8.0 Hz, J2–3 9.0 Hz, H-2 Glc), 3.06 (t, 1 H,
Ethyl-2-(2,3,4,6-tetra-O-acetyl-i- -gal-
D
actopyranosyloxy) [2,3-b](11,12-benzo-1,4,7,-
10,13,16-hexaoxacyclooctadeca-11-ene)-2,3-
dideoxy-4,6-di-O-methyl-h- -glucopyranoside
D
(30).—The same procedure and work-up were
used as for the synthesis of 27 except for the
reaction time, which was 22 h. From 7 (0.15 g,
0.298 mmol), after chromatography on a silica
gel column (1:1 EtOAc–n-hexane), the target
compound 30 (0.12 g, 47%) was obtained as a
J3–4 ꢀJ4–5 9.0 Hz, H-4 Glc), 2.15–1.9 (4 s,
+
’
each 3 H, 4×OAc); EIMS: m/z 843 [M] .
1
Ethyl-2-(2,3,4,6-tetra-O-acetyl-i-
D
-gal-
colourless gum: [h]D +39.3° (c 1, CHCl3); H
actopyranosyloxy) [2,3-b](11,12-benzo-1,4,7,-
10,13,16-hexaoxacyclooctadeca-11-ene)-4,6-
NMR (CDCl3): l 6.89 (bs, 4 H, catechol), 5.35
(d, 1 H, J4–5 3.5 Hz, H-4 Gal), 5.15 (t, 1 H,
H-2 Gal), 4.98 (dd, 1 H, J2–3 10.0 Hz, J3–4 3.5
Hz, H-3 Gal), 4.87 (d, 1 H, H-1 Glc), 4.52 (d,
1 H, J1–2 8.0 Hz, H-1 Gal), 4.2–3.65 (m, 24 H,
10×OCH2, H-6% Glc, H-5, -6, -6% Gal), 3.68
(dd, 1 H, J5–6 6.5 Hz, J6–6% 14.0 Hz, H-6 Glc),
3.56 (m, 1 H, H-5 Glc), 3.55 (t, 1 H, J2–3 9.5
Hz, H-3 Glc), 3.5 (s, 3 H, OCH3), 3.37 (s, 3 H,
OCH3), 3.35 (dd, 1 H, J1–2 3.7 Hz, H-2 Glc),
O-benzylidene-2,3-dideoxy-h- -glucopyran-
D
oside (28).—The same procedure and work-up
were used as for the synthesis of 27 except for
the reaction time, which was 2 h. From the
acceptor 16 (0.16 g, 0.284 mmol) the target
compound 28 (60 mg, 22%) was first recov-
ered as a colourless gum after HPLC on a
silica gel column (20 mm i.d., 1:2 EtOAc–n-
1
hexane): [h]D +28.8° (c 1, CHCl3); H NMR
3.21 (bt, 1 H, J3–4 9.0 Hz, H-4 Glc), 2.2–1.9 (4
+
’
(CDCl3): l 7.5–7.3 (m, 5 H, Ar), 6.9 (bs, 4 H,
catechol), 5.5 (s, 1 H, Hb), 5.36 (d, 1 H, J4–5
3.0 Hz, H-4 Gal), 5.18 (t, 1 H, H-2 Gal), 5.0
(dd, 1 H, J2–3 10.0 Hz, J3–4 3.5 Hz, H-3 Gal),
4.96 (d, 1 H, H-1 Glc), 4.55 (d, 1 H, J1–2 8.0
Hz, H-1 Gal), 4.3–3.6 (m, 26 H, 10×OCH2,
H-5, -6, -6% Glc, H-5, -6, -6% Gal), 3.54 (t, 1 H,
J4–5 9.0 Hz 9 Hz, H-4 Glc), 3.52 (t, 1 H, J3–4
9.5 Hz, H-3 Glc), 3.45 (dd, 1 H, J1–2 3.5 Hz,
s, each 3 H, 4×OAc); EIMS: m/z 832 [M] .
Ethyl-2-(2,3,4,6-tetra-O-acetyl-i- -galac-
D
topyranosyloxy) 4,6-di-O-acetyl-[2,3-b](11,12-
benzo-1,4,7,10,13,16-hexaoxacyclooctadeca-11-
ene)-2,3-dideoxy-h- -glucopyranoside (31).—
D
From 29 (0.10 g, 0.124 mmol) after reaction
with Ac2O (1 mL) for 24 h at rt in pyridine (10
mL) and a catalytical amount of 4-DMAP,
usual work-up, and chromatography (1:1
EtOAc–n-hexane) on a silica gel column, the
peracetylated derivative 31 (0.110 g, ꢀ 99%)
was isolated as a colourless gum: [h]D +32.8°
J2–3 9.5 Hz, H-2 Glc), 2.2–1.9 (4 s, each 3 H,
+
’
4×OAc); EIMS: m/z 892 [M] ; then, with
1:1 EtOAc–n-hexane as eluent, ethyl-2-
1
(2,3,4,6-tetra-O-acetyl-b-
D
-galactopyranosyl-
(c 1, CHCl3); H NMR (CDCl3): l 6.88 (bs, 4
oxy) [2,3 - b](11,12 - benzo - 1,4,7,10,13,16-
hexaoxacyclooctadeca-11-ene)-2,3-dideoxy-a-
H, catechol), 5.38 (d, 1 H, J4–5 3.0 Hz, H-4
Gal), 5.17 (t, 1 H, H-2 Gal), 5.01 (dd, 1 H,
J2–3 10.0 Hz, J3–4 3.5 Hz, H-3 Gal), 4.97 (t, 1
H, J4–5 9.0 Hz, H-4 Glc), 4.95 (d, 1 H, H-1
D-glucopyranoside (29) (0.10 g, 44%) was iso-
lated as a colourless gum: [h]D +15.4° (c 1,

B. Dumont-Hornebeck et al. / Carbohydrate Research 321 (1999) 214–227
225
-6% Glc, H-3, -5, -6, -6% Gal), 3.6 (dd, 1 H, J1–2
3.5 Hz, J2–3 9.5 Hz, H-2 Glc), 3.49 (t, 1 H,
J3–4ꢀJ4–5 9.0 Hz, H-4 Glc); 13C NMR (1:1
CD3CN–D2O): l 148.4 (C-1, -2 catechol),
122.6 (C-4, -5 catechol), 114.0 (C-3, -6 cate-
chol), 103.7 (C-1 Gal), 96.4 (C-1 Glc), 80.9
(C-5 Gal), 78.8 (C-2 Glc), 75.9 (C-3 Glc),
72.65 (C-4 Glc), 72.1 (OCH2), 71.6 (C-4 Gal),
71.3, 70.7 (OCH2), 70.6 (C-3 Gal), 69.8
(OCH2), 69.5 (C-2 Gal), 69.3, 69.1, 68.6
(OCH2), 67.3 (OCH2 spacer), 61.8 (C-6 Gal),
61.4 (C-6 Glc); LSIHRMS: calcd for
C28H44O16Na 659.2527, found m/z 659.2525;
tret (HPLC) 7.1 min (4:1 water–MeCN).
Glc), 4.52 (d, 1 H, J1–2 8.0 Hz, H-1 Gal), 4.25
(dd, 1 H, J5–6% 4.5 Hz, H-6% Glc), 4.2–3.7 (m,
24 H, 10×OCH2, H-5 Glc, H-5, -6, -6% Gal),
4.03 (dd, 1 H, J1–2 2.0 Hz, J6–6% 12.5 Hz, H-6
Glc), 3.67 (t, 1 H, J3–4 8.5 Hz, H-3 Glc), 3.45
(dd, 1 H, J1–2 3.0 Hz, J2–3 8.5 Hz, H-2 Glc),
2.2–1.9 (m, 18 H, 6×OAc); EIMS: m/z 802
+
’
[M] .
Ethyl-2-(i- -galactopyranosyloxy) [2,3-b]-
D
(11,12-benzo-1,4,7,10,13,16-hexaoxacyclooct-
adeca-11-ene)-4,6-O-benzylidene-2,3-dideoxy-
h- -glucopyranoside (32).—To a stirred soln
D
of 28 (40 mg, 44.8 mmol) in MeOH (10 mL)
was added MeONa (25 mg) at rt. The reaction
was monitored by TLC (95:5 EtOAc–EtOH)
and was stopped after 24 h by addition of
Dowex H+ beads (ꢀ0.5 mL). The suspension
was stirred again for 10 min and filtered to
yield, after evaporation of solvents, the galac-
toside 32 (28 mg, \99%) as a pale yellow
Ethyl-2-(i- -galactopyranosyloxy) [2,3-b]-
D
(11,12-benzo-1,4,7,10,13,16-hexaoxacycloocta-
deca-11-ene)-2,3-dideoxy-4,6-di-O-methyl-h-
D-glucopyranoside (34).—The same procedure
was used as for the synthesis of 32. From 30
(0.10 g, 0.12 mmol) the deprotected galac-
toside 34 (77 mg, 96%) was obtained after the
1
gum: [h]D +18.1° (c 0.2, CH3CN); H NMR
same work-up as a colourless gum: [h]D
+
selected values (D2O): l 7.53–7.4 (m, 5 H,
Ar), 7.03 (bs, 4 H, catechol), 5.63 (s, 1 H, Hb),
5.17 (d, 1 H, H-1 Glc), 4.48 (d, 1 H, J1–2 8.4
Hz, H-1 Gal), 3.55 (dd, 1 H, J1–2 3.5 Hz, J2–3
9.5 Hz, H-2 Glc); 13C NMR (1:1 CD3CN–
D2O): l 149.0 (C-1, -2 catechol), 137.7 (C-1
Bz), 130.2 (C-4 Bz), 129.4 (C-2, -3 Bz), 127.1,
122.6 (C-4, -5 catechol), 114.6 (C-3, -6 cate-
chol), 103.8 (C-1 Gal), 102.2 (OCHO), 97.9
(C-1 Glc), 81.9 (C-4 Glu), 80.1 (C-5 Gal), 78.5
(C-2 Glc), 75.9 (C-3 Glc), 73.8 (C-3 Gal), 72.4
(OCH2), 71.6 (C-2 Gal), 71.1, 71.0, 70.6, 70.1,
70.0 (OCH2), 69.6 (C-4 Gal), 69.3 (OCH2),
67.8 (OCH2 spacer), 63.2 (C-5 Glc), 61.8 (C-6
Gal), 61.6 (C-6 Glc); LSIHRMS: calcd for
C35H48O16Na 747.2840, found m/z 747.2846;
tret (HPLC) 10.7 min (4:1 water–MeCN).
21.5° (c 0.4, CH3CN); 1H NMR (1:1 CD3CN–
D2O): l 7.0 (bs, 4 H, catechol), 5.06 (d, 1 H,
H-1 Glc), 4.73 (dd, 1 H, J2–3 10.5 Hz, H-2
Gal), 4.35 (d, 1 H, J1–2 8.5 Hz, H-1 Gal),
4.21–3.52 (m, 29 H, 10×OCH2, H-3, -5, -6,
-6% Glc, H-3, -4, -5, -6, -6% Gal), 3.48 (s, 3 H,
OCH3), 3.41 (dd, 1 H, J1–2 3.9 Hz, J2–3 10.0
Hz, H-2 Glc), 3.33 (s, 3 H, OCH3), 3.17 (t, 1
H, J3–4 ꢀJ4–5 9.5 Hz, H-4 Glc); 13C NMR (1:1
CD3CN–D2O): l 149.3 (C-1, -2, catechol),
115.1 (C-3, -6, catechol), 112.7 (C-4, -5, cate-
chol), 103.9 (C-1 Gal), 96.9 (C-1 Glc), 82.0
(C-5 Gal), 80.2 (C-2, -4 Glc), 76.0 (C-3 Glc),
73.9 (C-5 Glc), 73.1, 71.8 (OCH2 crown),
71.45, 71.2 (OCH2 crown), 71.7 (C-4 Gal),
70.5 (C-3 Gal), 70.4, 70.1, 69.7 (OCH2 crown),
69.6 (C-2 Gal), 69.3 (OCH2 crown), 67.5
(OCH2 spacer), 61.9 (C-6 Glc), 61.2 (C-6 Gal),
61.1 (OCH3), 59.4 (OCH3); LSIHRMS: calcd
for C30H48O16Na 687.2840, found m/z
687.2849; (HPLC) 7.8 min (73:27 water–
MeCN).
Ethyl-2-(i-
(11,12-benzo-1,4,7,10,13,16-hexaoxacycloocta-
deca-11-ene)-2,3-dideoxy-h- -glucopyranoside
D-galactopyranosyloxy) [2,3-b]-
D
(33).—The same procedure was used as for
the synthesis of 32 except for the reaction
time, which was 6 h. From 31 (0.10 g, 0.11
mmol), after the same work-up, the depro-
tected galactoside 33 (72 mg, 86%) was ob-
tained as a colourless wax: [h]D +27.2° (c 0.2,
Methyl [2,3-b](11,12-benzo-1,4,7,10,13,16-
hexaoxacyclooctadeca-11-ene)-4-O-[2-(2,3,-
4,6-tetra-O-acetyl-i-
D
-galactopyranosyloxy)-
-glucopyran-
1
CH3CN); H NMR (1:1 CD3CN–D2O): l
ethane-1-yl]-2,3,6-trideoxy-h-
D
7.03 (bs, 4 H, catechol), 5.15 (d, 1 H, H-1
Glc), 4.4 (d, 1 H, J1–2 7.0 Hz, H-1 Gal),
4.29–3.57 (m, 28 H, 10×OCH2, H-3, -5, -6,
oside (35).—The same procedure was used as
for the synthesis of 28. From 24 (0.30 g, 0.634
mmol) the galactoside 35 (0.255 g, 50%) was

226
B. Dumont-Hornebeck et al. / Carbohydrate Research 321 (1999) 214–227
l 6.87 (bs, 4 H, catechol), 5.37 (d, 1 H, J4–5
3.5 Hz, H-4 Gal), 4.99 (dd, 1 H, H-2 Gal), 4.9
(dd, 1 H, J2–3 10.0 Hz, J3–4 3.5 Hz, H-3 Gal),
4.79 (d, 1 H, H-1 Glc), 4.47 (d, 1 H, J1–2 8.0
Hz, H-1 Gal), 4.24–3.62 (m, 25 H, 10×
OCH2, H-5, -6%, -5, -6, -6%), 3.57 (t, 1 H, H-3
Glc), 3.44 (dd, 1 H, J5–6 2.5 Hz, J6–6% 12.5 Hz,
H-6 Glc), 3.4 (s, 3 H, OCH3), 3.32 (t, 1 H, H-4
Glc), 3.23 (dd, 1 H, J1–2 3.5 Hz, J2–3 8.5 Hz,
H-2 Glc), 2.2–1.9 (4 s, each 3 H, 4×OAc);
obtained after chromatography on silica gel
(1:1“2:1 EtOAc–n-hexane) as a colourless
1
gum: [h]D +31.3° (c 1, CHCl3); H NMR
(CDCl3): l 6.90 (bs, 4 H, catechol), 5.39 (d, 1
H, J4–5 3.0 Hz, H-4 Gal), 5.21 (dd, 1 H, H-2
Gal), 5.01 (dd, 1 H, J2–3 10.0 Hz, J3–4 3.0 Hz,
H-3 Gal), 4.73 (d, 1 H, H-1 Glc), 4.52 (d, 1 H,
J1–2 8.0 Hz, H-1 Gal), 4.22-3.6 (m, 24 H,
10×OCH2, H-5, -6,-6% Glc, H-5, -6, -6% Gal),
3.56 (t, 1 H, H-3 Glc), 3.38 (s, 3 H, OCH3),
3.37 (dd, 1 H, J1–2 3.5 Hz, J2-3 9.5, H-2 Glc),
2.91 (t, 1 H, J3–4 ꢀJ4–5 9.5 Hz, H-4 Glc),
2.2–1.9 (4 s, each 3 H, 4×OAc), 1.25 (d, 3 H,
J5–6 6.5 Hz, H-6 Glc); LSIHRMS: calcd for
C37H54O17Na 825.3157, found m/z 825.3163.
Methyl [2,3-b](11,12-benzo-1,4,7,10,13,16-
hexaoxacyclooctadeca-11-ene)-6-bromo-4-O-
+
+
’
EIMS m/z 843 [M] , 816 [M–N2+H] .
Methyl [2,3-b](11,12-benzo-1,4,7,10,13,16-
hexaoxacyclooctadeca-11-ene)-4-O-[2-(i-
galactopyranosyloxy)-ethane-1-yl]-2,3,6-tri-
deoxy-h- -glucopyranoside (38).—The same
D
-
D
procedure was used as for the synthesis of 32
except for the reaction time, which was 5 h.
From the precursor 35 (0.11 g, 0.137 mmol)
the deprotected galactoside 38 (87 mg, 99%)
was obtained after usual work-up as a colour-
[2-(2,3,4,6-tetra-O-acetyl-i-
osyloxy)-ethane-1-yl]-2,3,6-trideoxy-h-
D
-galactopyran-
-glu-
D
copyranoside (36).—The same procedure was
used as for the synthesis of 27 except for the
reaction time, which was 4 h. From 25 (0.37 g,
0.671 mmol) the galactoside 36 (0.255 g, 43%)
was obtained after chromatography on silica
gel (1:1 EtOAc–n-hexane) as a colourless
1
less gum: [h]D +43.1° (c 0.5, CH3CN); H
NMR (D2O): l 7.0 (bs, 4 H, catechol), 4.86 (d,
1 H, H-1 Glc), 4.35 (d, 1 H, H-1 Gal), 4.2–
3.55 (m, 24 H, 10×OCH2, H-3, -5, -6, -6%
Gal), 3.65 (m, 1 H, H-5 Glc), 3.58 (d, 1 H,
J3–4ꢀJ4–5 3.5 Hz, H-4 Gal), 3.49 (t, 1 H,
J1–2ꢀJ2–3 8.0 Hz, H-2 Gal), 3.488 (t, 1 H,
H-3 Glc), 3.43 (dd, 1 H, J1–2 3.0 Hz, J2–3 10.0
Hz, H-2 Glc), 3.35 (s, 3 H, OCH3), 3.03 (t, 1
H, J3–4 9.0 Hz, J4–5 10.0 Hz, H-4 Glc), 1.26 (d,
1
gum: [h]D +37.6° (c 1, CHCl3); H NMR
(CDCl3): l 6.87 (bs, 4 H, catechol), 5.36 (d, 1
H, J4–5 3.5 Hz, H-4 Gal), 5.19 (dd, 1 H, H-2
Gal), 4.98 (dd, 1 H, J2–3 10.0 Hz, J3–4 3.5 Hz,
H-3 Gal), 4.8 (d, 1 H, H-1 Glc), 4.49 (d, 1 H,
J1–2 8.0 Hz, H-1 Gal), 4.22–3.73 (m, 25 H,
10×OCH2, H-6, -6% Glc, H-5, -6, -6% Gal),
3.67 (m, 1 H, J5–6 7.0 Hz, J5–6% 2.5 Hz, H-5
Glc), 3.61 (t, 1 H, H-3 Glc), 3.4 (s, 3 H,
OCH3), 3.37 (dd, 1 H, J1–2 3.0 Hz, J2–3 9.0
Hz, H-2 Glc), 3.28 (t, 1 H, J3–4 ꢀJ4–5 9.0 Hz,
H-4 Glc), 2.2–1.9 (4 s, each 3 H, 4×OAc);
13
3 H, J5–6 6.5 Hz, CH3); C NMR (1:1 D2O–
CD3CN): l 149.25, 149.2 (C-1, -2, catechol),
122.8 (C-4, -5, catechol), 115.05, 114.9 (C-3,
-6, catechol), 103.9 (C-1 Gal), 97.9 (C-1 Glc),
84.8 (C-5 Glc), 81.8 (C-5 Gal), 80.4 (C-2 Glc),
76.0 (C-3 Glc), 73.9 (C-4 Glc), 73.0, 72.9
(OCH2), 71.9 (C-4 Gal), 71.5, 71.3, 70.15, 70.1
(OCH2), 69.8 (C-3 Gal), 69.6, 69.55 (OCH2),
67.4 (C-2 Gal), 62.1 (OCH2), 61.9 (C-6 Gal),
55.6 (OCH3), 17.9 (C-6 Glc); FABMS: calcd
for C29H46O15 634, found m/z 635 [M+H]+,
79
+
’
81
+
’
EIMS: m/z 880 [ BrM] and 882 [ BrM] .
Methyl 6-azido-[2,3-b](11,12-benzo-1,4,7,-
10,13,16-hexaoxacyclooctadeca-11-ene)-4-O-
[M–C6H10O5+Na]+,
379
[M–
[2-(2,3,4,6-tetra-O-acetyl-i-
osyloxy)-ethane-1-yl]-2,3,6-trideoxy-h-
D
-galactopyran-
-glu-
495
C11H18O8+Na]+; LSIHRMS: calcd for
C29H46O15Na 657.2734, found m/z 657.2730;
tret (HPLC) 6.9 min (73:27 water–MeCN).
Micro-organisms and culture conditions.—
The flocculent yeast strain used throughout
the present study was K. bulgaricus (ATCC
96631). The growth of the yeast was per-
formed in a liquid medium composed of 4.0%
D
copyranoside (37).—The same procedure was
used as for the synthesis of 27 except for the
reaction time, which was 4 h. From 26 (0.23 g,
0.448 mmol) the galactoside 37 (0.214 g, 57%)
was obtained after chromatography on silica
gel (1:1“2:1 EtOAc–n-hexane) as a colour-
less gum: [h]D +50.4° (c 1, CHCl3);); IR
1
(NaCl): w 2099 cm−1 (N3); H NMR (CDCl3):
D-glucose, 0.4% bactopeptone (Difco, MI,

B. Dumont-Hornebeck et al. / Carbohydrate Research 321 (1999) 214–227
227
USA), 0.1% KH2PO4, 0.02% MgSO4·7H2O,
and 0.02% CaCl2 (Prolabo, France). The cells
were cultured under aerobic conditions at
20 °C in a 2 L capacity fermentor containing
1.5 L of medium. The aeration was 20 L/h
and the agitation 220 rpm.
mixing 150 mL of a two-fold serial dilution of
oligosaccharidic derivatives in Helm’s acetate
buffer with 150 mL of a lectinic solution titre
4. The mixture was incubated for 60 min at rt
after mild agitation, then 150 mL of an EDTA
deflocculated yeast cell suspension were
added. After 1 h of incubation at rt, yeast
flocculation was estimated by visual reading
or microscope observation. The MIC of the
flocculation corresponds to the tube in which
flocculation is visible. The control of inhibi-
tion was achieved in the same way, but with
Extraction of the galactose-specific lectin Kb
CWL1.—The
D
-galactose lectin Kb CWL1
was extracted from whole cells harvested at
the beginning of the stationary growth phase
according to Al-Mahmood et al. [5]. After
growth in the 2 L fermentor, K. bulgaricus
cells were harvested by centrifugation at 3000g
for 10 min at 4 °C, washed with Helm’s buffer
(150 mM CH3CO2Na, 3.75 mM CaCl2, 3.0
mM NaN3) at pH 5.0, treated with a 0.2 M
D
-galactose, methyl b- -galactoside and lac-
D
tose [11].
D-galactose solution and extensively washed
References
with 0.01 M phosphate buffer (pH 7.0). Cells
were then suspended at a concentration of 4%
(w/v) in PBS buffer (137 mM NaCl, 2.7 mM
KCl, 8.0 mM Na2HPO4, 1.5 mM KH2PO4, 0.5
MgCl2, 1.0 mM CaCl2) at pH 7.4 supple-
mented with 5 mM EDTA and incubated at
37 °C for 90 min under moderate stirring.
After centrifugation at 3000g for 10 min, the
supernatant containing the lectin was collected
and dialysed at 4 °C for at least 48 h against
distilled water, then freeze dried. The residual
flocculating activity of the yeast was assessed
in Helm’s acetate buffer.
Flocculation inhibition tests.—Non-floccu-
lating yeast cells obtained after lectin extrac-
tion were washed with distilled water, then
with 10 mM EDTA aqueous solution, and
twice with distilled water. Washed cells (about
2.5×106 cells in 1 mL) were suspended in 10
mL Helm’s acetate buffer (pH 4.5). Inhibition
of flocculation was assayed in glass tubes by
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