Syntheses of an α-D-Gal-(1→6)-β-D-Gal diglyceride, as lipase substrate

Article abstract of DOI:10.1016/j.carres.2006.01.021

Two different routes were explored to afford 3-O-(6-O-α-d- galactopyranosyl-β-d-galactopyranosyl)-1,2-di-O-dodecanoyl-sn-glycerol. In the first one, the key step was the glycosylation of the 3-O-(2,3,4-tri-O- benzyl-β-d-galactopyranosyl)-1,2-O-isopropylid

Full text of DOI:10.1016/j.carres.2006.01.021

Carbohydrate Research 341 (2006) 695–704
Syntheses of an a-D-Gal-(1!6)-b-D-Gal diglyceride,
as lipase substrate
a
b
b
a,
*
´ ´
`
Dominique Lafont, Frederic Carriere, Francine Ferrato and Paul Boullanger
a
ˆ
Laboratoire de Chimie Organique II, UMR CNRS 5181, Universite´ Lyon 1, Batiment 308, 43 Bd du 11 Novembre 1918,
F 69622 Villeurbanne, France
bLaboratoire d’Enzymologie Interfaciale et de Physiologie de la Lipolyse, UPR CNRS 9025, IBSM, 31 chemin Joseph Aiguier,
F 13402 Marseille, France
Received 21 December 2005; received in revised form 16 January 2006; accepted 17 January 2006
Available online 3 February 2006
Abstract—Two different routes were explored to afford 3-O-(6-O-a-D-galactopyranosyl-b-D-galactopyranosyl)-1,2-di-O-dodecano-
yl-sn-glycerol. In the first one, the key step was the glycosylation of the 3-O-(2,3,4-tri-O-benzyl-b-D-galactopyranosyl)-1,2-O-iso-
propylidene-sn-glycerol acceptor with 2-pyridyl 2,3,4,6-tetra-O-benzyl-1-thio-b-D-galactopyranoside as the donor. In the second
one, the key step was the coupling of 2,3,4-tri-O-acetyl-6-O-(2,3,4,6-tetra-O-benzyl-a-D-galactopyranosyl)-D-galactopyranosyl tri-
chloroacetimidate donor with 1,2-O-isopropylidene-sn-glycerol. Even though the number of steps was the same in both pathways,
the first one afforded a better overall yield (12.4%) than the second one (6.5%). This eight-step synthesis allowed the preparation of
the expected glycolipid, which was used as substrate for recombinant GPLRP2 galactolipase using the monomolecular film
technique.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Keywords: Digalactosyldiglyceride (DGDG); Glycosylation; Lipase
1. Introduction
lipase-related protein 2 (PLRP2) as the enzyme from
human pancreatic juice displaying the major galacto-
lipase activity.3 In order to investigate the biochemical
properties and the substrate specificity of PLRP2, we
synthesized previously two monogalactosyl diglycerides
epimers at C-2 of the glycerol moiety.3,4 Then, using
the monomolecular film technique, we reported the
activity of human and guinea pig PLRP2s on 1,2-di-O-
dodecanoyl-3-O-b-D-galactopyranosyl-sn-glycerol and
its epimer.
The most abundant membrane lipids in plants are
galactoglycerolipids, which constitute about 75% of
the total membranes lipids in leaves.1 Monogalactosyl
diacylglycerol (MGDG) and digalactosyl diacylglycerol
(DGDG) are the major galactolipids found in higher
plants where they represent up to 50% and 20% of the
chloroplast lipids, respectively. In both families, a b-D-
galactopyranosyl moiety is linked to a diacylglycerol;
in DGDG, a second a-D-galactopyranosyl residue is
linked at OH-6 of the first galactopyranosyl unit.
The present paper is devoted to the chemical synthesis
of a digalactosyl diacylglycerol, analogous with the
monogalactosyl diacylglycerol described previously.3
Ten years ago, Anderson et al.2 demonstrated that the
lipolysis of galactolipids was not restricted to herbi-
vores. They reported that human pancreatic juice and
duodenal contents were able to hydrolyze digalactosyl
glycerolipids. Recently, we have identified pancreatic
2. Results and discussion
Digalactosyl glycerides with various chain lengths have
been widely studied since they are easily obtained from
natural sources. Among the recent papers dealing with
*
Corresponding author. Tel.: +33 4 72 43 11 62; e-mail: Paul.
Boullanger@univ-lyon1.fr
0008-6215/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2006.01.021

696
D. Lafont et al. / Carbohydrate Research 341 (2006) 695–704
such compounds, the isolation of DGDG derivatives
bearing poly-unsaturated lipid chains,5–7 poly-unsatu-
rated and saturated lipid chains8,9 or saturated lipid
chains only can be mentioned.10 All these compounds
have been fully characterized by 1H, 13C NMR and mass
spectrometry. To our knowledge, there are only two
examples of synthetic digalactosyl diglycerides described
in the literature. The synthesis of 3-O-(6-O-a-D-galacto-
pyranosyl-b-D-galactopyranosyl)-1,2-di-O-stearoyl-sn-
glycerol by Gent and Gigg in 1975 was the first prepara-
tion of a digalactosyl diglyceride.11,12 Very recently,
Tanaka et al.13 reported the synthesis of a series of the
aforementioned compounds from a common precursor,
in order to study the structure–inhibition relationship of
the latter on human lanosterol synthase. The present
paper deals with our own approaches to the synthesis
of these biologically relevant derivatives.
monogalactosyl glycerol acceptor was synthesized and
then reacted with an appropriate a-D-galactopyranosyl
donor. In the second approach, the a-Gal(1!6)Gal
disaccharide was prepared first, before coupling with
the glycerol moiety.
The first pathway involves the glycosylation of 1,2-O-
isopropylidene-sn-glycerol with
a
galactopyranosyl
donor able to induce the b-anomeric stereochemistry,
without epimerization of the glycerol moiety. As reported
previously,3 the imidate methodology developed by
Schmidt,14 allowed to achieve this goal contrary to use
galactosyl bromides, which give rise to epimerization
of the glycerol moiety.15 Therefore, a trichloroacetimido-
yl glycosylation donor was prepared as follows. 1,2:3,4-
Di-O-isopropylidene-a-D-galactopyranose (1)16 was pro-
tected at OH-6 with a p-methoxyphenyl group via a
Mitsunobu reaction, to afford the 6-O-p-methoxyphenyl
ether 2. After acetal cleavage in acidic conditions and
acetylation, product 3 was obtained as an a,b-anomeric
Two different strategies were attempted toward the
synthesis of compound 15 (Chart 1). In the first one, a
R4O
R3O
1
2
3
4
R1-R2 = R3-R4 = C(CH3)2, R6 = H
OR6
R1-R2 = R3-R4 = C(CH3)2, R6 = C6H4OCH3
R1 = R2 = R3 = R4 = Ac, R6 = C6H4OCH3
R1= C(=NH)CCl3, R2 = R3 = R4 = Ac, R6 = C6H4OCH3
O
OR1
OR2
R4O
R3O
5
6
7
8
9
Ra-Rb = C(CH3)2, R2 = R3 = R4 = Ac, R6 = C6H4OCH3
Ra-Rb = C(CH3)2, R2 = R3 = R4 = Bn, R6 = C6H4OCH3
Ra-Rb = C(CH3)2, R2 = R3 = R4 = Bn, R6 = H
Ra-Rb = C(CH3)2, R2 = R3 = R4 = R6 = Ac
OR6
O
O
O
OR2
ORa
RbO
Ra-Rb = C(CH3)2, R2 = R3 = R4 = H, R6 = TBDMS
10 Ra-Rb = C(CH3)2, R2 = R3 = R4 = Bn, R6 = TBDMS
BnO
BnO
OBn
N
S
11
BnO
R4'O
R3'O
OR6'
O
12 Ra-Rb = C(CH3)2, R2 = R3 = R4 = R2' = R3' = R4' = R6'= Bn
13 Ra = Rb = H, R2 = R3 = R4 = R2' = R3' = R4' = R6'= Bn
14 Ra = Rb = COC11H23, R2 = R3 = R4 = R2' = R3' = R4' = R6'= Bn
15 Ra = Rb = COC11H23, R2 = R3 = R4 = R2' = R3' = R4' = R6'= H
19 Ra-Rb = C(CH3)2, R2 = R3 = R4 = Ac, R2' = R3' = R4' = R6'= Bn
R4O R2'O
O
O
R3O
O
OR2
ORa
RbO
R4'O
R3'O
OR6'
O
16 R1-R2 = R3-R4 = C(CH3)2, R2' = R3' = R4' = R6'= Bn
17 R1 = R2 = R3 = R4 = Ac, R2' = R3' = R4' = R6'= Bn
18 R1 = C(=NH)CCl3, R2 = R3 = R4 = Ac, R2' = R3' = R4' = R6'= Bn
R4O
2'
R O
O
O
R3O
OR1
OR2
Chart 1.

D. Lafont et al. / Carbohydrate Research 341 (2006) 695–704
697
mixture. Regioselective cleavage of the anomeric acetate
with hydrazine acetate, followed by reaction with
trichloroacetonitrile under basic conditions afforded
the imidate 4. Glycosylation of 1,2-O-isopropylidene-
sn-glycerol with imidate 4 led to the glycoside 5 in
72% yield. A change of protective groups to compound
6 was effected by O-deacetylation and benzylation
(benzyl chloride and powdered potassium hydroxide in
dimethylsulfoxide). Finally, the p-methoxyphenyl group
was removed with ammonium cerium nitrate in aceto-
nitrile to afford 7. Another route to compound 7 involved
the use of the known 3-O-(2,3,4,6-tetra-O-acetyl-b-D-
galactopyranosyl)-1,2-O-isopropylidene-sn-glycerol (8)3
as starting material. This compound was O-deacetylated
ride 16 in 74% yield, after purification. Treatment of the
disaccharide 16 under acidic condition (80% aqueous
trifluoroacetic acid) and O-reacetylation afforded the
product 17 in 62% yield as a a,b-anomeric mixture. The
anomeric acetate was selectively removed using benzyl
amine in tetrahydrofurane, and the hemiacetal (obtained
in 70% yield) was transformed into the imidate 18 (89%
yield) by treatment with trichloracetonitrile and DBU
in dichloromethane. Glycosylation of 1,2-O-isopropyl-
idene-sn-glycerol with the imidate 18, promoted by
catalytic trimethylsilyl trifluoromethanesulfonate, in
dichloromethane at ꢀ30 ꢁC afforded the expected deriv-
ative 19 in 55% yield. Compound 12 was then obtained in
75% yield, after O-deacetylation and O-benzylation. As a
summary, the first route to 15, via compound 7 inter-
mediate, afforded the expected derivative with a 12.4%
overall yield, whereas the second one, via compound
18 intermediate, displayed a 6.5% overall yield only.
We further checked that the a-Gal(1!6)-b-Gal
diglyceride (DGDG) obtained was recognized and
hydrolysed by a galactolipase. Figure 1 shows the
hydrolysis of the DGDG by the guinea pig pancreatic
lipase-related protein 2 (GPLRP2) measured using the
monomolecular film technique. The maximum enzyme
activity on DGDG was measured at a surface pressure
of 5 mN/m, whereas the activities of the same enzyme
on monogalactosyldiglyceride (MGDG), 1,2-dicaprine
(1,2-DC10) and phosphatidylglycerol (DiC12PG) were
maximum at higher surface pressures. The activity on
´
by the Zemplen method and selectively silylated at 6-OH
using tert-butyldimethylsilyl chloride and imidazole in
dimethylformamide. The free hydroxyl groups of 9 were
benzylated to afford compound 10 in 66% yield. Finally,
the silyl ether was cleaved with 1 N tetrabutylammo-
nium fluoride in tetrahydrofurane to provide the alcohol
7 in 85% yield. It could be mentioned that the overall
yield to 7 of the latter pathway (from 2,3,4,6-tetra-O-
acetyl-D-galactopyranosyl trichloroacetimidate and 1,2-
O-isopropylidene-sn-glycerol)3 is much more better than
the preceding one (37.3% vs 16.3%). Glycosylation of
acceptor 7 requires a donor able to induce the a-ano-
meric stereochemistry in smooth conditions. Trichloro-
acetimidoyl donors are sometimes of lower efficiency
than thioglycosides in the induction of 1,2-cis stereo-
chemistry with primary alcohol acceptors. On the other
hand, the activation of thioglycosides often requires
promoters that are dangerous and difficult to handle.
This prompted us to use the methodology developed
by Mereyala et al.17 with 2-pyridyl 2,3,4,6-tetra-O-benz-
yl-1-thio-b-D-galactopyranoside (11) as the glycosyl
donor and methyl iodide as the promoter. Thus, the
reaction of acceptor 7 and donor 11 afforded the a-gly-
coside 12, although in a modest yield (60%) that can be
explained by the formation of small amounts of the b-
disaccharide by-product, difficult to separate by column
chromatography. The acetal function was cleaved using
85% aqueous acetic acid and, after usual work up, the
crude reaction product was treated by catalytic sodium
methylate in methanol in order to remove the traces of
monoacetylated derivatives formed during the acetic
acid treatment. Reaction of diol 13 with dodecanoyl
chloride and pyridine afforded the pure perbenzyl diga-
lactosyl didodecanoylglyceride 14 in 92% yield. Finally,
debenzylation was realized by hydrogen transfer, using
cyclohexene and 20% palladium hydroxide in absolute
ethanol. The deprotected product 15 was obtained in
75% yield after purification by column chromatography.
The second pathway to compound 15 involved the
coupling of 1,2:3,4-di-O-isopropylidene-a-D-galactopyr-
anose (1) and 2-pyridyl 2,3,4,6-tetra-O-benzyl-1-thio-b-
D-galactopyranoside (11)17 to give the a-linked disaccha-
Figure 1. Variations with surface pressure of guinea pig pancreatic
lipase-related protein 2 (GPLRP2) lipolytic activities on monomole-
cular lipid films of 15 (DGDG), 3-O-b-D-galactopyranosyl-1,2-di-O-
dodecanoyl-sn-glycerol (MGDG),3 1,2-dicaprin (1.2DC10) and 1,2-di-
dodecanoyl phosphatidylglycerol (DiC12PG).

698
D. Lafont et al. / Carbohydrate Research 341 (2006) 695–704
MGDG is maximum at 12 mN/m whereas the activity
on DGDG is almost abolished at this surface pressure.
This observation suggests that the presence of an addi-
tional galactose residue, when compared to MGDG,
decreases the enzyme interaction with the lipid–water
interface when the molecular packing of the substrate
molecules is increased. One hypothesis is that the
increased surface density of the hydrophilic moiety of
DGDG impairs the adsorption of the enzyme, which is
mainly driven by hydrophobic interactions. Validation
of this hypothesis will require however the simultaneous
measurement of lipase adsorption and activity to com-
plete these preliminary experiments. The detailed kinetic
characterization of the galactolipase action on DGDG
and MGDG studied by the monolayer technique, will
be published separately.
In summary, we have tested two different routes to
digalactosyl diglycerides. The best results were obtained
when the glycerol residue was b-linked to the galactose,
before the formation of the a-(1!6) linkage between the
two D-galactose moieties. It is afore to mention that the
syntheses of the titled saccharide, reported in this paper,
can be compared favourably to those reported earlier in
the literature. In the first route, the improvement is dis-
played in terms of the number of steps and yields;11,12 in
the second one, in terms of the number of steps.13
3.2. 1,2:3,4-Di-O-isopropylidene-6-O-p-methoxyphenyl-
a-D-galactopyranose (2)
Diisopropylazodicarboxylate (1.10 mL, 5.55 mmol) was
added dropwise at 0 ꢁC under argon to a soln of alcohol
1 (0.775 g, 4.30 mmol), triphenylphosphine (1.45 g,
5.53 mmol) and 4-methoxyphenol (1.56 g, 12.57 mmol)
in THF (15 mL). The mixture was stirred for 30 min
at 0 ꢁC, then at 80 ꢁC for 4 h. After cooling, the soln
was concentrated, Et2O (100 mL) was added and the
soln was washed twice with 15% aq NaOH
(2 · 20 mL), then with brine (2 · 10 mL) and dried
(Na2SO4). Evaporation of the solvent afforded a solid
residue, which was recrystallized from absolute EtOH
to afford compound 2 as a white solid (1.30 g, 83%);
mp 71 ꢁC (EtOH); [a]D ꢀ77.6 (c 1.0, CHCl3); Rf 0.55
(1:3 EtOAc–petroleum ether); 1H NMR (CDCl3): d
6.92–6.79 (m, 4H, C6H4), 5.58 (d, 1H, J1,2 5.0 Hz, H-
1), 4.65 (dd, 1H, J2,3 7.9, J3.4 2.3 Hz, H-3), 4.38–4.34
(m, 2H, H-2, H-4), 4.16–4.06 (m, 3H, H-5, H-6a, H-
6b), 3.76 (s, 3H, OCH3), 1.51, 1.47, 1.36, 1.34 (4s,
12H, 2C(CH3)2); 13C NMR (CDCl3): d 154.1, 152.8,
115.9, 114.6 (C6H4), 109.3, 108.6 (2C(CH3)2), 96.4 (C-
1), 71.0, 70.7, 70.4 (C-2, C-3, C-5), 67.4 (C-6), 66.2 (C-
4), 55.6 (OCH3), 26.0, 25.0, 24.5 (2C(CH3)2). Anal.
Calcd for C19H26O7 (366.40): C, 62.28; H, 7.15. Found:
C, 62.04; H, 7.27.
3. Experimental
3.3. 1,2,3,4-Tetra-O-acetyl-6-O-p-methoxyphenyl-a-D-
galactopyranose (3)
3.1. Materials and methods
Compound 2 (1.25 g, 3.41 mmol) was dissolved in 80%
aq AcOH (25 mL) and the soln was heated for 12 h at
80 ꢁC. After cooling to rt, the soln was concentrated
under diminished pressure and the residue was coeva-
porated twice from toluene (2 · 15 mL). The crude
product was acetylated overnight in a 2:1 pyridine–
Ac2O mixture (40 mL). After concentration, the residue
was purified by column chromatography (1:1 EtOAc–
petroleum ether) to afford a mixture of a,b-anomers
(1.40 g, 90%) from which the pure a-anomer could be
recovered by recrystallization from EtOH; mp 98 ꢁC
(EtOH); [a]D +70.2 (c 1.0, CHCl3); Rf 0.80 (1:1
Pyridine was dried by boiling with CaH2 prior to distil-
lation. Dichloromethane was washed twice with water,
dried with CaCl2 and distilled over P2O5. MeOH was
distilled from magnesium. Tetrahydrofuran was distilled
over sodium-benzophenone. Pyridine, THF and CH2Cl2
˚
were stored over 4 A molecular sieves and MeOH over
˚
3 A molecular sieves. Recombinant GPLRP2 was pro-
duced in Aspergillus orizae.18 Melting points were deter-
mined on a Buchi apparatus and were uncorrected. Thin
¨
layer chromatography was performed on aluminium
sheets coated with Silica gel 60 F254 (E. Merck). Com-
pounds were visualized by spraying the TLC plates with
dilute 15% aq H2SO4, followed by charring at 150 ꢁC for
a few minutes. Column chromatography was performed
on Silica-gel Geduran Si 60 (E. Merck). Optical rota-
tions were recorded on a Perkin–Elmer 241 polarimeter
1
EtOAc–petroleum ether); H NMR (CDCl3): d 6.81 (s,
4H, C6H4), 6.42 (d, 1H, J1,2 2.4, H-1), 5.68 (br s, 1H,
H-4), 5.42–5.37 (m, 2H, H-3, H-2), 4.47 (bdd, 1H,
J5,6a 5.9, J5,6b 7.3 Hz, H-5), 4.04 (dd, 1H, J6a,6b 9.5 Hz,
H-6a), 3.88 (dd, 1H, H-6b), 3.76 (s, 3H, OCH3), 2.16,
2.10, 2.04, 2.02 (4s, 12H, 4CH3CO); 13C NMR (CDCl3):
d 170.0, 169.9, 169.8, 169.0 (4CH3CO), 154.4, 152.1,
115.9, 114.6 (C6H4), 89.8 (C-1), 69.4 (C-5), 67.8, 67.5
(C-2, C-3), 67.4 (C-6), 66.6 (C-4), 55.6 (OCH3), 20.8,
20.6, 20.5 (4CH3COO). Anal. Calcd for C21H26O11
(454.42): C, 55.50; H, 5.77. Found: C, 55.46; H, 5.75.
1
in a 1 dm cell at 21 ꢁC. H and 13C NMR spectra were
recorded with a Bruker AC-200 spectrometer (working
at 200 MHz and 50 MHz) or a Bruker DRX 500 (work-
ing at 500 MHz and 125 MHz), respectively, with Me4Si
as internal standard. Elemental analyses were performed
by the ‘Laboratoire Central d’Analyses du CNRS’
(Vernaison, France). Mass spectra were recorded on a
Finnigan Mat 95 XL apparatus in FAB mode.
1
Selected data for b-anomer (in mixture with a): H
NMR (CDCl3): d 5.76 (d, 1H, J1,2 8.2, H-1), 5.62 (br

D. Lafont et al. / Carbohydrate Research 341 (2006) 695–704
699
d, 1H, J3,4 3.4 Hz, H-4), 5.37 (dd, 1H, J2,3 10.4 Hz, H-2),
5.14 (dd, 1H, H-3), 3.76 (s, 3H, OCH3).
ing to ꢀ20 ꢁC. A soln of TMSOTf (10 lL, 0.046 mmol)
in CH2Cl2 (0.5 mL) was introduced through a syringe
during a period of 1 h. The mixture was stirred for 4 h
at ꢀ20 ꢁC and then neutralized by the addition of
Et3N (20 lL). After filtration over Celite and dilution
with CH2Cl2 (50 mL), the organic phase was washed
with satd aq NaHCO3 and dried (Na2SO4). Concentra-
tion of the soln gave a residue, which was purified by
column chromatography (1:2 petroleum ether–EtOAc)
affording compound 5 as an oil (0.520 g, 72%); [a]D
ꢀ14.3 (c 1.0, CHCl3); Rf 0.60 (1:1 EtOAc–petroleum
3.4. 2,3,4-Tri-O-acetyl-6-O-p-methoxyphenyl-a-D-galacto-
pyranosyl trichloroacetimidate (4)
Hydrazine acetate (0.320 g, 3.47 mmol) was added to a
soln of 3 (1.38 g, 3.03 mmol) in dry DMF and the mix-
ture was heated at 50 ꢁC for 1 h. The soln was cooled to
rt, EtOAc (40 mL) was added and the organic extract
was washed twice with brine (2 · 10 mL). After drying
(Na2SO4) and concentration, the residue was purified
by column chromatography (1:1 EtOAc–petroleum
ether) to afford the expected hemiacetal as a 5:2 a,b-ano-
meric mixture (0.780 g, 62%); oily material; Rf 0.49–0.40
(1:2 EtOAc–petroleum ether). a-Anomer, 1H NMR
(CDCl3): d 6.82 (s, 4H, C6H4), 5.63–5.58 (m, 2H, H-4,
H-1), 5.47–5.21 (m, 2H, H-2, H-3), 4.63 (br dd, 1H,
J4,5 1.0, J5,6a 6.1, J5,6b 6.1 Hz, H-5), 4.13–3.90 (m, 2H,
H-6a, H-6b), 3.76 (s, 3H, OCH3), 2.12, 2.10, 2.01 (3s,
9H, 3CH3CO); 13C NMR (CDCl3): d 170.7, 170.5,
170.3 (3CH3CO), 154.2, 152.3, 115.8, 114.1 (C6H4),
90.5 (C-1), 68.8, 68.6 (C-3, C-5), 67.6 (C-2), 67.2 (C-6),
66.8 (C-4), 55.6 (OCH3), 20.7, 20.6, 20.5 (3CH3COO).
Selected data for b-anomer: 13C NMR (CDCl3): d
95.6 (C-1).
1
ether); H NMR (CDCl3): d 6.82 (s, 4H, C6H4), 5.57
(br d, 1H, J3,4 3.3, J4,5 0.7 Hz, H-4), 5.24 (dd, 1H, J1,2
7.8, J2,3 10.5 Hz, H-2), 5.06 (dd, 1H, H-3), 4.62 (d,
1H, H-1), 4.28 (m, 1H, H-2gl), 4.13–3.89 (m, 5H, H-5,
H-6a, H-6b, H-1agl, H-3agl), 3.82 (dd, 1H, J1bgl,2gl 6.0,
J1agl,1bgl 8.2 Hz, H-1bgl), 3.77 (s, 3H, CH3O), 3.68 (dd,
1H, J2gl,3bgl 6.0, J3agl,3bgl 10.5 Hz, H-3bgl), 2.11, 2.08,
2.00 (3s, 9H, 3CH3CO), 1.43, 1.35 (2s, 6H, C(CH3)2);
13C NMR (CDCl3): d 170.2, 170.1, 169.5 (3CH3CO),
154.4, 152.3, 115.9, 114.7 (C6H4), 109.4 (C(CH3)2),
101.5 (C-1), 74.3 (C-2gl), 71.7 (C-5), 71.1 (C-3), 69.2
(C-3gl), 68.9 (C-2), 67.5 (C-4), 66.5 (C-6), 66.2 (C-1gl),
55.7 (OCH3), 26.6, 25.2 (C(CH3)2), 20.7, 20.6, 20.5
(3CH3COO). Anal. Calcd for C25H34O12 (526.522): C,
57.02; H, 6.51. Found: C, 56.64; H, 6.19.
DBU (0.275 mL, 1.84 mmol) was added to a soln of
the hemiacetal (0.780 g, 1.89 mmol) and trichloroaceto-
nitrile (1.2 mL, 12.0 mmol) in dry CH2Cl2 (5.7 mL)
and the mixture was stirred for 4 h at rt. After concen-
tration under diminished pressure, the residue was puri-
fied by column chromatography (1:2 EtOAc–petroleum
ether) to afford the pure imidate 4 as an oily material
(0.790 g, 77%); [a]D +66.3 (c 1.0, CHCl3); Rf 0.50 (1:2
EtOAc–petroleum ether); 1H NMR (CDCl3): d 8.67
(s, 1H, NH), 6.79 (s, 4H, C6H4), 6.63 (d, 1H, J1,2 3.2,
H-1), 5.74 (dd, 1H, J3,4 2.9 Hz, J4,5 0.8, H-4), 5.49 (dd,
1H, J2,3 10.8 Hz, H-3), 5.39 (dd, 1H, H-2), 4.56 (ddd,
1H, J5,6a 6.4, J5,6b 7.3 Hz, H-5), 4.08 (dd, 1H, J6a,6b
9.7 Hz, H-6a), 3.91 (dd, 1H, H-6b), 3.76 (s, 3H,
OCH3), 2.05, 2.04, 2.03 (3 s, 9H, 3CH3CO); 13C NMR
(CDCl3): d 170.9, 170.0, 169.9 (3CH3CO), 160.8
(OC(NH)CCl3), 154.4, 152.0, 115.8, 114.6 (C6H4), 93.6
(C-1), 90.8 (CCl3), 69.4 (C-5), 67.7, 67.6 (C-2, C-3),
67.0 (C-4), 66.1 (C-6), 55.5 (OCH3), 20.9, 20.6, 20.5
(3CH3COO).
3.6. 3-O-(2,3,4-Tri-O-benzyl-6-O-p-methoxyphenyl-b-D-
galactopyranosyl)-1,2-O-isopropylidene-sn-glycerol (6)
Compound 5 (0.500 g, 0.95 mmol) was stirred overnight
in MeOH (25 mL) in the presence of a chip of MeONa.
After concentration, the crude product was dissolved in
Me2SO (2 mL) and added dropwise to a cold (0 ꢁC) mix-
ture of BnCl (0.440 mL, 3.82 mmol) and powdered
KOH (0.375 g, 6.68 mmol) in Me2SO (2.5 mL). The
mixture was stirred for 16 h at rt, then poured into ice
and the product was extracted with CH2Cl2
(2 · 30 mL). The organic phase was washed once with
water (15 mL), then dried and concentrated under
diminished pressure. The residue was purified by column
chromatography (5:2 petroleum ether–EtOAc) affording
the pure product 6 as an oil (0.450 g, 71%); [a]D ꢀ6.0 (c
1
1.0, CHCl3); Rf 0.58 (2:5 EtOAc–petroleum ether); H
NMR (CDCl3): d 7.43–7.25 (m, 15H, 3C6H5), 6.87 (s,
4H, C6H4), 5.02 and 4.69 (2d, 2H, J 11.6 Hz, CH2Ph),
5.00 and 4.85 (2d, J 10.6 Hz, CH2Ph), 4.87 and 4.80
(2d, J 12.1 Hz, CH2Ph), 4.50 (d, 1H, J1,2 7.6 Hz, H-1),
4.39 (m, 1H, H-2gl), 4.15–3.60 (m, 13H, H-2, H-3, H-
4, H-5, H-6a, H-6b, H-1agl, H-1bgl, H-3agl, H-3bgl,
OCH3), 1.48, 1.42 (2s, 6H, C(CH3)2); 13C NMR
(CDCl3): d 154.4, 152.3, 115.9, 114.7 (C6H4), 138.8,
138.6, 138.4, 128.6, 128.5, 128.4, 128.3, 128.2, 127.7,
127.6 (3C6H5), 109.4 (C(CH3)2), 104.4 (C-1), 82.2 (C-
3), 79.5 (C-2), 75.3, 74.7, 73.3 (3CH2Ph), 74.4 (C-2gl),
73.2, 73.0 (C-4, C-5), 70.4 (C-3gl), 67.2, 66.9 (C-6, C-1gl),
Anal. Calcd for C21H24Cl3NO11 (556.762): C, 45.30;
H, 4.35; N, 2.52. Found: C, 45.10; H, 4.46; N, 2.85.
3.5. 3-O-(2,3,4-Tri-O-acetyl-6-O-p-methoxyphenyl-b-D-
galactopyranosyl)-1,2-O-isopropylidene-sn-glycerol (5)
1,2-O-Isopropylidene-sn-glycerol (0.270 g, 1.89 mmol),
˚
imidate 4 (0.740 g, 1.36 mmol) and 4 A activated mole-
cular sieves (0.500 g) were added to alcohol-free CH2Cl2
(10 mL). The mixture was flushed with argon while cool-

700
D. Lafont et al. / Carbohydrate Research 341 (2006) 695–704
55.8 (OCH3), 27.0, 25.5 (C(CH3)2). Anal. Calcd for
C40H46O9 (670.768): C, 71.62; H, 6.91. Found: C,
71.33; H, 6.99.
cerol (8),3 was treated at 0 ꢁC with a mixture of tert-
butyldimethysilyl chloride (0.823 g, 5.56 mmol) and
imidazole (0.707 g, 10.38 mmol) in dry DMF (10 mL).
Stirring was maintained for 1 h at 0 ꢁC, then overnight
at rt. The reaction mixture was diluted with CHCl3,
washed successively with 10% aq HCl (10 mL), then
with satd aq NaHCO3 and finally with water (10 mL).
The organic extract was dried (Na2SO4), concentrated
under diminished pressure and the residue was coevapo-
rated twice from toluene. After purification by column
chromatography (10:1 CHCl3–EtOH), the pure product
9 was recovered as an oil (1.88 g, 80%); [a]D ꢀ12.7 (c 2.0,
CHCl3); Rf 0.80 (10:1 CHCl3–EtOH); 1H NMR
(CDCl3): d 4.34 (dddd, 1H, J1agl,2gl 6.4, J1bgl,2gl 6.2,
J2gl,3agl 5.8, J2gl,3bgl 5.2 Hz, H-2gl), 4.30 (d, 1H, J1,2
7.4 Hz, H-1), 4.07 (dd, 1H, J1agl,1bgl 8.2 Hz, H-1agl),
4.05 (br d, 1H, J3,4 3.2 Hz, J4,5 1.0 Hz, H-4), 3.94 (dd,
1H, J5,6a 5.4, J6a,6b 10.5 Hz, H-6a), 3.93 (dd, 1H,
J3agl,3bgl 10.6 Hz, H-3agl), 3.86 (dd, 1H, J5,6b 5.6 Hz,
H-6b), 3.84 (dd, 1H, H-1bgl), 3.70 (dd, 1H, J2,3 9.8 Hz,
H-2), 3.64 (dd, 1H, H-3bgl), 3.58 (dd, 1H, H-3), 3.50
(ddd, 1H, H-5), 1.44, 1.36 (2s, 6H, C(CH3)2), 0.90
(s, 9H, (CH3)3CSi), 0.09 (s, 6H, (CH3)2Si); 13C
NMR (CDCl3): d 109.5 (C(CH3)2), 103.8 (C-1), 75.1
(C-5), 74.4 (C-2gl), 73.8 (C-3), 71.2 (C-2), 70.4 (C-3gl),
68.7 (C-4), 66.6 (C-1gl), 62.3 (C-6), 26.8, 25.3
(C(CH3)2), 25.9 ((CH3)3CSi), 18.2 ((CH3)3CSi), ꢀ5.3
((CH3)2Si).
3.7. 3-O-(2,3,4-Tri-O-benzyl-b-D-galactopyranosyl)-1,2-
O-isopropylidene-sn-glycerol (7)
Method A. Compound 6 (0.430 g, 0.64 mmol) was dis-
solved in MeCN (8.5 mL); water (2.2 mL) and NaHCO3
(0.555 g, 6.60 mmol) were added and the mixture was
cooled to 0 ꢁC, before the addition of ammonium cer-
ium nitrate (0.935 g, 1.70 mmol). The mixture was
stirred for 1 h at 0 ꢁC, then concentrated under dimini-
shed pressure and extracted with CHCl3. The organic
phase was washed with water, dried and concentrated
again; then, the crude product was purified by column
chromatography (5:2 petroleum ether–EtOAc) affording
the pure product 7 as an oil (0.235 g, 69%); [a]D ꢀ16.6 (c
1
1.0, CHCl3); Rf 0.65 (2:5 EtOAc–petroleum ether); H
NMR (CDCl3): d 7.35–7.30 (m, 15H, 3C6H5), 4.97 and
4.68 (2d, 2H, J 11.8 Hz, CH2Ph), 4.92 and 4.74 (2d, J
11.0 Hz, CH2Ph), 4.83 and 4.78 (2d, J 10.5 Hz, CH2Ph),
4.40 (d, 1H, J1,2 7.5 Hz, H-1), 4.34 (dddd, 1H, J1agl,2gl
6.3, J1bgl,2gl 6.2, J2gl,3agl 5.0, J2gl,3bgl 6.2 Hz, H-2gl),
4.07 (dd, 1H, J1agl,1bgl 8.3 Hz, H-1agl), 3.98 (dd, 1H,
J3agl,3bgl 10.3 Hz, H-3agl), 3.85 (dd, 1H, J2,3 9.8 Hz, H-
2), 3.82 (dd, 1H, H-1bgl), 3.80–3.79 (m, 1H, H-4), 3.77
(dd, 1H, J5,6a 6.3, J6a,6b 10.3 Hz, H-6a), 3.61 (dd, 1H,
H-3bgl), 3.54 (dd, 1H, J3,4 3.2 Hz, H-3), 3.50 (dd, 1H,
J5,6b 5.4 Hz, H-6b), 3.38 (bdd, 1H, H-5), 1.64 (m, 1H,
OH), 1.41, 1.36 (2s, 6H, C(CH3)2); 13C NMR (CDCl3):
d 138.7, 138.4, 138.3, 128.7, 128.5, 128.4, 128.2, 128.0,
127.8, 127.7 (3C6H5), 109.5 (C(CH3)2), 104.5 (C-1),
82.3 (C-3), 79.6 (C-2), 75.3, 74.3, 73.4 (3CH2Ph), 74.9
(C-5), 74.7 (C-2gl), 73.0 (C-4), 70.8 (C-3gl), 67.1 (C-1gl),
61.9 (C-6), 26.9, 25.5 (C(CH3)2). Anal. Calcd for
C33H40O8 (564.65): C, 70.19; H, 7.14. Found: C, 70.05;
H, 6.95.
Method B. A 1 N soln of Bu4NF in THF (1.3 mL) was
added dropwise at 0 ꢁC under argon to a soln of sily-
lated derivative 10 (0.679 g, 1.00 mmol) in THF
(20 mL). The mixture was stirred for 1 h at 0 ꢁC, then
poured into satd aq NaHCO3 (60 mL). After concentra-
tion under diminished pressure, the product was ex-
tracted with CH2Cl2 (4 · 20 mL). The organic phase
was dried, concentrated and purified by column chroma-
tography as described above. Pure product 7 was recov-
ered in 85% yield.
3.9. 3-O-(2,3,4-Tri-O-benzyl-6-O-tert-butyldimethylsilyl-
b-D-galactopyranosyl)-1,2-O-isopropylidene-sn-glycerol
(10)
Sodium hydride (60% in oil, 0.700 g, 17.5 mmol) was
added portionwise to a stirred soln of compound 9
(1.25 g, 3.06 mmol) in dry THF (15 mL) under argon.
After 1 h, benzyl bromide (1.65 mL, 13.79 mmol) and
Bu4NI (0.185 g, 0.50 mmol) were added and the mixture
was stirred for 16 h at rt. Sodium hydride (0.200 g,
5.00 mmol) and Bu4NI (0.185 g, 0.50 mmol) were added
again and the mixture was stirred at 50 ꢁC for 16 h.
After cooling to rt, the excess of benzyl bromide was
destroyed by careful addition of MeOH at 0 ꢁC
(0.5 mL) and the mixture was concentrated. The residue
was diluted with EtOAc (80 mL), the organic phase was
washed with water (2 · 15 mL), dried (Na2SO4) and
concentrated. After purification by column chromato-
graphy (5:1 petroleum ether–EtOAc), the pure product
10 was recovered as a solid (1.37 g, 66%); mp 63–
64 ꢁC; [a]D +10.0 (c 1.0, CHCl3); Rf 0.60 (5:1 petroleum
ether–EtOAc); 1H NMR (CDCl3): d 7.36–7.29 (m, 15H,
3C6H5), 4.99–4.61 (m, 6H, 3CH2C6Ph), 4.38 (d, 1H, J1,2
7.8 Hz, H-1), 4.32 (dddd, 1H, J1agl,2gl 6.6, J1bgl,2gl 6.1,
J2gl,3agl 4.7, J2gl,3bgl 7.0 Hz, H-2gl), 4.06 (dd, 1H, J1agl,1bgl
8.5 Hz, H-1agl), 4.00 (dd, 1H, J3agl,3bgl 10.1 Hz, H-3agl),
3.87 (dd, 1H, H-1bgl), 3.87 (br d, 1H, J3,4 3.2,
3.8. 3-O-(6-O-tert-Butyldimethylsilyl-b-D-galactopyranos-
yl)-1,2-O-isopropylidene-sn-glycerol (9)
3-O-b-D-Galactopyranosyl-1,2-O-isopropylidene-sn-gly-
cerol (1.53 g, 5.19 mmol), obtained quantitatively by
´
Zemplen O-deacetylation of 3-O-(2,3,4,6-tetra-O-acet-
yl-b-D-galactopyranosyl)-1,2-O-isopropylidene-sn-gly-

D. Lafont et al. / Carbohydrate Research 341 (2006) 695–704
701
J4,5 < 1.0 Hz, H-4), 3.83 (dd, 1H, J2,3 10.2 Hz, H-2), 3.69
(br d, 2H, J5,6a, J5,6b 6.6 Hz, H-6a, H-6b), 3.55 (dd, 1H,
H-3bgl), 3.52 (dd, 1H, H-3), 3.37 (br dd, 1H, H-5), 1.41,
1.36 (2s, 6H, C(CH3)2), 0.89 (s, 9H, (CH3)3CSi), 0.06 (s,
6H, (CH3)2Si); 13C NMR (CDCl3): d 138.9, 138.9, 138.7,
128.5, 128.4, 128.3, 128.2, 128.2, 127.7, 127.6 (3C6H5),
109.3 (C(CH3)2), 104.4 (C-1), 82.3 (C-3), 79.6 (C-2),
75.3 (C-4), 75.3, 74.8, 73.2 (3CH2Ph), 74.4 (C-2gl), 73.7
(C-5), 70.3 (C-3gl), 67.2 (C-1gl), 61.8 (C-6), 27.0,
25.6 (C(CH3)2), 26.0 ((CH3)3CSi), 18.3 ((CH3)3CSi),
ꢀ5.2, ꢀ5.3 ((CH3)2Si). Anal. Calcd for C39H54O8Si
(678.908): C, 68.99; H, 8.02. Found: C, 68.99; H, 8.05.
(C(CH3)2). Anal. Calcd for C67H74O13 (1087.262): C,
74.01; H, 6.86. Found: C, 73.68; H, 6.95.
3.11. 3-O-[6-O-(2,3,4,6-Tetra-O-benzyl-a-D-galacto-
pyranosyl)-2,3,4-tri-O-benzyl-b-D-galactopyranosyl)]-
sn-glycerol (13)
Product 12 (0.326 g, 0.30 mmol) was stirred for 16 h at rt
in 85% aq AcOH (6 mL). After concentration under
diminished pressure and coevaporation from toluene
(2 · 15 mL), the residue was treated for 2 h by a cata-
lytic amount of MeONa in MeOH (25 mL). The soln
was concentrated again, the residue was dissolved in
CH2Cl2 (100 mL) and the organic phase was washed
with water (10 mL), dried (Na2SO4) and concentrated.
The crude product was purified by column chromato-
graphy (2:1 EtOAc–petroleum ether) to provide the pure
product 13 as a white crystalline material (0.251 g, 80%);
mp 103–104 ꢁC; [a]D +24.0 (c 1.0, CHCl3); Rf 0.75 (2:1
EtOAc–petroleum ether); 1H NMR (CDCl3): d 7.34–
7.23 (m, 35H, 7C6H5), 4.94 and 4.56 (2d, 2H, J
11.6 Hz, CH2Ph), 4.93 and 4.74 (2d, 2H, J 11.6 Hz,
CH2Ph), 4.92 and 4.63 (2d, 2H, J 12.0 Hz, CH2Ph),
3.10. 3-O-[6-O-(2,3,4,6-Tetra-O-benzyl-a-D-galacto-
pyranosyl)-2,3,4-tri-O-benzyl-b-D-galactopyranosyl)]-
1,2-O-isopropylidene-sn-glycerol (12)
2-Pyridyl
2,3,4,6-tetra-O-benzyl-1-thio-b-D-galacto-
pyranoside (11)17 (0.651 g, 1.027 mmol) and alcohol 7
(0.580 g, 1.027 mmol) were dissolved in CH2Cl2 (4 mL)
˚
containing activated crushed 4 A molecular sieves
(0.200 g). Methyl iodide (120 lL, 1.93 mmol) was added,
and the mixture was stirred at 50 ꢁC under argon for
48 h. Methyl iodide was added again (120 lL,
1.93 mmol) and stirring was maintained for 2 days.
After cooling to rt and filtration, the soln was concen-
trated and the residue was directly purified by column
chromatography (1:2 EtOAc–petroleum ether). Product
12 was recovered as a solid (0.650 g, 60% yield); mp
78 ꢁC; [a]D +21.0 (c 1.0, CHCl3); Rf 0.65 (1:2 EtOAc–
4.83 (d, 1H, J1 ;2 3.2 Hz, H-10), 4.80 (m, 2H, CH2Ph),
4.77 and 4.68 (2d, 2H, J 11.0 Hz, CH2Ph), 4.67 and
4.60 (2d, 2H, J 11.0 Hz, CH2Ph), 4.47 and 4.23 (2d,
2H, J 11.6 Hz, CH2Ph), 4.33 (d, 1H, J1,2 7.7 Hz, H-1),
4.00–3.30 (m, 17H, H-1agl, H-1bgl, H-2gl, H-3agl, H-
3bgl, H-2, H-3, H-4, H-5, H-6a, H-6b, H-20, H-30, H-
40, H-50, H-60a, H-60b); 13C NMR (CDCl3): d 138.9,
138.8, 138.7, 138.5, 137.9, 128.6, 128.5, 128.4, 128.3,
128.2, 128.0, 127.8, 127.7, 127.6 (7C6H5), 104.5 (C-1),
98.6 (C-10), 82.2 (C-3), 79.5 (C-2), 79.0 (C-30), 76.5 (C-
20), 75.4, 74.9, 74.7, 73.8, 73.7, 73.3, 72.9 (7 CH2Ph),
75.0 (C-40), 74.1 (C-4), 73.6 (C-5), 72.9 (C-3gl), 70.9
(C-2gl), 69.6 (C-50), 69.0 (C-60), 67.8 (C-6), 63.6 (C-1gl).
Anal. Calcd for C64H70O13 (1047.20): C, 73.40; H,
6.74. Found: C, 73.22; H, 7.07.
0
0
1
petroleum ether); H NMR (CDCl3): d 7.37–7.27 (m,
35H, 7C6H5), 4.92 and 4.55 (2d, 2H, J 11.3 Hz, CH2Ph),
4.89 and 4.75 (2d, 2H, J 11.4 Hz, CH2Ph), 4.87 and 4.71
(2d, 2H, J 11.7 Hz, CH2Ph), 4.80 and 4.59 (2d, 2H, J
11.7 Hz, CH2Ph), 4.77 (d, 1H, J1 ;2 3.6 Hz, H-10),
4.75–4.74 (m, 2H, CH2Ph), 4.72 and 4.62 (2d, 2H, J
11.9 Hz, CH2Ph), 4.47 and 4.23 (2d, 2H, J 11.6 Hz,
CH2Ph), 4.32 (d, 1H, J1,2 7.6 Hz, H-1), 4.25 (dddd,
1H, J1agl,2gl 6.3, J1bgl,2gl 6.3 Hz, J2gl,3agl 4.4, J2gl,3bgl
0
0
3.12. 3-O-[6-O-(2,3,4,6-Tetra-O-benzyl-a-D-galacto-
pyranosyl)-2,3,4-tri-O-benzyl-b-D-galactopyranosyl)]-
1,2-di-O-dodecanoyl-sn-glycerol (14)
6.9 Hz, H-2gl), 4.02 (dd, 1H, J2 ;3 10.3 Hz, H-20), 3.98
0
0
0
0
(dd, 1H, J1agl,1bgl 8.2 Hz, H-1agl), 3.97 (br d, 1H, J3 ;4
2.9 Hz, H-40), 3.93 (dd, 1H, J3agl,3bgl 10.5 Hz, H-3agl),
3.92 (dd, 1H, H-30), 3.88 (br dd, 1H, J5 ;6 a 6.3, J5 ;6 b
0
0
0
0
A soln of dodecanoyl chloride (0.162 mL, 0.700 mmol)
in CH2Cl2 (1 mL) was added dropwise for 1 h to a soln
of diol 13 (0.209 g, 0.200 mmol) and pyridine (0.327 mL)
in CH2Cl2 (2 mL). The mixture was stirred overnight at
rt, then water (40 lL) was added and stirring was main-
tained for 3 h. The mixture was diluted with CH2Cl2
(50 mL) and the organic phase was washed with satd
aq NaHCO3 (10 mL), dried (Na2SO4) and concentrated.
The residue was coevaporated twice from toluene
(2 · 15 mL). The mixture was purified by column chro-
matography (1:3 EtOAc–petroleum ether). Compound
14 was obtained as a crystalline material (0.257 g,
92%); mp 47–48 ꢁC; [a]D +25.1 (c 1.0, CHCl3); Rf 0.65
6.3 Hz, H-50), 3.82 (dd, 1H, H-1bgl), 3.80 (m, 1H, H-
4), 3.78 (d, 1H, J2,3 9.8 Hz, H-2), 3.74 (dd, 1H, J5,6a
,
3.8 Hz, J6a,6b 8.2 Hz, H-6a), 3.57–3.47 (m, 6H, H-3bgl,
H-3, H-5, H-6b, H-60a, H-60b), 1.37, 1.32 (2s, 6H,
C(CH3)2). 13C NMR (CDCl3): d 138.9, 138.9, 138.8,
138.7, 138.1, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1,
128.0, 127.9, 127.8, 127.7, 127.6 (7C6H5), 109.3
(C(CH3)2), 104.3 (C-1), 98.6 (C-10), 82.2 (C-3), 79.5
(C-2), 79.2 (C-30), 76.5 (C-20), 75.3, 74.9, 74.6, 73.7,
73.7, 73.2, 73.0 (7 CH2Ph), 75.0 (C-40), 74.4 (C-2gl),
74.1 (C-4), 73.2 (C-5), 70.2 (C-3gl), 69.7 (C-50), 69.1
(C-60), 67.3 (C-6), 67.2 (C-1gl), 61.9 (C-6), 27.1, 25.6

702
D. Lafont et al. / Carbohydrate Research 341 (2006) 695–704
(1:3 EtOAc–petroleum ether); 1H NMR (CDCl3): d
7.37–7.27 (m, 35H, 7C6H5), 5.24 (dddd, 1H, J1agl,2gl
3.5, J1bgl,2gl 6.9, J2gl,3agl 4.0, J2gl,3bgl 4.9 Hz, H-2gl),
4.95 and 4.58 (2d, 2H, J 11.5 Hz, CH2Ph), 4.93 and
4.75 (2d, 2H, J 11.0 Hz, CH2Ph), 4.92 and 4.62 (2d,
2H, J 11.5 Hz, CH2Ph), 4.83 and 4.65 (2d, 2H, J
12.0 Hz, CH2Ph), 4.80–3.79 (m, 2H, CH2Ph), 4.79 (d,
29.2, 29.0, 28.7, 24.5, 22.3 (CH2 alkyl chains), 13.5
(2CH3). HRMS m/z calcd for [M+Li]: 787.5032; found
787.5039.
3.14. 6-O-(2,3,4,6-Tetra-O-benzyl-a-D-galactopyranos-
yl)-1,2:3,4-di-O-isopropylidene-a-D-galactopyranose (16)
1H, J1 ;2 3.5 Hz, H-10), 4.77 and 4.67 (2d, 2H, J
12.0 Hz, CH2Ph), 4.51 and 4.40 (2d, 2H, J 11.7 Hz,
CH2Ph), 4.36 (dd, 1H, J1agl,1bgl 12.0 Hz, H-1agl), 4.31
(d, 1H, J1,2 7.6 Hz, H-1), 4.21 (dd, 1H, H-1bgl), 4.05
0
0
Methyl iodide (240 lL, 3.85 mmol) was added to a
mixture of 1,2:3,4-di-O-isopropylidene-a-D-galactopyran-
ose (1) (0.520 g, 2.00 mmol), 2-pyridyl 2,3,4,6-tetra-O-
benzyl-1-thio-b-D-galactopyranoside (11) (1.20 g, 1.89
(dd, 1H, J2 ;3 10.0 Hz, H-20), 4.03–4.00 (m, 2H, H-3agl,
0
0
˚
mmol), and activated 4 A molecular sieves in dry
H-40), 3.94 (dd, 1H, J3 ;4 2.8 Hz, H-30), 3.91 (bdd, 1H,
0
0
CH2Cl2 (8 mL) and the mixture was stirred for 3 days
at 50 ꢁC. After a new addition of CH3I (120 lL, 1.92
mmol), the mixture was stirred for 1 day at 60 ꢁC. After
filtration and concentration, the residue was purified by
column chromatography (1:3 EtOAc–petroleum ether)
affording the pure product 16 as an oil. (1.10 g, 74%)
and a 1:6 a,b-mixture (0.130 g, 9%). [a]D +7.0 (c 1.0,
CHCl3) [lit.19 [a]D +10.5 (c 1.0, CHCl3)]; Rf 0.80 (1:3
EtOAc–petroleum ether); 1H NMR (CDCl3): d 7.41–
7.27 (m, 20H, 4C6H5), 5.53 (d, 1H, J1,2 5.0, H-1), 5.03
J4 ;5 0.8, J5 ;6 a 6.5, J5 ;6 b 6.4 Hz, H-50), 3.84 (dd, 1H,
J3,4 2.8 Hz, H-4), 3.80 (dd, 1H, J2,3 9.8 Hz, H-2), 3.78–
3.76 (m, 1H, H-6a), 3.63 (dd, 1H, J3agl,3bgl 10.2 Hz, H-
3bgl), 3.60–3.55 (m, 4H, H-5, H-6b, H-60a, H-60b), 3.50
(dd, 1H, H-3), 2.33–2.23 (m, 4H, 2CH2CO), 1.64–1.50
(m, 4H, 2COCH2CH2), 1.40–1.20 (m, 36H, 18 CH2 alkyl
chains), 0.89 (t, 6H, 2CH3 alkyl chains); 13C NMR
(CDCl3): d 173.4, 173.1 (2COC11H23), 138.8, 138.7,
138.6, 138.0, 128.5, 128.4, 128.3, 128.0, 127.9, 127.8,
127.7, 127.6, 127.5 (7C6H5), 104.3 (C-1), 98.6 (C-10),
82.0 (C-3), 79.3 (C-2), 79.1 (C-30), 76.4 (C-20), 75.2,
74.9, 74.7, 73.7, 73.6, 73.2, 72.9 (7CH2Ph), 75.0 (C-40),
74.0 (C-4), 73.3 (C-5), 70.2 (C-2gl), 69.7 (C-50), 69.1
(C-60), 68.00 (C-3gl), 67.4 (C-6), 62.9 (C-1gl), 34.9,
34.2, 32.0, 29.8, 29.6, 29.5, 29.4, 29.3, 29.2, 25.0, 22.8
(CH2 alkyl chains), 14.3 (2CH3). Anal. Calcd for
C88H114O15 (1411.192): C, 74.86; H, 8.14. Found: C,
74.54; H, 8.46.
0
0
0
0
0
0
(d, 1H, J1 ;2 3.4 Hz, H-10), 4.96 and 4.59 (2d, J
11.5 Hz, CH2Ph), 4.86 and 4.74 (2d, J 11.7 Hz, CH2Ph),
4.77 (s, 2H, CH2Ph), 4.57 (dd, 1H, J2,3 2.3, J3,4 7.9 Hz,
H-3), 4.50 and 4.42 (2d, J 11.8 Hz, CH2Ph), 4.33 (dd,
1H, J4,5 1.8 Hz, H-4), 4.31 (dd, 1H, H-2), 4.08–3.50
(m, 9H, H-5, H-6a, H-6b, H-20, H-30, H-40, H-50, H-
60a, H-60b), 1.54, 1.44, 1.34, 1.31 (4s, 12H, 2(CH3)2C);
13C NMR (CDCl3): d 139.1, 138.9, 138.2, 128.5, 128.4,
128.3, 127.9, 127.8, 127.6, 127.5 (4C6H5), 109.3, 108.6
(2C(CH3)2), 97.7 (C-10), 96.4 (C-1), 79.1 (C-30), 76.6
(C-20), 75.1 (C-40), 74.9, 73.5, 73.2, 72.8 (4CH2Ph),
71.0, 70.8, 70.8 (C-2, C-3, C-5), 69.3 (C-50), 68.8 (C-
60), 66.5 (C-6), 66.0 (C-4), 26.3, 26.2, 25.1, 24.7
(2C(CH3)2).
0
0
3.13. 3-O-(6-O-a-D-Galactopyranosyl-b-D-galacto-
pyranosyl)-1,2-di-O-dodecanoyl-sn-glycerol (15)
A mixture of 14 (0.200 g, 0.14 mmol), freshly distilled
cyclohexene (2 mL) and Pd(OH)2 (50 mg) in EtOH
(4 mL) was heated for 5 h at 80 ꢁC. After cooling and fil-
tration on Celite, the soln was concentrated and the res-
idue was purified by column chromatography (7:3:1
EtOAc–EtOH–water) to afford the pure product 15 as
an amorphous solid (0.083 g, 75%); [a]D +41.7 (c 1.0,
Selected values for b-anomer: 13C NMR (CDCl3): d
104.8 (C-10), 96.5 (C-1).
3.15. 1,2,3,4-Tetra-O-acetyl-6-O-(2,3,4,6-tetra-O-benzyl-
a-D-galactopyranosyl)-D-galactopyranose (17)
1
CHCl3); Rf 0.75 (7:3:1 EtOAc–EtOH–water); H NMR
Compound 16 (1.050 g, 1.34 mmol) was stirred for
30 min in 80% aq TFA. The mixture was concentrated
under diminished pressure and coevaporated three times
from toluene (3 · 15 mL). The residue was acetylated
overnight in a 2:1 pyridine–Ac2O mixture (15 mL).
The soln was concentrated under diminished pressure
and the crude residue was purified by column chroma-
tography (2:3 EtOAc–petroleum ether). Compound 17
(CDCl3+CD3OD): d 5.24–5.18 (m, 1H, H-2gl), 4.87
(dd, 1H, J1 ;2 3.4 Hz, H-10), 4.36 (dd, 1H, J1agl,2gl 3.0,
J1agl,1bgl 12.0 Hz, H-1agl), 4.20 (dd, 1H, J1bgl,2gl 6.7 Hz,
H-1bgl), 4.17 (d, 1H, J1,2 7.1 Hz, H-1), 3.96–3.65 (m,
12H, H-3agl, H-3bgl , H-4, H-5, H-6a, H-6b, H-20, H-
30, H-40, H-50, H-60a, H-60b), 3.56–3.45 (m, 2H, H-2,
H-3), 2.33–2.26 (m, 4H, 2CH2CO), 1.65–1.50 (m, 4H,
2COCH2CH2), 1.34–1.24 (m, 36H, 18 CH2 alkyl chains),
0.84 (t, 6H, 2CH3 alkyl chains); 13C NMR (CDCl3): d
173.7, 173.4 (2COC11H23), 103.6 (C-1), 99.7 (C-10),
72.8 (C-3), 72.7 (C-5), 70.9 (C-2), 70.5 (C-2gl), 70.0 (C-
30), 69.4 (C-40, C-50), 68.6 (C-20), 67.7 (C-4), 67.4 (C-
3gl), 65.8 (C-6), 62.5 (C-1gl), 61.3 (C-60), 33.9, 31.5,
0
0
1
was obtained as an oily a,b-mixture (0.725 g, 62%). H
NMR (CDCl3), selected values: d 7.39–7.27 (m, 20H,
4C6H5), 6.38 (d, 1H, J1,2 2.4, H-1a), 5.63 (d, 1H, J1,2
8.2, H-1b), 5.58 (m, 1H, H-4a), 5.48 (br d, 1H, J3,4
3.2 Hz, H-4b), 5.44–5.34 (m, 2H, H-2a, H-3a), 5.32

D. Lafont et al. / Carbohydrate Research 341 (2006) 695–704
703
(dd, 1H, J2,3 10.5 Hz, H-2b), 5.08 (dd, 1H, H-3b); 13C
3.17. 3-O-[2,3,4-Tri-O-acetyl-6-O-(2,3,4,6-tetra-O-
benzyl-a-D-galactopyranosyl)-b-D-galactopyranosyl)]-
1,2-O-isopropylidene-sn-glycerol (19)
NMR (CDCl3), selected values: d 98.8 (C-10 b), 98.3
(C-10 a), 92.3 (C-1 b), 89.9 (C-1 a). Anal. Calcd for
C48H54O15,H2O (888.95): C, 64.85; H 6.35. Found: C,
64.70; H, 6.20.
A mixture of imidate 18 (0.360 g, 0.37 mmol), 1,2-O-iso-
propylidene-sn-glycerol (68 lL, 0.55 mmol) and acti-
˚
3.16. 2,3,4-Tri-O-acetyl-6-O-(2,3,4,6-tetra-O-benzyl-a-D-
galactopyranosyl)-D-galactopyranosyl trichloroacetimi-
date (18)
vated 4 A molecular sieves (0.400 g) in dry CH2Cl2
(4 mL) was stirred for 15 min under argon, before cool-
ing to ꢀ30 ꢁC. A soln of trimethylsilyl trifluoromethane-
sulfonate (10 lL) in CH2Cl2 (0.5 mL) was added
dropwise and the mixture was stirred for 3 h at
ꢀ30 ꢁC. The mixture was neutralized by the addition
of Et3N (20 lL) and allowed to reach rt. After filtration
on Celite, the solvent was evaporated and the product
was purified by column chromatography (1:1 petroleum
ether–EtOAc). A second column chromatography (1:20
MeOH–CH2Cl2) was necessary in order to remove the
trichloroacetamide formed during the reaction. The
pure product 19 was obtained as an amorphous solid
(0.155 g, 55%); [a]D +15.9 (c 1.0, CHCl3); Rf 0.37 (1:2
EtOAc–petroleum ether), Rf 0.75 (1:20 MeOH–
CH2Cl2); 1H NMR (CDCl3): d 7.38–7.27 (m, 20H,
4C6H5), 5.42 (br d, 1H, J3,4 3.5, J4,5 0.9 Hz, H-4), 5.16
(dd, 1H, J1,2 7.9, J2,3 10.4 Hz, H-2), 4.99 (dd, 1H, H-
3), 4.92 and 4.59 (2d, 2H, J 11.6 Hz, CH2Ph), 4.82 and
4.72 (2d, 2H, J 11.6 Hz, CH2Ph), 4.77 and 4.66 (2d,
Compound 17 (0.650 g, 0.746 mmol) was stirred for 16 h
in THF (3 mL) in the presence of benzylamine
(0.127 mL, 1.17 mmol). When TLC showed that all the
starting material has been consumed (16 h), the mixture
was diluted with CH2Cl2 (100 mL), and the organic
phase was successively washed with 1 N HCl, satd aq
NaHCO3, then with water and dried over Na2SO4. After
filtration and concentration, the residue was purified by
column chromatography (3:4 EtOAc–petroleum ether).
The hemiacetal was obtained as a 5:1 a,b-anomeric mix-
ture (0.450 g, 70%); Rf 0.45–55 (3:4 EtOAc–petroleum
ether); 13C NMR (a anomer): (CDCl3): d 170.6, 170.5,
170.1 (3CH3CO), 138.9, 138.6, 138.5, 137.8, 128.6,
128.5, 128.4, 128.3, 128.1, 127.9, 127.7, 127.6 (4 C6H5),
98.8 (C-10), 90.5 (C-1), 78.9 (C-30), 76.7 (C-20), 75.1
(C-40), 74.8, 73.6, 73.5, 73.2 (4 CH2Ph), 69.9 (C-5),
69.4 (C-60), 69.3 (C-50), 68.8 (C-3), 68.1 (C-6), 67.8,
67.5 (C-2, C-4), 20.9, 20.8, 20.7 (3CH3COO).
0
0
2H, J 11.9 Hz, CH2Ph), 4.77 (d, 1H, J1 ;2 3.7 Hz, H-
10), 4.48 and 4.42 (2d, 2H, J 11.7 Hz, CH2Ph), 4.41 (d,
1H, H-1), 4.18 (dddd, 1H, J1agl,2gl 6.3, J1bgl,2gl 6.0 J2gl,3agl
Selected values for b-anomer: 13C NMR (CDCl3): d
99.2 (C-10), 95.6 (C-1).
0
0
4.1, J2gl,3bgl 6.3 Hz, H-2gl), 4.02 (dd, 1H, J2 ;3 10.0 Hz,
DBU (0.10 mL, 3.19 mmol) was added to a soln of
hemiacetal (0.430 g, 0.52 mmol) and trichloroacetonit-
rile (0.32 mL) in CH2Cl2. The mixture was stirred for
5 h, and concentrated under diminished pressure. The
residue was purified by column chromatography (1:3
EtOAc–petroleum ether). A pure fraction of the a-imi-
date was recovered first (0.400 g, 79%) followed by an
a,b-anomeric mixture (0.052 g, 10%). The pure product
18 was obtained as an oil; [a]D +66.1 (c 1.0, CHCl3); Rf
0.48 (1:3 EtOAc–petroleum ether); 1H NMR (CDCl3): d
8.60 (s, 1H, NH), 7.38–7.27 (m, 20H, 4C6H5), 6.61 (d,
1H, J1,2 3.4 Hz, H-1), 5.62 (br d, 1H, J3,4 3.0 Hz, H-4),
5.47 (dd, 1H, J3,4 10.8 Hz, H-3), 5.37 (dd, 1H, H-2),
4.92–4.39 (m, 10H, 4CH2Ph, H-10, H-5), 4.01 (dd, 1H,
H-20), 3.97–3.92 (m, 4H, H-40, H-5, H-50, H-1agl), 3.89
(dd, 1H, J3 ;4 2.6 Hz, H-30), 3.82 (dd, 1H, J3agl,3bgl
10.4 Hz, H-3agl), 3.76 (dd, 1H, J1agl,1bgl 8.2 Hz, H-
1bgl), 3.68 (dd, 1H, J5,6a 6.0, J6a,6b 10.4 Hz, H-6a),
0
0
0
0
3.61 (dd, 1H, J5,6b 6.6 Hz, H-6b), 3.55 (dd, 1H, J5 ;6 a
6.6, J6 a;6 b 9.5 Hz, H-60a), 3.51 (dd, 1H, H-3bgl), 3.46
0
0
(dd, 1H, J5 ;6 b 6.0 Hz, H-60b), 2.07, 2.07, 1.99 (3s, 9H,
3CH3COO), 1.40, 1.33 (2s, 6H, C(CH3)2); 13C NMR
(CDCl3): d 170.4, 170.2, 169.7 (3COCH3), 138.8, 138.6,
138.4, 138.1, 128.8, 128.7, 128.5, 128.3, 128.2, 128.1,
128.0, 127.9 (4 C6H5), 109.3 (C(CH3)2), 101.6 (C-1),
99.3 (C-10), 79.3 (C-30), 76.8 (C-20), 75.5 (C-40), 75.1,
74.0, 73.8, 73.7 (4 CH2Ph), 74.6 (C-2gl), 72.2 (C-5),
71.4 (C-3), 70.3 (C-50), 69.6 (C-60), 69.5 (C-3gl), 69.4
(C-2), 68.2 (C-4), 67.4 (C-6), 66.7 (C-1gl), 26.7, 25.3
(C(CH3)2), 20.9, 20.7, 20.7 (3CH3CO). Anal. Calcd for
C52H62O16 (943.016): C, 66.23; H, 6.63. Found: C,
66.02; H, 6.75.
0
0
J1 ;2 3.6, J2 ;3 10.1 Hz, H-20), 3.99–3.92 (m, 2H, H-40,
0
0
0
0
H-50), 3.85 (dd, 1H, J3 ;4 2.5 Hz, H-30), 3.72 (dd, 1H,
J5,6a 6.2, J6a,6b 10.6 Hz, H-6a), 3.59 (dd, 1H, J5,6b
0
0
0
0
0
0
6.2 Hz, H-6b), 3.52 (d, 2H, J5 ;6 a ¼ J5 ;6 b 6.5 Hz, H-
60a, H-60b), 2.09, 2.04, 2.03 (3s, 9H, 3CH3COO); 13C
NMR (CDCl3): d 170.2, 170.1, 169.9 (3COCH3), 161.2
(OC(NH)CCl3), 139.0, 138.8, 138.6, 138.2, 128.5,
128.4, 128.3, 128.2, 127.9, 127.7, 127.6 (4C6H5), 98.3
(C-10), 93.8 (C-1), 91.0 (CCl3), 78.9 (C-30), 76.5 (C-20),
75.2 (C-40), 74.9, 73.4, 73.4, 73.3 (4 CH2Ph), 70.2 (C-
5), 69.7 (C-50), 68.8 (C-60), 68.1, 67.8 (C-2, C-3), 67.3
(C-4), 66.5 (C-6), 20.8, 20.7, 20.6 (3CH3CO).
3.18. Galactolipase activity measurements
The hydrolysis of DGDG, MGDG, diglyceride and
phospholipids by GPLRP2 was measured using
the monomolecular film technique as previously
described.3

704
D. Lafont et al. / Carbohydrate Research 341 (2006) 695–704
11. Gent, P. A.; Giggs, R. JCS, Perkin Trans. 1 1975, 1521–
References
1524.
12. Manthorpe-Gent, P. A.; Giggs, R. Methods Carbohydr.
Chem. 1980, 8, 305–311.
13. Tanaka, R.; Sakano, Y.; Nagatsu, A.; Shibuya, M.;
Ebizuka, Y.; Goda, Y. Bioorg. Med. Chem. Lett. 2005,
15, 159–162.
1. Do¨rmann, P.; Benning, C. Trends Plant Sci. 2002, 7, 112–
118.
2. Anderson, L.; Bratt, C.; Arnoldsson, K. C.; Herslof, B.;
Olsson, N. U.; Sternby, B.; Nilsson, A. J. Lipid Res. 1995,
36, 1392–1400.
14. Schmidt, R. R. Angew. Chem., Int. Ed. Engl. 1986, 25,
212–235.
3. Sias, B.; Ferrato, F.; Grandval, P.; Lafont, D.; Boullanger,
`
P.; De Caro, A.; Leboeuf, B.; Verger, R.; Carriere, F.
15. Shingu, Y.; Nishida, Y.; Dohi, H.; Kobayashi, K. Org.
Biomol. Chem. 2003, 1, 2518–2521.
16. Tipson, R. S. Methods Carbohydr. Chem. 1962, 2, 246–
250.
17. Mereyala, H. B.; Reddy, G. V. Tetrahedron 1991, 47,
6435–6448.
Biochemistry 2004, 43, 10138–10148.
4. Lafont, D.; Boullanger, P. Unpublished results.
5. Hiraga, Y.; Kaku, K.; Omoda, D.; Sugihare, K.; Hosoya,
H.; Hino, M. J. Nat. Prod. 2002, 65, 1494–1496.
6. Hisamatsu, Y.; Goto, N.; Sekigushi, M.; Hasegawa, K.;
Shigemori, H. J. Nat. Prod. 2005, 68, 600–603.
7. Wegner, C.; Hamburger, M.; Kunert, O.; Haslinger, E.
Helv. Chim. Acta 2000, 83, 1454–1464.
8. Murakami, N.; Morimoto, T.; Imamura, H.; Nagatsu, A.;
Sakakibara, J. Tetrahedron 1994, 50, 1993–2002.
9. Jung, J. H.; Lee, H.; Kang, S. S. Phytochemistry 1996, 42,
447–452.
`
18. Hjorth, A.; Carriere, F.; Cudrey, C.; Wo¨ldike, H.; Boel, E.;
Lawson, D. M.; Ferrato, F.; Cambillau, C.; Dodson, G. G.;
Thim, L.; Verger, R. Biochemistry 1993, 32, 4702–
4707.
19. Milat, M.-L.; Amvam Zollo, P.; Sinay, P. Carbohydr. Res.
¨
1982, 100, 263–271.
10. Murakami, N.; Morimoto, T.; Imamura, H.; Ueda, T.;
Nagai, S. I. Chem. Pharm. Bull. 1991, 39, 2277–2281.