Syntheses of α-d-galactosamine neoglycolipids

Article abstract of DOI:10.1016/j.carres.2006.02.013

Several N-acetyl-α-d-galactosamine neoglycolipids, as well as hydrophobized T and T_N antigen analogues, were prepared for embedment onto liposomes. Three different lipidic structures were used for the anchoring, that is cholesterol, 1,3-bis(undecyloxy)propan-2-ol and 1,3-bis(3,7,11,15-tetramethylhexadecyloxy)propan-2-ol. Oligoethyleneglycol spacers were used to link the carbohydrate and the hydrophobic moieties; their lengths were varied in order to obtain model compounds for the selective recognition by sialyl transferases involved in cancer processes. Glycosylation reactions were optimized to sluggish amphiphilic acceptor alcohols, in order to reach good 1,2-cis-stereoselectivities and acceptable yields. This aim was achieved by using 3,4,6-tri-O-acetyl-2-azido-2-deoxy-d-galactopyranosyl trichloroacetimidate as the donor, trimethylsilyl trifluoromethanesulfonate as the promoter and diethyl ether or mixtures of diethyl ether and dichloromethane as solvents.

Full text of DOI:10.1016/j.carres.2006.02.013

Carbohydrate Research 341 (2006) 823–835
Syntheses of a-D-galactosamine neoglycolipids
Nicolas Laurent, Dominique Lafont and Paul Boullanger*
Laboratoire de Chimie Organique II-Glycochimie, Unite´ Mixte de Recherche UCBL-CNRS 5181, Universite´ Lyon 1,
Ecole Supe´rieure de Chimie Physique Electronique de Lyon, 43 Bd du 11 Novembre 1918, F-69622 Villeurbanne, France
Received 28 December 2005; accepted 17 February 2006
Available online 3 March 2006
Abstract—Several N-acetyl-a-D-galactosamine neoglycolipids, as well as hydrophobized T and TN antigen analogues, were prepared
for embedment onto liposomes. Three different lipidic structures were used for the anchoring, that is cholesterol, 1,3-bis(undecyl-
oxy)propan-2-ol and 1,3-bis(3,7,11,15-tetramethylhexadecyloxy)propan-2-ol. Oligoethyleneglycol spacers were used to link the car-
bohydrate and the hydrophobic moieties; their lengths were varied in order to obtain model compounds for the selective recognition
by sialyl transferases involved in cancer processes. Glycosylation reactions were optimized to sluggish amphiphilic acceptor alcohols,
in order to reach good 1,2-cis-stereoselectivities and acceptable yields. This aim was achieved by using 3,4,6-tri-O-acetyl-2-azido-2-
deoxy-D-galactopyranosyl trichloroacetimidate as the donor, trimethylsilyl trifluoromethanesulfonate as the promoter and diethyl
ether or mixtures of diethyl ether and dichloromethane as solvents.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Keywords: Neoglycolipids; N-Acetyl-a-D-galactosamine; Glycosylation; Hydrophobic anchor
1. Introduction
a-D-GalNAc-Thr). Further galactosylation at C-3
affords the core 1 saccharidic structures (T antigens:
b-D-Gal-(1!3)-a-D-GalNAc-Ser (or Thr)). At this
stage, the C-6 position of D-galactose can be glycosyl-
ated by means of a b-(1!6)-D-GlcNAc transferase.
After chain extension with poly-N-acetyllactosamine,
Mucins are transmembrane O-glycoproteins found in
mucus.1,2 The biosynthesis of epithelial mucins is initi-
ated by the linkage of N-acetyl-a-D-galactosamine to
serine or threonine (TN antigens: a-D-GalNAc-Ser or
Me
OH
HO
HO
OH
OH
HO
HO
OH
Me
O
O
O
CHMe2
O
OH
AcHN
AcHN
O
O
O
O
O
OR
OC11H23
4
1 R=
OC11H23
O
OH
HO
HO
OPhy
OPhy
Me
O
2 R=
3 R=
O
O
Me
AcHN
O
NH2
NH
OC11H23
OC11H23
CHMe2
O
5
O
O
O
O
*
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.2006.02.013

824
N. Laurent et al. / Carbohydrate Research 341 (2006) 823–835
the chain growth is ended by attachment of sialic acid,
L-fucose or D-galactose. In tumour cells, the biosynthesis
of complex glycans is inhibited by the underexpression of
several glycosyltransferases and the overexpression
of sialyl transferases. As a consequence, the high levels
of short oligosaccharides (such as TN and T antigens),
acting as substrates for sialyl transferases, result in the
accumulation of STN, a-2,3 ST or a-2,6 ST antigens.1,3
Antitumour vaccines were prepared by covalent cou-
pling of the synthetic aforementioned antigens to immu-
nogenic proteins.4–6 In the same perspective, the aim of
this work was the preparation of N-acetyl-a-D-galactos-
amine neoglycolipids 1–4 and that of a TN antigen ana-
logue 5 suitable for embedment onto liposomes. The
resulting vesicles could constitute targets for the sialyl
transferases involved in cancer processes.
For that purpose, we intended the synthesis of three
families of neoglycolipids built on distinct lipid struc-
tures: 1,3-bis(undecyloxy)propan-2-ol (6), 1,3-bis(3,7,
11,15-tetramethylhexadecyloxy)propan-2-ol (7) and cho-
lesterol. The oligoethyleneglycol spacer between the lipid
and saccharide moieties was set to bring a favourable
hydrophilic–hydrophobic balance on the molecule for a
good embedment onto liposomes. The length of the latter
was chosen with regard to our preceding results,7 that is
monoethyleneglycol for the less hydrophobic structure
(8) and triethyleneglycol for the more hydrophobic ones
(9 and 10).
OPhy
OC11H23
OPhy
RO
O
O
O
RO
HO
OPhy
OPhy
OC11H23
7
9
R=H
13 R=THP
17a R=Ra
17b R=Rb
2 R=Rc
6 R=H
8 R=CH2CH2OH
Phy=
3
11 R=CH2CN
12 R=CH2CH2NH2
RO
NHZ
RO
NHFmoc
OC11H23
OC11H23
OC11H23
OC11H23
RO
O
O
NH
O
Y
O
8 R=H
16a R=Ra
16b R=Rb
1 R=Rc
15 R=H, Z=Fmoc
19a R=Ra, Z=Fmoc
19b R=Rb, Z=Fmoc
22b R=Rb, Z=H
5 R=Rc, Z=H
14 R=H, Y=OH
20a R=Ra, Y=OBn
20b R=Rb, Y=OBn
21b R=Rb, Y=OH
OR6
R4O
R3O
Me
Me
O
CHMe2
Z2N
RO
O
O
O
Ra. NZ2=N3, R3=R4=R6=Ac
10 R=H
18a R=Ra
18b R=Rb
18d R=Rd
3 R=Rc
Rb. NZ2=NHAc, R3=R4=R6=Ac
Rc. NZ2=NHAc, R3=R4=R6=H
Rd. NZ2=N3, R3=H, R4-R6=PhCH<
Me
RO
RO
OR
OR6
OR4
Me
O
O
CHMe2
O
OR
Z2N
O
O
O
O
23 NZ2=N3, R=Ac, R4-R6=PhCH<
24 NZ2=N3, R=Ac, R4=R6=H
25 NZ2=NHAc, R=R4=R6=Ac
4 NZ2=NHAc, R=R4=R6=H

N. Laurent et al. / Carbohydrate Research 341 (2006) 823–835
825
2. Results and discussion
mixtures of diethyl ether and dichloromethane were
used as the solvent (diethyl ether is known to favour
the formation of a-glycosides and dichloromethane
was often necessary to enhance solubility at low temper-
ature), catalytic amounts of trimethylsilyl trifluoro-
methanesulfonate were used as the promoter. Acceptor
alcohol 8 was thus reacted with an excess of donor
(1.5 equiv) to afford 16a (62%) as an inseparable 3:1
a/b-anomeric mixture. After reduction of the azido
function (nickel chloride–sodium borohydride) and
N-acetylation, pure a isomer 16b could be separated
in 64% yield. Fully deprotected 1 was quantitatively
To our knowledge, there were no preparations of neo-
glycolipids of N-acetyl-D-galactosamine reported in the
literature. However, several long chain aglycons (e.g.,
9-hydroxynonanoic acid methyl ester) were glycosylated
with 2-azido-2-deoxy-D-galactopyranosyl chlorides or
bromides and various promoters favouring the a-stereo-
selectivity.8–10 The most challenging concern in such
reactions is the selection of a donor/promoter system
able to react with sluggish acceptors and to induce the
formation of 1,2-cis-glycosides in high yields and with
a high stereoselectivity. Latest advancements in the field
have been reviewed recently.11,12 In the D-galactosamine
series, the 1,2-cis-stereoselectivity is usually obtained
with a donor bearing a non-participating azido group
at C-2 and an anomeric leaving group such as b-chlo-
ride,13,14 a-bromide15,16 or b-imidate.17–19 Other meth-
ods, starting from 2-acetamido-2-deoxy-a-D-galacto-
pyranosyl imidate20 or 2-nitro-D-galactal21,22 have also
been reported recently.
´
obtained by Zemplen O-deacetylation of 16b.
Similarly, glycosylation of alcohol 9 afforded 17a (4:1,
a/b mixture, 91%) from which it was possible to separate
a small amount of pure a-isomer for analytical purposes.
When reduction of the azido function and N-acetylation,
in the same conditions as above, was applied to 17a (a/b
mixture), compound 17b was obtained as an a/b mixture
(84%) from which the a anomer could be separated in
51% yield. Finally, 17b was de-O-acetylated to afford
the fully deprotected derivative 2.
1,3-Bis(undecyloxy)propan-2-ol (6) was prepared in a
different way than that already reported.23 Racemic 1,2-
O-isopropylidene glycerol was reacted at the OH-free
position with undecyl bromide, before hydrolysis of
the acetal function in acidic conditions. The diol, thus
obtained, was regioselectively alkylated at the primary
position with undecyl bromide and dibutyltin oxide24
to afford the expected derivative 623 in 75% overall yield.
This three-step synthesis is more time consuming than
the reaction of undecyl alcohol with epichlorohydrin,23
but it affords a much more better yield. Alcohol 6 was
then converted to compound 8 (58%) by reaction with
2-tetrahydropyranyloxyethyl chloride in phase transfer
conditions, followed by hydrolysis of the tetrahydropyr-
anyl group in acidic medium. The amino compound 12
(70%) was obtained from alcohol 6, by reaction with
bromoacetonitrile and sodium hydride, followed by
reduction of the nitrile intermediate 11 by hydrogena-
tion (10% Pd/C).
1,3-Bis(3,7,11,15-tetramethylhexadecyl)propan-2-ol (7)
was prepared in 32% overall yield by reaction of phytol
(3,7,11,15-tetramethyl-2-hexadecen-1-ol) with epichloro-
hydrin and sodium hydride in THF, followed by
hydrogenation of the crude intermediate in the presence
of platinum oxide. Reaction of 7 with tetrahydropyranyl
triethyleneglycol monotosylate25 and NaH in refluxing
THF afforded the intermediate 13, further converted
into alcohol 9 by removal of the tetrahydropyranyl
group in mild acidic conditions.
With the monocholesteryl triethyleneglycol acceptor
10,7 the results were very similar: the glycosylation led
to 18a (4:1, a/b mixture, 89%), reduction and N-acetyl-
ation afforded 18b from which the pure a-isomer could
be separated in 56% yield. O-Deacetylation gave quanti-
tatively the expected neoglycolipid 3.
Glycosylation of lipopeptide 15 in similar conditions
was less efficient. Compound 19a was obtained in 35%
yield only, and the reaction required a stoichiometric
amount of promoter (TMSOTf). Furthermore, the stereo-
selectivity in favour of the a isomer was lower (2:1). The
reverse procedure reported by Schmidt and Toepfer28
did not afford any improvement. Therefore, glycosyla-
tion of a protected serine, before condensation of the
lipid moiety, was considered as another route to 19b.
Thus, glycosylation of benzyl N-(9H-fluoren-9-ylmeth-
oxycarbonyl)-L-serinate24 with 3,4,6-tri-O-acetyl-2-
azido-2-deoxy-D-galactopyranosyl trichloroacetimidate
afforded the known glycoside 20a29 (a-anomer, 72%).
The azido function was then converted to acetamide as
described previously (20b). The benzyl ester was re-
moved by hydrogenolysis to afford 21b; then, reaction
of the latter with the amine 12 in the presence of N-
ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ)
afforded 19b in 43% yield. Compound 19b could also
be obtained in 47% yield by reduction-acetylation of
19a. The Fmoc group was removed by treatment of
19b with morpholine to afford 22b (76%), which was
Fmoc-L-serine 1426 was reacted with the amino inter-
mediate 12 in usual conditions (DCC, HOBt in DMF)
to afford the C-lipidic aminoacid 15 in 59% yield.
For glycosylation reactions, 3,4,6-tri-O-acetyl-2-azido-
2-deoxy-D-galactopyranosyl trichloroacetimidate27 (1:4,
a/b mixture) was used as the donor, diethyl ether or
fully O-deacetylated to 5 in Zemplen conditions.
´
In order to synthesize a neoglycolipid bearing the same
disaccharidic determinant as T antigens, the azido deriv-
ative 18a was O-deacetylated and reacted with benzalde-
hyde dimethyl acetal in the presence of p-toluenesulfonic
acid to afford the 4,6-O-benzylidene derivative 18d

826
N. Laurent et al. / Carbohydrate Research 341 (2006) 823–835
(69%). Glycosylation of the latter with 2,3,4,6-tetra-O-
acetyl-D-galactopyranosyl trichloroacetimidate30 and a
catalytic amount of trimethylsilyl trifluoromethanesulfo-
nate in dichloromethane at ꢀ20 ꢁC afforded the disac-
charidic neoglycolipid 23 (71%). Removal of the
benzylidene group (borontrifluoride etherate/ethaneth-
iol) afforded compound 24, which was then O-acetylated.
After reduction of the azido function (hydrogenation,
10% Pd/C) and N-acetylation, compound 25 (70%) was
3.2. 2-O-[2-(2-Acetamido-2-deoxy-a-D-galactopyranosyl-
oxy)ethyl]-1,3-bis(undecyloxy)propan-2-ol (1)
A soln of compound 16b (0.441 g, 0.57 mmol) in MeOH
(28 mL) containing a chip of sodium was stirred over-
night at rt. After neutralization with Amberlyst IR 120
(H+) and filtration, the solvent was evaporated under
diminished pressure to afford 1 (0.367 g, 94%) as an
1
amorphous solid; [a]D +30.3 (c 1.0, CHCl3); H NMR
´
quantitatively O-deacetylated in Zemplen conditions to
afford the expected compound 4.
(CDCl3): d 6.63 (d, 1H, J2,NH 9.7 Hz, NH), 4.91 (d,
1H, J1,2 3.7 Hz, H-1), 4.72 (s, 1H, OH), 4.62 (ddd, 1H,
J2,3 11.4 Hz, H-2), 4.00 (dd, 1H, J3,4 2.7, J4,5 1.0 Hz,
H-4), 3.87 (dd, 1H, H-3), 3.89–3.76 (m, 6H, H-6a,
H-6b, OCH2CH2O), 3.68–3.62 (m, 2H, H-5, CH(CH2O-
C11H23)2), 3.55–3.39 (m, 8H, 2CH2OCH2C10H21), 2.96
(s, 1H, OH), 2.05 (s, 3H, CH3CON), 1.60–1.48 (m,
4H, 2OCH2CH2C9H19), 1.35–1.15 (m, 32H, 16CH2 alkyl
chains), 0.89 (t, 6H, J 6.5 Hz, 2CH3 alkyl chains); 13C
NMR (CDCl3): d 172.7 (CH3CO), 97.9 (C-1), 78.2
(CH(CH2OC11H23)2), 71.7, 70.8 (CH(CH2OCH2-
C10H21)2), 70.4, 70.1, 69.5 (C-3, C-4, C-5), 69.0, 67.7
(OCH2CH2O), 62.4 (C-6), 50.6 (C-2), 31.9, 29.7, 29.6,
29.5, 29.4, 26.1, 22.7 (CH2 alkyl chains), 23.1
(CH3CON), 14.1 (CH3 alkyl chains). Anal. Calcd for
C35H69NO9Æ0.5H2O (656.95): C, 63.99; H, 10.74; N,
2.13. Found: C, 63.74; H, 10.69; N, 2.19.
As a conclusion, it can be mentioned that glycosyl-
ation reactions with highly hydrophobic acceptor alco-
hols do not result in yields and stereoselectivities as
high as those reported in the literature. Nevertheless,
five neoglycolipids (1–5) in which the hydrophilic moie-
ties are building blocks of T and TN antigens have been
prepared by chemical synthesis in quite acceptable
yields. They will be used as substrates of sialyl transfer-
ases, specific to the OH-6 position of N-acetyl-a-D-
galactosamine (ST6GalNAc transferases). The amphi-
philic character of these neoglycolipids will allow them
to be embedded onto liposomes or adsorbed on immuno-
titration plates, for enzymatic measurements. The roles
of the hydrophobic anchors in the adsorption process
and the shifts in specificities of the enzymes towards
the different haptens at an interface are expected to give
further insights into the biochemistry of some cancer cell
surfaces.
3.3. 15,19,23,27-Tetramethyl-10-(3,7,11,15-tetramethyl-
hexadecyloxymethyl)-3,6,9,12-tetraoxaoctacosyl 2-acet-
amido-2-deoxy-a-D-galactopyranoside (2)
3. Experimental
Prepared from 17b (0.369 g, 0.33 mmol), as described
for 1, to afford 2 (0.318 g, 97%) as a colourless oil;
[a]D +25.8 (c 1.0, CHCl3); 13C NMR (CDCl3): d 172.8
(CH3CO), 97.9 (C-1), 78.5 (CH(CH2OPhy)2), 70.9,
70.8, 70.6, 70.4, 70.0, 69.7, 67.3 ((OCH2CH2)3,
CH(CH2OCH2C19H39)2), 71.0, 70.4, 69.6 (C-3, C-4,
C-5), 62.6 (C-6), 50.8 (C-2), 39.4, 37.6, 37.5, 37.4, 37.3,
36.8, 36.7, 32.8, 30.0, 28.0, 25.3, 24.8, 24.5, 23.3, 22.8,
22.7, 19.8, 19.7, 19.7 (CH, CH2, CH3 phytyl chains,
CH3CON). Anal. Calcd for C57H113NO11Æ0.5H2O
(997.49): C, 68.63; H, 11.52; N, 1.40. Found: C, 68.31;
H, 11.10; N, 1.42.
3.1. General methods
Pyridine was dried by refluxing with CaH2 prior to distil-
lation. Dichloromethane was washed twice with water,
dried with CaCl2 and distilled from CaH2. Tetrahydrofu-
ran was distilled from sodium-benzophenone. Acetoni-
trile was distilled from CaH2. Methanol was distilled
from magnesium. Pyridine, THF and CH2Cl2 were stored
˚
˚
over 4 A molecular sieves; CH3CN and MeOH over 3 A
molecular sieves. Melting points were determined on a
Buchi apparatus and are uncorrected. Thin layer chroma-
¨
tography was performed on aluminium sheets coated
with Silica Gel 60 F254 (E. Merck). Compounds were
visualized by spraying the TLC plates with dilute 15%
aq H2SO4, followed by charring at 150 ꢁC for a few min-
utes. Column chromatography was performed on Silica-
gel Geduran Si 60 (E. Merck). Optical rotations were
recorded on a Perkin Elmer 241 polarimeter in a 1 dm cell
at 21 ꢁC. 1H and 13C NMR spectra were recorded with a
Bruker AC-200 spectrometer working at 200 and
50 MHz, respectively, with Me4Si as internal standard.
Elemental analyses were performed by the ‘Laboratoire
Central d’Analyses du CNRS’ (Vernaison, France).
3.4. 8-(Cholest-5-en-3b-yloxy)-3,6-dioxaoctyl 2-acet-
amido-2-deoxy-a-D-galactopyranoside (3)
Prepared from 18b (0.705 g, 0.83 mmol), as described
for 1, to afford 3 (0.609 g, 98%) as a colourless oil;
1
[a]D +10.6 (c 1.0, CHCl3); H NMR (CDCl3): d 6.79
(d, 1H, J2,NH 7.7 Hz, NHAc), 5.36–5.34 (m, 1H,
H-6chol), 4.90 (d, 1H, J1,2 3.1 Hz, H-1), 4.31–4.27 (m,
1H, H-2), 4.04–4.02 (m, 1H, H-4), 3.87–3.83 (m, 4H,
H-3, H-5, H-6a, H-6b), 3.67–3.63 (m, 12H, (OCH2-
CH2)3), 3.20–3.18 (m, 1H, H-3chol), 2.34–0.68 (m, 43H,
cholesterol), 2.06 (s, 3H, CH3CON); 13C NMR (CDCl3):

N. Laurent et al. / Carbohydrate Research 341 (2006) 823–835
827
d 172.8 (CH3CO), 140.8 (C-5chol), 121.7 (C-6chol), 97.9
3.7. 1,3-Bis(undecyloxy)propan-2-ol (6)
(C-1), 79.6 (C-3chol), 70.9, 70.5, 69.9, 67.2 ((OCH2-
CH2)3), 70.0, 69.9, 69.4 (C-3, C-4, C-5), 61.9 (C-6), 56.8
(C-14chol), 56.2 (C-17chol), 50.5 (C-9chol), 50.2 (C-2),
42.4 (C-13chol), 39.8 (C-12chol), 39.5 (C-24chol), 39.0
(C-4chol), 37.2 (C-1chol), 36.9 (C-10chol), 36.2 (C-22chol),
35.8 (C-20chol), 32.0 (C-7chol), 31.9 (C-8chol), 28.4
(C-2chol), 28.3 (C-16chol), 28.0 (C-25chol), 24.3 (C-15chol),
23.9 (C-23chol), 23.2 (C-27chol), 22.8 (C-26chol), 22.6
(CH3CON), 21.1 (C-11chol), 19.4 (C-19chol), 18.8 (C-
21chol), 11.9 (C-18chol). Anal. Calcd for C41H71NO9Æ
1.5H2O (749.01): C, 65.74; H, 9.89; N, 1.87. Found: C,
65.64; H, 9.79; N, 1.87.
Racemic 1,2-O-isopropylidene glycerol (3.30 g, 25
mmol) was added to freshly prepared 50% aq NaOH
(50 mL) and the mixture was stirred for 1 h at 80 ꢁC.
Undecyl bromide (15.0 mL) and Bu4NBr (1.30 g,
4.0 mmol) were successively added and heating was
maintained for 7 h. After cooling to rt, the mixture
was poured into water (150 mL) and extracted with
CH2Cl2 (3 · 50 mL). Then, the combined organic
extracts were washed with water (2 · 25 mL), dried
(Na2SO4) and concentrated. The crude product was dis-
solved in a mixture of EtOH (20 mL) and 2 N aq HCl
(30 mL) and refluxed for 1 h. After evaporation of
EtOH, the aq phase was neutralized with satd aq
NaHCO3 and extracted with CHCl3. The organic
extract was dried and concentrated before purification
by column chromatography (3:1 EtOAc–petroleum
ether) to afford pure 1-O-undecyl glycerol (5.31 g,
86%). A mixture of the latter (2.74 g, 11.12 mmol) and
dibutyltin oxide (3.33 g, 13.38 mmol) was refluxed in
dry toluene for 5 h; water was removed by azeotropic
distillation. The solvent was removed under diminished
pressure and the residue was carefully dried. The prod-
uct was dissolved in DMF (90 mL), undecyl bromide
(3.45 mL, 15.40 mmol) and CsF (4.56 g, 30.00 mmol)
were added and the mixture was stirred for 24 h at rt.
Then, EtOAc (60 mL) and water (1.5 mL) were added
and the mixture was stirred for 1 h, before filtration
and concentration. The residue was redissolved in
CH2Cl2 (100 mL), washed with water (2 · 25 mL) and
concentrated before purification by column chromato-
graphy (1:8 EtOAc–petroleum ether) to afford product
6 (3.87 g, 87%) as a solid; mp 35 ꢁC (lit.23 mp 38 ꢁC);
1H NMR (CDCl3): d 4.02–3.91 (m, 1H, CHOH), 3.55–
3.40 (m, 8H, 2CH2OCH2C10H21), 2.59 (d, 1H, J
3.9 Hz, OH), 1.64–1.50 (m, 4H, 2OCH2CH2C9H19),
1.40–1.20 (m, 32H, 16CH2 alkyl chains), 0.87 (t, 6H,
J 6.7 Hz, 2CH3 alkyl chains); 13C NMR (CDCl3): d
71.9, 71.7 (CH(CH2OCH2C10H21)2), 69.5 (CHOH),
31.9, 29.7, 29.5, 29.4, 26.1, 22.7 (CH2 alkyl chains),
14.1 (CH3 alkyl chains).
3.5. 8-(Cholest-5-en-3b-yloxy)-3,6-dioxaoctyl 2-acet-
amido-2-deoxy-3-O-b-D-galactopyranosyl-a-D-galacto-
pyranoside (4)
Prepared from 25 (0.273 g, 0.23 mmol), as described for
1, to afford 4 (0.199 g, 96%) as a white solid; mp 213–
214 ꢁC; [a]D +24.2 (c 1.1, 3:1 CHCl3–MeOH); 1H
NMR (CDCl3): d 5.17–5.15 (m, 1H, 6chol), 4.65 (d, 1H,
J1,2 3.7 Hz, H-1), 4.28 (dd, 1H, J2,3 10.7 Hz, H-2), 4.11
(d, 1H, J1 ;2 7.3 Hz, H-10), 4.08–4.06 (m, 1H, H-4),
3.97–3.95 (m, 1H, H-40), 3.68–3.30 (m, 21H, H-3, H-5,
H-6a, H-6b, H-20, H-30, H-50, H-60a, H-60b,
(OCH2CH2)3), 3.21–3.19 (m, 1H, H-3chol), 2.30–0.65
(m, 43H, H cholesterol), 1.79 (s, 3H, CH3CON); 13C
NMR (CDCl3): d 172.8 (CH3CO), 140.5 (C-5chol),
121.6 (C-6chol), 105.0 (C-10), 97.7 (C-1), 79.5 (C-3chol),
78.4 (C-3), 74.9 (C-50), 73.1 (C-30), 70.9 (C-20, C-5),
70.6, 70.2, 70.1, 70.0, 67.0, 66.4 ((OCH2CH2)3), 68.8,
68.6 (C-4, C-40), 61.3 (C-6, C-60), 56.6 (C-14chol), 56.0
(C-17chol), 50.1 (C-9chol), 48.2 (C-2), 42.2 (C-13chol),
39.6 (C-12chol), 39.4 (C-24chol), 38.8 (C-4chol), 37.0
(C-1chol), 36.7 (C-10chol), 36.0 (C-22chol), 35.6 (C-20chol),
31.7 (C-7chol), 31.7 (C-8chol), 28.1 (C-2chol), 28.0
(C-16chol), 27.8 (C-25chol), 24.1 (C-15chol), 23.6 (C-23chol),
22.5 (CH3CON), 22.5 (C-27chol), 22.3 (C-26chol), 20.9
(C-11chol), 19.1 (C-19chol), 18.5 (C-21chol), 11.6 (C-18chol).
Anal. Calcd for C47H81NO14ÆH2O (902.141): C, 62.57; H,
9.28; N, 1.55. Found: C, 62.67; H, 9.28; N, 1.43.
0
0
3.6. 4-Undecyloxymethyl-3,6-dioxaheptadecyl O-(2-acet-
amido-2-deoxy-a-D-galactopyranosyl)-L-serinamide (5)
3.8. 1,3-Bis(3,7,11,15-tetramethylhexadecyloxy)propan-
2-ol (7)
Prepared from 22b (0.034 g, 0.040 mmol), as described
for 1, to afford 5 (0.028 g, 95%) as a colourless oil; [a]D
+36.5 (c 1.0, CHCl3); 13C NMR (1:1 CDCl3–CD3OD):
d 172.7 (CH3CO), 170.8 (CONHSer), 98.5 (C-1), 78.1
(CH(CH2OC11H23)2), 71.7, 70.6, 69.2, 68.7, 68.9
(CH(CH2OCH2C10H21)2, b-CH2Ser, NHCH2CH2O, C-
3, C-4, C-5), 60.8 (C-6), 55.4 (a-CHSer), 50.4 (C-2), 39.5
(NHCH2CH2O), 32.1, 29.9, 29.7, 29.6, 26.9, 22.8 (CH2
alkyl chains), 22.8 (CH3CON), 14.1 (CH3 alkyl chains).
MS FAB (M+H)+: calcd 734.5531; found, 734.5531.
A soln of phytol (20.0 mL, 57.32 mmol) in THF (32 mL)
was added dropwise (80 min) at rt to a mixture of 60%
NaH in oil (5.00 g, 125 mmol), THF (40 mL) and
HMPA (3.2 mL). The mixture was refluxed for 1 h, then
cooled to 0 ꢁC, before dropwise addition of epichloro-
hydrin (2.2 mL, 27.8 mmol) in THF (16 mL) for
40 min. The mixture was refluxed for 16 h, then cooled
to rt and poured into satd aq NH4Cl and extracted with
EtOAc (3 · 75 mL). The organic phase was dried
(Na2SO4) and concentrated under diminished pressure.

828
N. Laurent et al. / Carbohydrate Research 341 (2006) 823–835
After filtration on a short column of silica-gel (1:10
EtOAc–petroleum ether), the product was dissolved in
EtOAc (50 mL) and the soln was hydrogenated (4 atm)
in the presence of PtO2 (0.400 g) for 6 h at rt. After fil-
tration on Celite, and concentration under diminished
pressure, the residue was purified by column chromato-
graphy (1:1 EtOAc–petroleum ether) to afford the
expected compound 7 (5.85 g, 32%) as an oily material;
Rf 0.53; 1H NMR (CDCl3): d 3.98–3.90 (m, 1H,
HOCH), 3.53–3.39 (m, 8H, 4OCH2), 2.46 (d, 1H, J
4.1 Hz, OH), 1.56–1.08 (m, 48H, 20CH2, 8CH phytyl
chains), 0.89–0.84 (m, 30H, 10CH3 phytyl chains); 13C
NMR (CDCl3): d 72.0, 70.0 (CH(CH2OCH2C19H39)2),
69.5 (CHOH), 39.4, 37.4, 37.3, 36.7, 36.6, 32.8, 29.9,
28.0, 25.4, 24.9, 24.5, 24.4, 22.8, 22.7, 19.8, 19.7 (CH2,
CH, CH3 phytyl chains). Anal. Calcd for C43H88O3
(653.16): C, 79.07; H, 13.58. Found: C, 79.31; H, 13.65.
soln was stirred at rt for 45 min. Then, NaHCO3
(0.300 g, 3.57 mmol) was added to the mixture, before
addition of EtOAc (50 mL). The organic extract was
washed with water, dried (MgSO4), concentrated and
purified by column chromatography (1:2 EtOAc–petro-
leum ether) to afford product 9 (0.588 g, 81%) as a
1
colourless oil; Rf 0.43; H NMR (CDCl3): d 3.80–3.73
(m, 1H, CH(CH2OPhy)2), 3.70–3.59 (m, 12H, (CH2-
CH2O)3), 3.51–3.44 (m, 8H, 2CH2OCH2C19H39), 1.56–
1.08 (m, 48H, 20CH2, 8CH phytyl chains), 0.89–0.85
(m, 30H, 10CH3 phytyl chains); 13C NMR (CDCl3): d
78.4 (CH(CH2OPhy)2), 72.7, 70.9, 70.6, 70.4, 70.0, 69.7
(CH(CH2OCH2C19H39)2, (CH2CH2O)2, OCH2CH2OH),
61.7 (CH2OH), 39.4, 37.5, 37.4, 37.3, 36.7, 36.6, 32.8,
29.9, 28.0, 24.8, 24.5, 24.4, 24.1, 22.7, 22.6, 19.8,
19.7 (CH2, CH, CH3 phytyl chains). Anal. Calcd for
C49H100O6 (785.31): C, 74.94; H, 12.83. Found: C,
74.93; H, 13.09.
3.9. 2-O-(2-Hydroxyethyl)-1,3-bis(undecyloxy)propan-
2-ol (8)
3.11. 4-Undecyloxymethyl-3,6-dioxaheptanenitrile (11)
Freshly prepared 50% aq NaOH (4 mL) was added
dropwise with stirring to a soln of alcohol 6 (1.00 g,
Sodium hydride (600 mg, 15 mmol, 60% in oil) was
slowly added under Ar to a soln of the alcohol 6
(1.20 g, 3.00 mmol) in CH3CN (7 mL). After stirring
for 2 h, the mixture was cooled to ꢀ20 ꢁC and bromo-
acetonitrile (1.56 mL, 24.00 mmol) was added slowly.
Stirring was maintained overnight at ꢀ20 ꢁC, then the
reaction mixture was allowed to reach rt and concen-
trated. After addition of CH2Cl2 (50 mL), the salts were
removed by centrifugation and filtration. After evapora-
tion of the solvent and purification by column chroma-
tography (1:6 EtOAc–petroleum ether), compound 11
(1.015 g, 77%) was obtained as a colourless oil; Rf
0.75; 1H NMR (CDCl3): d 4.50 (s, 2H, OCH2CN),
3.92–3.82 (m, 1H, OCH), 3.55 (d, 4H, J 4.8 Hz,
2CH2OC11H23), 3.44 (t, 4H, J 6.6 Hz, 2OCH2C10H21),
1.65–1.50 (m, 4H, 2OCH2CH2C9H19), 1.37–1.17 (m,
32H, 16CH2 alkyl chains), 0.88 (t, 6H, J 6.3 Hz, 2CH3
alkyl chains); 13C NMR (CDCl3): d 116.7 (CN), 78.8
(OCH), 71.8, 71.2 (CH(CH2OCH2C10H21)2), 56.1
(CH2CN), 31.9, 29.7, 29.6, 29.5, 29.4, 26.1, 22.7 (CH2
alkyl chains), 14.1 (CH3 alkyl chains). Anal. Calcd for
C27H53NO3 (439.40): C, 73.75; H, 12.15; N, 3.19.
Found: C, 73.72; H, 12.08; N, 3.22.
2.50 mmol),
2-(2-chloroethoxy)tetrahydro-2H-pyran
(1.10 mL, 7.50 mmol) and Bu4NHSO4 (0.085 g, 0.25
mmol) in CH2Cl2 (4 mL). The mixture was stirred at
65 ꢁC for 5 days. After dilution with CH2Cl2 (40 mL),
the organic extract was washed with water
(3 · 10 mL), dried and concentrated under diminished
pressure, before purification by column chromatogra-
phy (1:6 EtOAc–petroleum ether, 0.5% NEt3) to afford
the expected tetrahydropyranyl derivative. The latter
was dissolved in a 1:1 MeOH–CH2Cl2 mixture (20 mL)
and treated during 2 h with 12 N aq HCl (0.2 mL). After
neutralization with NaHCO3 (0.730 g), the mixture was
evaporated to dryness before addition of EtOAc. After
filtration and concentration, the residue was purified
by column chromatography (1:3 EtOAc–petroleum
ether) to afford 8 (0.645 g, 58%) as an oily material; Rf
1
0.58; H NMR (CDCl3): d 3.82–3.70 (m, 5H, CHOH,
OCH2CH2OH), 3.58–3.44 (m, 8H, 2CH2OCH2C10H21),
1.64–1.50 (m, 4H, 2OCH2CH2C9H19), 1.38–1.18 (m,
32H, 16CH2 alkyl chains), 0.87 (t, 6H, J 6.7 Hz, 2CH3
alkyl chains); 13C NMR (CDCl3): d 78.6 (OCH), 72.3
(OCH2CH2OH), 71.8, 71.3 (CH(CH2OCH2C10H21)2),
62.1 (OCH2CH2OH), 31.9, 29.6, 29.5, 29.4, 26.1, 22.7
(CH2 alkyl chains), 14.1 (CH3 alkyl chains). Anal. Calcd
for C27H56O4 (444.73): C, 72.92; H, 12.69. Found: C,
72.85; H, 12.87.
3.12. 2-O-(2-Aminoethyl)-1,3-bis(undecyloxy)propan-2-ol
(12)
A mixture of compound 11 (0.900 g, 2.05 mmol) in
EtOH (25 mL) and 12 N aq HCl (1 mL) containing
10% Pd/C (108 mg) was stirred under hydrogen
(10 atm) for 4 h before neutralization with NEt3
(15 mL). After filtration and evaporation, petroleum
ether was added to the mixture. Triethylammonium
chloride was filtered off and the organic phase was
washed with water (2 · 5 mL), dried (Na2SO4) and con-
3.10. 15,19,23,27–Tetramethyl-10-(3,7,11,15-tetrameth-
ylhexadecyloxymethyl)-3,6,9,12-tetraoxaoctacosan-1-ol
(9)
Product 13 (0.800 g, 0.920 mmol) was dissolved in
0.3:10:10 12 N aq HCl–MeOH–CH2Cl2 (9 mL) and the

N. Laurent et al. / Carbohydrate Research 341 (2006) 823–835
829
centrated to dryness. After purification by column chro-
matography (10:1 CHCl3–MeOH), product 12 (0.758 g,
83%) was obtained as a colourless oil; Rf 0.50; 1H NMR
(CDCl3): d 3.65–3.60 (m, 3H, OCH, OCH2CH2NH2),
3.51–3.40 (m, 8H, 2CH2OCH2C10H21), 2.85 (t, 2H, J
4.9 Hz, CH2NH2), 1.70 (s, 2H, NH2), 1.65–1.50 (m,
4H, 2OCH2CH2C9H19), 1.43–1.23 (m, 32H, 16CH2 alkyl
chains), 0.88 (t, 6H, J 6.4 Hz, 2CH3 alkyl chains); 13C
NMR (CDCl3): d 78.1 (OCH), 72.4 (OCH2CH2NH2),
71.6, 70.8 (CH(CH2OCH2C10H21)2), 42.1 (OCH2CH2-
NH2), 31.9, 29.6, 29.5, 29.3, 26.1, 22.7 (CH2 alkyl
chains), 14.1 (CH3 alkyl chains). Anal. Calcd for
C27H57NO3 (443.75): C, 73.08; H, 12.95; N, 3.16.
Found: C, 72.74; H, 12.93; N, 3.14.
for 10 min at 0 ꢁC, then allowed to reach rt and stirred
for 6 h. After dilution with CH2Cl2 and filtration on Cel-
ite, the organic extract was washed with 0.1 N HCl, then
with satd aq NaHCO3 and water. After drying (Na2SO4)
and concentration, the crude product was purified by
column chromatography (2:1 EtOAc–petroleum ether)
to afford 15 (0.404 g, 59%) as an amorphous solid; [a]D
+2.4 (c 1.0, CHCl3); Rf 0.60; 13C NMR (1:1 CDCl3–
CD3OD): d 170.8 (CONH), 156.4 (NHCOOFmoc),
143.3, 140.8 (aromatic CFmoc), 127.1, 126.5, 124.5,
119.3 (aromatic CHFmoc), 77.5 (OCH(CH2OC11H23)2),
71.1, 69.9, 68.0, 66.6 (CH(CH2OCH2C10H21)2, OCH2-
CH2NH,CH2Fmoc), 61.8 (b-CH2Ser), 56.5 (a-CHSer),
46.6 (C-9Fmoc), 39.2 (OCH2CH2NH), 33.1, 31.4, 29.0,
28.9, 28.8, 25.5, 25.0, 24.4, 22.1 (CH2 alkyl chains),
13.2 (CH3 alkyl chains). Anal. Calcd for C45H72N2O7
(753.06): C, 71.77; H, 9.64; N, 3.72. Found: C, 71.50;
H, 9.55; N, 3.86.
3.13. 15,19,23,27-Tetramethyl-10-(3,7,11,15-tetramethyl-
hexadecyloxymethyl)-1-tetrahydropyranyloxy-3,6,9,12-
tetraoxaoctacosane (13)
A mixture of alcohol 7 (1.30 g, 0.200 mmol) and NaH
(60% in oil, 0.400 g, 10 mmol, washed with pentane) in
THF was refluxed for 2 h and then cooled to rt. A soln
of 1-tetrahydropyranyloxy-8-tosyloxy-3,6-dioxaoctane25
(3.90 g, 10 mmol) in THF (70 mL) was added dropwise
over 2 h and the mixture was refluxed for 22 h. After
cooling to rt and addition of water (50 mL), THF was
removed under diminished pressure and the mixture
was extracted with CH2Cl2 (3 · 30 mL). The organic
layer was washed with water, dried (Na2SO4), concen-
trated and purified by column chromatography (1:5
EtOAc–petroleum ether) to afford product 13 (1.06 g,
60%) as an amorphous solid; Rf 0.52; 1H NMR
(CDCl3): d 4.64–4.62 (m, 1H, CH THP), 3.92–3.84 (m,
1H, CH(CH2OPhy)2), 3.82–3.74 (m, 2H, OCH2 THP),
3.71–3.61 (m, 12H, (CH2CH2O)3), 3.54–3.42 (m, 8H,
2CH2OCH2C19H39), 1.71–1.08 (m, 54H, 3CH2 THP,
20CH2, 8CH phytyl chains), 0.89–0.85 (m, 30H,
10CH3 phytyl chains); 13C NMR (CDCl3): d 98.9 (CH
THP), 78.5 (CH(CH2OPhy)2), 78.5, 70.9, 70.8, 70.6,
70.0, 69.8, 69.4, 67.3, 66.7, 62.2 (CH(CH2OCH2-
C19H39)2, (CH2CH2O)3, CH2O THP), 39.4, 37.5,
37.4, 37.3, 36.8, 36.7, 32.8, 30.6, 29.9, 28.0, 25.5,
24.8, 24.5, 24.4, 24.0, 22.8, 22.7, 19.8, 19.7 (CH2 THP,
CH2, CH, CH3 phytyl chains). Anal. Calcd for
C54H108O7 (869.43): C, 74.60; H, 12.52. Found: C,
74.63; H, 12.66.
3.15. 2-O-[2-(3,4,6-Tri-O-acetyl-2-azido-2-deoxy-D-
galactopyranosyloxy)ethyl]-1,3-bis(undecyloxy)propan-
2-ol (16a)
Trimethylsilyltrifluoromethane sulfonate (64 lL, 0.331
mmol) in Et2O (0.5 mL) was added dropwise (30 min)
to a cooled (ꢀ30 ꢁC) soln of 8 (0.650 g, 1.46 mmol)
and 3,4,6-tri-O-acetyl-2-azido-2-deoxy-D-galactopyran-
osyl trichloroacetimidate (1:4 a/b-anomeric mixture,
1.037 g, 2.18 mmol) in 3:1 Et2O–CH2Cl2 (15 mL), con-
˚
taining crushed activated 4 A molecular sieves. After
2 h at ꢀ30 ꢁC, the mixture was allowed to reach rt and
stirring was maintained for 18 h. The mixture was then
neutralized by addition of NEt3 (100 lL), diluted with
CH2Cl2 (50 mL), washed with satd aq NaHCO3
(2 · 10 mL), dried (Na2SO4) and concentrated under
diminished pressure. After purification by column chro-
matography (1:3 EtOAc–petroleum), compound 16a
(0.686 g, 62%) was obtained as a 3:1 a/b-anomeric mix-
ture. A small amount of the a anomer was isolated in a
pure form as a colourless oil; [a]D +83.4 (c 1.0, CHCl3);
1
Rf 0.58; H NMR (CDCl3): d 5.45–5.44 (m, 1H, H-4),
5.39 (dd, 1H, J2,3 10.9 Hz, J3,4 3.2 Hz, H-3), 5.11 (d,
1H, J1,2 3.4 Hz, H-1), 4.31 (br t, 1H, J5,6a, J5,6b 6.7 Hz,
H-5), 4.13 (dd, 1H, J6a,6b 11.1 Hz, H-6a), 4.06 (dd, 1H,
H-6b), 3.88–3.77 (m, 4H, OCH2CH2O), 3.75–3.72 (m,
1H, CH(CH2OC11H23)2), 3.62 (dd, 1H, H-2), 3.51–3.40
(m, 8H, 2CH2OCH2C10H21), 2.14, 2.05, 2.04 (3s, 9H,
3CH3CO), 1.64–1.50 (m, 4H, 2OCH2CH2C9H19), 1.35–
1.18 (m, 32H, 16CH2 alkyl chains), 0.88 (t, 6H, J
6.3 Hz, 2CH3 alkyl chains); 13C NMR (CDCl3): d
170.2, 170.0, 169.7 (3CH3CO), 98.1 (C-1), 78.5
(CH(CH2OC11H23)2), 71.6, 70.9 (CH(CH2OCH2-
C10H21)2), 69.3, 67.8 (OCH2CH2O), 70.9, 68.1, 67.8
(C-3, C-4, C-5), 61.5 (C-6), 57.4 (C-2), 31.9, 29.6, 29.5,
29.3, 26.1, 22.7 (CH2 alkyl chains), 20.7, 20.6
(CH3COO), 14.1 (CH3 alkyl chains). Anal. Calcd for
3.14. 4-Undecyloxymethyl-3,6-dioxaheptadecyl N-[(9H-
fluoren-9-ylmethoxy)carbonyl]-L-serinamide (15)
A mixture of amine 12 (0.406 g, 0.910 mmol), N-(9H-flu-
oren-9-ylmethoxycarbonyl)-L-serine24 14 (0.310 g, 0.940
mmol) and 1-hydroxybenzotriazole (0.141 g, 1.04 mmol)
in dry DMF (4 mL) was cooled to 0 ꢁC. A soln of dic-
yclohexylcarbodiimide (0.203 g, 0.980 mmol) in DMF
(5 mL) was added dropwise and the mixture was stirred

830
N. Laurent et al. / Carbohydrate Research 341 (2006) 823–835
C39H71N3O11 (757.99): C, 61.80; H, 9.44; N, 5.54.
Found: C, 61.78; H, 9.46; N, 5.57.
purification by column chromatography (1:2 EtOAc–
petroleum ether), compound 17a (4:1 a,b-anomeric mix-
ture, 0.840 g, 91%) was obtained as a colourless oil. A
small amount (0.200 g) of pure a-isomer was recovered
as an amorphous solid, after a second chromatography;
[a]D +53.0 (c 1.0, CHCl3); Rf 0.76 (0.70 for b-isomer);
1H NMR (CDCl3): d 5.46 (dd, 1H, J3,4 3.3 Hz, J4,5
1.0 Hz, H-4), 5.39 (dd, 1H, J2,3 10.9 Hz, H-3), 5.09 (d,
1H, J1,2 3.5 Hz, H-1), 4.30 (br dd, 1H, J5,6a 6.5 Hz,
J5,6b 7.0 Hz, H-5), 4.14 (dd, 1H, J6a,6b 11.0 Hz H-6a),
4.06 (dd, 1H, H-6b), 3.88–3.39 (m, 22H, H-2, OCH-
(CH2OPhy)2, (OCH2CH2)3, 2CH2OCH2C19H39), 2.15,
2.06, 2.05 (3s, 9H, 3CH3COO), 1.60–1.08 (m, 48H,
20CH2, 8CH alkyl chains), 0.89–0.85 (m, 30H, 10CH3
phytyl chains); 13C NMR (CDCl3): d 170.3, 170.0,
169.8 (3CH3CO), 98.3 (C-1), 78.4 (CH(CH2OPhy)2),
70.9, 70.8, 70.7, 70.6, 70.1, 69.9, 69.7, 67.8 ((OCH2-
CH2)3, CH(CH2OCH2C19H39)2), 68.2, 67.6, 66.6 (C-3,
C-4, C-5), 61.6 (C-6), 57.4 (C-2), 39.4, 37.5, 37.4, 37.3,
36.8, 36.7, 32.8, 29.9, 28.0, 25.3, 24.8, 24.5, 24.4, 22.8,
22.7, 19.8, 19.7 (CH, CH2, CH3 phytyl chains), 20.7,
20.6 (3CH3COO). Anal. Calcd for C61H115N3O13
(1098.58): C, 66.69; H, 10.55; N, 3.82. Found: C,
67.04; H, 10.66; N, 4.02.
3.16. 2-O-[2-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-
D-galactopyranosyloxy)ethyl]-1,3-bis(undecyloxy)propan-
2-ol (16b)
A soln of NaBH4 in EtOH (0.300 mol/L) was added
dropwise to a stirred soln of NiCl2Æ6H2O (2.371 g,
10.00 mmol), B(OH)3 (0.927 g, 15.00 mmol) and azido-
glycoside 16a (0.758 g, 1.00 mmol) in EtOH (50 mL),
until persistency of the dark colour. After 1.5 h, the mix-
ture was concentrated under diminished pressure and
the residue was dissolved in a 3:1 pyr–Ac2O mixture
(10 mL). After stirring for 18 h at rt, the mixture was
concentrated under diminished pressure before addition
of CH2Cl2, washing with 3% aq KHSO4 (10 mL), then
water (10 mL). After drying (Na2SO4) and concentra-
tion, the residue was purified by column chromatogra-
phy (2:1 EtOAc–petroleum ether) to afford the
a-product 16b (0.495 g, 64%) as an amorphous solid;
1
[a]D +44.7 (c 1.0, CHCl3); Rf 0.50; H NMR (CDCl3):
d 6.13 (d, 1H, J2,NH 9.7 Hz, NH), 5.38 (dd, 1H, J3,4
3.4 Hz, J4,5 1.0 Hz, H-4), 5.16 (dd, 1H, J3,4 11.2 Hz,
H-3), 4.89 (d, 1H, J1,2 3.6 Hz, H-1), 4.62 (ddd, 1H, H-
2), 4.25 (br t, 1H, J5,6a, J5,6b 6.5 Hz, H-5), 4.15–4.05
(m, 2H, H-6a, H-6b), 3.88–3.73 (m, 4H, OCH2CH2O),
3.70–3.61 (m, 1H, CH(CH2OC11H23)2), 3.50–3.40 (m,
8H, 2CH2OCH2C10H21), 2.16, 2.05, 1.98, 1.96 (4s,
12H, 3CH3COO, 1CH3CON), 1.62–1.47 (m, 4H,
2OCH2CH2C9H19), 1.40–1.15 (m, 32H, 16CH2 alkyl
chains), 0.88 (t, 6H, J 6.3 Hz, 2CH3 alkyl chains); 13C
NMR (CDCl3): d 170.7, 170.4, 170.2 (4CH3CO), 98.1
(C-1), 78.3 (CH(CH2OC11H23)2), 71.7, 70.8 (CH(CH2O-
CH2C10H21)2), 69.8, 68.1 (OCH2CH2O), 68.7, 67.4, 66.8
(C-3, C-4, C-5), 61.9 (C-6), 47.5 (C-2), 31.9, 29.6, 29.5,
29.3, 26.1, 22.7 (CH2 alkyl chains), 23.1 (CH3CON),
20.7, 20.6 (3CH3COO), 14.1 (CH3 alkyl chains). Anal.
Calcd for C41H75NO12 (774.03): C, 63.62; H, 9.77; N;
1.81. Found: C, 63.11; H, 9.82; N, 1.94.
3.18. 15,19,23,27-Tetramethyl-10-(3,7,11,15-tetramethyl-
hexadecyloxymethyl)-3,6,9,12-tetraoxaoctacosyl 2-acet-
amido-3,4,6-tri-O-acetyl-2-deoxy-a-D-galactopyranoside
(17b)
Prepared as described for 16b from the a/b-anomeric
mixture 17a (0.840 g, 0.765 mmol). Purification by col-
umn chromatography with EtOAc afforded 17b as a
a/b-anomeric mixture (0.715 g, 84%). This mixture was
chromatographed again (2:1 petroleum ether–acetone)
to afford the pure a-anomer (0.435 g, 51%) as a colour-
1
less oil; [a]D +29.1 (c 1.0, CHCl3); Rf 0.42; H NMR
(CDCl3): d 6.25 (d, 1H, J2,NH 7.7 Hz, NHAc), 5.39
(dd, 1H, J3,4 3.1 Hz, J4,5 1.0 Hz, H-4), 5.19 (dd, 1H,
J3,2 11.3 Hz, H-3), 4.90 (d, 1H, J1,2 3.6 Hz, H-1), 4.61
(ddd, 1H, H-2), 4.24 (br dd, 1H, J5,6a 6.0 Hz, J5,6b
7.1 Hz, H-5), 4.14 (dd, 1H, J6a,6b 11.0 Hz H-6a), 4.06
(dd, 1H, H-6b), 3.85–3.43 (m, 21H, OCH(CH2OPhy)2,
(OCH2CH2)3, 2CH2OCH2C19H39), 2.17, 2.05, 1.99,
1.97 (4s, 12H, 3CH3COO, CH3CON), 1.63–1.08 (m,
48H, 20CH2, 8CH phytyl chains), 0.89–0.85 (m, 30H,
10CH3 phytyl chains); 13C NMR (CDCl3): d 170.8,
170.4, 170.3, 170.2 (4CH3CO), 98.3 (C-1), 78.5
(CH(CH2OPhy)2), 70.9, 70.8, 70.7, 69.9, 69.7, 67.8
((OCH2CH2)3, CH(CH2OCH2C19H39)2), 68.6, 67.5,
66.8 (C-3, C-4, C-5), 61.9 (C-6), 47.6 (C-2), 39.4, 37.5,
37.4, 37.3, 36.8, 36.7, 32.8, 30.0, 28.0, 24.8, 24.5, 24.4,
24.0, 22.8, 22.6, 19.8, 19.7 (CH, CH2, CH3 phytyl
chains), 23.2 (CH3CON), 20.8, 20.7 (3CH3COO). Anal.
Calcd for C63H119NO14 (1114.62): C, 67.89; H, 10.76; N,
1.26. Found: C, 67.32; H, 10.70; N, 1.33.
3.17. 15,19,23,27-Tetramethyl-10-(3,7,11,15-tetramethyl-
hexadecyloxymethyl)-3,6,9,12-tetraoxaoctacosyl 3,4,6-
tri-O-acetyl-2-azido-2-deoxy-D-galactopyranoside (17a)
Trimethylsilyl trifluoromethanesulfonate (12 lL, 0.062
mmol) was added to a cooled (ꢀ30 ꢁC) mixture of alco-
hol 9 (0.660 g, 0.840 mmol) and 3,4,6-tri-O-acetyl-2-azi-
do-2-deoxy-D-galactopyranosyl
trichloroacetimidate
(1:5 a/b-anomeric mixture, 0.520 g, 1.09 mmol) in dry
Et2O (7 mL). The mixture was then allowed to reach
rt and after 40 min, it was neutralized by addition of
i-Pr2NEt (15 lL). After evaporation, the residue was
dissolved in CH2Cl2 (50 mL), washed with 0.1 N aq
HCl (3 · 5 mL), then water (2 · 5 mL), dried (MgSO4)
and concentrated under diminished pressure. After

N. Laurent et al. / Carbohydrate Research 341 (2006) 823–835
831
3.19. 8-(Cholest-5-en-3b-yloxy)-3,6-dioxaoctyl 3,4,6-tri-
O-acetyl-2-azido-2-deoxy-D-galactopyranoside (18a)
3.21. 8-(Cholest-5-en-3b-yloxy)-3,6-dioxaoctyl 2-azido-
4,6-O-benzylidene-2-deoxy-a-D-galactopyranoside (18d)
Trimethylsilyl trifluoromethanesulfonate (18 lL, 0.093
mmol) was added to a cooled (ꢀ30 ꢁC) mixture of
8-(cholest-5-en-3b-yloxy)-3,6-dioxa-1-octanol 107 (1.32 g,
2.52 mmol) and 3,4,6-tri-O-acetyl-2-azido-2-deoxy-D-
galactopyranosyl trichloroacetimidate (1:5 a/b-ano-
meric mixture, 0.930 g, 1.95 mmol), in dry Et2O
(18 mL). The mixture was allowed to reach rt and, after
30 min, it was neutralized by addition of i-Pr2NEt
(100 lL). After evaporation of the solvent, the residue
was dissolved in CH2Cl2 (50 mL) and the soln was
washed with 0.1 N aq HCl (3 · 5 mL), then water
(2 · 5 mL), dried (MgSO4) and concentrated under
diminished pressure, before purification by column
chromatography (2:3 EtOAc–petroleum ether), to afford
18a (1.45 g, 89%) as an inseparable 4:1 a/b-anomeric
The a/b-anomeric mixture 18b (0.705 g, 0.847 mmol)
obtained from 18a was quantitatively O-deacetylated
with catalytic MeONa in MeOH (20 mL). After neutral-
ization with Amberlyst IR 120 (H+) and evaporation,
the crude product was treated for 4 h at 40 ꢁC with benz-
aldehyde dimethyl acetal (250 lL, 1.46 mmol) in MeCN
(4.1 mL) in the presence of p-TsOH (0.005 g). After neu-
tralization with satd aq NaHCO3 (0.5 mL), concentra-
tion and coevaporation from toluene, the residue was
directly purified by column chromatography (1:4
EtOAc–petroleum ether) to afford the pure a-anomer
18d (0.488 g, 69%) as an oily material; [a]D +53.2 (c
1
1.0, CHCl3); Rf 0.57; H NMR (CDCl3): d 7.49–7.38
(m, 5H, C6H5), 5.59 (s, 1H, CHPh), 5.39–5.35 (m, 1H,
H-6chol), 5.06 (d, 1H, J1,2 3.4 Hz, H-1), 4.33–4.29 (m,
1H, H-4), 4.28 (dd, 1H, J5,6a 1.3 Hz, J6a,6b 12.5 Hz,
H-6a), 4.25–4.21 (m, 1H, H-3), 4.10 (dd, 1H, J5,6b
1.9 Hz, H-6b), 3.88–3.62 (m, 13H, H-5, (OCH2CH2)3),
3.59 (dd, 1H, J2,3 10.3 Hz, H-2), 3.19 (m, 1H, H-3chol),
2.41–0.67 (m, 43H, H cholesterol); 13C NMR (CDCl3):
d 141.0 (C-5chol), 137.5, 129.3, 128.4, 126.3 (C6H5),
121.6 (C-6chol), 101.3 (CHC6H5), 98.8 (C-1), 79.6 (C-
1
mixture; Rf 0.46; H NMR (CDCl3): selected values d
5.10 (d, 0.8H, J1,2 3.5 Hz, H-1a), 4.77 (dd, 0.2H, J2,3
10.9 Hz, J3,4 3.2 Hz, H-3b), 4.50 (d, 0.2H, J1,2 8.0 Hz,
H-1b); 13C NMR (CDCl3): d 170.4, 170.1, 169.9
(3CH3CO), 141.0 (C-5chol), 121.6 (C-6chol), 102.5
(C-1b), 98.3 (C-1a), 79.5 (C-3chol).
3.20. 8-(Cholest-5-en-3b-yloxy)-3,6-dioxaoctyl 2-acet-
amido-3,4,6-tri-O-acetyl-2-deoxy-a-D-galactopyranoside
(18b)
3chol), 75.62 (C-4), 71.0, 70.8, 70.7, 70.2, 67.7, 67.4
((OCH2CH2)3), 69.4 (C-6), 67.5 (C-3), 62.8 (C-5), 60.8
(C-2), 56.8 (C-14chol), 56.2 (C-17chol), 50.2 (C-9chol),
42.4 (C-13chol), 39.8 (C-12chol), 39.6 (C-24chol), 39.2 (C-
Prepared as described for 16b from the a/b-anomeric
mixture 18a (1.45 g, 1.74 mmol). Purification by column
chromatography (2:1 petroleum ether–acetone) afforded
an unseparated 1:1 a/b-mixture (0.269 g, 18%) and the
pure a-derivative 18b (0.808 g, 56%) as a colourless oil;
4
chol), 37.2 (C-1chol), 36.9 (C-10chol), 36.2 (C-22chol),
35.8 (C-20chol), 32.0 (C-7chol), 31.9 (C-8chol), 28.4 (C-
chol), 28.3 (C-16chol), 28.0 (C-25chol), 24.3 (C-15chol),
2
23.9 (C-23chol), 22.9 (C-27chol), 22.6 (C-26chol), 21.1 (C-
11chol), 19.4 (C-19chol), 18.8 (C-21chol), 11.9 (C-18chol).
Anal. Calcd for C46H71N3O8ÆH2O (812.06): C, 68.03;
H, 9.06; N, 5.17. Found: C, 67.69; H, 8.82; N, 5.36.
1
[a]D +13.0 (c 1.0, CHCl3); Rf 0.57; H NMR (CDCl3):
d 6.18 (d, 1H, J2,NH 9.8 Hz, NHAc), 5.39–5.35 (m, 2H,
H-4, H-6chol), 5.19 (dd, 1H, J2,3 11.3 Hz, J3,4 3.1 Hz,
H-3), 4.90 (d, 1H, J1,2 3.4 Hz, H-1), 4.61 (ddd, 1H, H-
2), 4.24–4.20 (m, 1H, H-5), 4.13–4.07 (m, 2H, H-6a,
H-6b), 3.86–3.63 (m, 12H, (OCH2CH2)3), 3.18 (m, 1H,
H-3chol), 2.17, 2.05, 1.99, 1.98 (4s, 12H, 3CH3COO,
1CH3CON), 2.39–0.67 (m, 43H, H cholesterol); 13C
NMR (CDCl3): d 170.8, 170.4, 170.2 (4CH3CO), 140.9
(C-5chol), 121.6 (C-6chol), 98.1 (C-1), 79.6 (C-3chol),
70.9, 70.6, 69.9, 67.5, 67.3 ((OCH2CH2)3), 68.6, 67.4,
66.8 (C-3, C-4, C-5), 61.9 (C-6), 56.8 (C-14chol), 56.2
(C-17chol), 50.2 (C-9chol), 47.6 (C-2), 42.3 (C-13chol),
39.8 (C-12chol), 39.5 (C-24chol), 39.1 (C-4chol), 37.2
(C-1chol), 36.9 (C-10chol), 36.2 (C-22chol), 35.8 (C-20chol),
31.9 (C–7chol), 30.9 (C-8chol), 28.3 (C-2chol), 28.2
(C-16chol), 28.0 (C-25chol), 24.3 (C-15chol), 23.8 (C-23chol),
23.3 (C-27chol), 22.8 (C-26chol), 22.6 (CH3CON), 21.1
(C-11chol), 20.8, 20.7 (3CH3COO), 19.4 (C-19chol),
18.7 (C-21chol), 11.9 (C-18chol). Anal. Calcd for
C47H77NO12 (848.11): C, 66.56; H, 9.15; N, 1.65.
Found: C, 65.91; H, 9.32; N, 1.72.
3.22. 4-Undecyloxymethyl-3,6-dioxaheptadecyl O-(3,4,6-
tri-O-acetyl-2-azido-2-deoxy-D-galactopyranosyl)-N-
[(9H-fluoren-9-ylmethoxy)carbonyl]-L-serinamide (19a)
A mixture of the serine derivative 15 (0.376 g, 0.499
mmol),
3,4,6-tri-O-acetyl-2-azido-2-deoxy-D-galacto-
pyranosyl trichloroacetimidate (0.356 g, 0.749 mmol)
˚
and activated 4 A molecular sieves in 2:1 Et2O–CH2Cl2
(6.5 mL) was cooled to ꢀ18 ꢁC. A soln of TMSOTf
(30 lL, 0.155 mmol) in Et2O (0.1 mL) was added slowly
and stirring was continued for 2 h at ꢀ18 ꢁC, then 18 h
at rt. After neutralization with NEt3 (30 lL), filtration,
concentration and purification by column chromatogra-
phy (200:1 CHCl3–MeOH) product 19a (0.177 g, 35%)
was obtained as an unseparable 2:1 a/b-anomeric mix-
ture. A fraction containing the a-derivative only,
together with some trichloroacetamide, was separated
1
for NMR. H NMR (CDCl3): d 7.72 (d, 2H, J 7.5 Hz,
aromatic HFmoc), 7.62 (d, 2H, J 7.3 Hz, aromatic

832
N. Laurent et al. / Carbohydrate Research 341 (2006) 823–835
HFmoc), 7.41 (t, 2H, J 7.3 Hz, aromatic HFmoc), 7.33 (t,
2H, J 7.5 Hz, aromatic HFmoc), 7.08–7.04 (m, 1H,
NHCO), 5.86 (br d, 1H, NHCOOFmoc), 5.43 (br d,
1H, J3,4 3.0 Hz, H-4), 5.31 (dd, 1H, J2,3 10.3, H-3),
5.07 (d, 1H, H-1), 4.40–3.40 (m, 23H, H-2, H-5, H-6a,
H-6b, H-9Fmoc, a-CHSer, b-CH2Ser CH(CH2OC11H23)2,
2CH2OCH2C10H21, CH2Fmoc, OCH2CH2N), 2.15, 2.06,
2.01 (3s, 9H, 3CH3COO), 1.62–1.50 (m, 4H,
2OCH2CH2), 1.36–1.16 (m, 32H, 16CH2 alkyl chains),
0.88 (t, 6H, 2CH3 alkyl chains); 13C NMR (CDCl3): d
170.4, 170.0, 169.7, 168.9 (3CH3COO, 1CH2CON),
156.0 (NHCOOFmoc), 143.8, 141.3 (aromatic CFmoc),
127.8, 127.1, 125.1, 120.0 (aromatic CHFmoc), 98.5 (C-
1), 78.4 (OCH(CH2OC11H23)2), 71.7, 70.9, 68.9, 68.3,
(a-CHSer), 47.8 (C-2, C-9Fmoc), 40.1 (NHCH2CH2O),
32.3, 30.0, 29.9, 29.7, 29.6, 26.4, 23.1 (CH2 alkyl chains),
23.5 (CH3CON), 21.1, 21.0 (3CH3COO), 14.5 (CH3 alkyl
chains). Anal. Calcd for C59H91N3O15 (1082.37): C,
65.47; H, 8.47; N, 3.88. Found: C, 65.19; H, 8.50; N, 3.82.
3.23.2. Method b. A soln of 20b (0.106 g, 0.142 mmol)
in EtOAc (8 mL) was stirred for 18 h under hydrogen
(4 atm) in the presence of 10% Pd/C (58 mg). After fil-
tration and concentration, the intermediate 21b thus
obtained, was used directly in the next step. A soln of
EEDQ (0.023 g, 0.093 mmol) in CH2Cl2 (1 mL) was
added at 0 ꢁC to a soln of 21b (0.056 g, 0.085 mmol)
and 12 (0.045 g, 0.101 mmol) in CH2Cl2 (5 mL). Stirring
was maintained for 2 h at 0 ꢁC and then for 18 h at rt.
The mixture was diluted with CH2Cl2, washed with
5% aq NaCl, dried (MgSO4), concentrated and purified
by column chromatography (3:1 EtOAc–petroleum
ether) to afford product 19b (0.040 g, 43%) as an amor-
phous solid.
67.3 (CH(CH2OCH2C10H21)2, OCH2CH2NH, b-CH2Ser
,
CH2Fmoc) 68.4, 67.4, 67.1 (C-3, C-4, C-5), 61.6 (C-6),
57.7 (C-2), 54.5 (a-CHSer), 47.1 (C-9Fmoc), 40.0
(OCH2CH2NH), 30.0, 29.9, 29.7, 29.6, 26.4, 22.7 (CH2
alkyl chains), 21.0, 20.9, 20.7 (3CH3COO), 14.1 (CH3
alkyl chains).
3.23. 4-Undecyloxymethyl-3,6-dioxaheptadecyl O-(2-
acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-galactopyran-
osyl)-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-serinamide
(19b)
3.24. Benzyl O-(3,4,6-tri-O-acetyl-2-azido-2-deoxy-a-D-
galactopyranosyl)-N-[(9H-fluoren-9-ylmethoxy)-
carbonyl]-L-serinate (20a)
A
mixture of 3,4,6-tri-O-acetyl-2-azido-2-deoxy-D-
3.23.1. Method a. Compound 19b was prepared from
19a (0.177 g, 0.166 mmol), as described above for 16b
from 16a. After purification by column chromatography
(3:1 EtOAc–petroleum ether), 19b (0.085 g, 47%) was
obtained as an amorphous solid; [a]D +41.4 (c 1.0,
galactopyranosyl trichloroacetimidate (0.254 g, 0.530
mmol), benzyl N-[(9H-fluoren-9-ylmethoxy)carbonyl]-
L-serinate26 (0.492 g, 1.18 mmol) and crushed activated
˚
4 A molecular sieves (0.800 g) in 1:1 Et2O–CH2Cl2
(12 mL) was stirred under Ar and cooled to ꢀ30 ꢁC. A
soln of TMSOTf (38 lL, 0.197 mmol) in CH2Cl2
(0.1 mL) was added dropwise through a syringe and
the mixture was stirred for 30 min at ꢀ30 ꢁC. After addi-
tion of i-Pr2NEt (75 lL), the mixture was allowed to
reach rt. After dilution with CH2Cl2 (50 mL), filtration
over Celite, washing three times with 0.1 N aq HCl
(3 · 5 mL), then with water (5 mL), the organic phase
was dried (Na2SO4) and concentrated under diminished
pressure. After purification by column chromatography
(2:3 EtOAc–petroleum ether), product 20a (0.281 g,
72%) was obtained as an amorphous solid; [a]D +84 (c
1.0, CHCl3) [lit.29 [a]D +86.5 (c 1.0, CHCl3)]; 1H
NMR (CDCl3): selected values: d 7.72 and 7.63 (2d,
4H, J 7.5 Hz, aromatic HFmoc), 7.45–7.30 (m, 9H, 4 aro-
matic HFmoc, 5 aromatic HBn), 6.02 (d, 1H, J 8.1 Hz,
NHCOOFmoc), 5.41–5.39 (m, 1H, H-4), 5.27 (s, 2H,
CH2Ph), 4.88 (d, 1H, J1,2 3.2 Hz, H-1), 3.60 (dd, 1H,
J2,3 11.1 Hz, H-2), 2.16, 2.08, 1.97 (3s, 9H, 3CH3COO);
13C NMR (CDCl3): d 170.5, 170.0, 169.8 (3CH3CO),
163.6 (COOBn), 156.0 (NHCOOFmoc), 143.8, 141.3
(aromatic CFmoc), 135.0 (aromatic CBn), 128.7, 128.6,
127.8, 127.2, 125.2, 120.1 (aromatic CHFmoc, CHBn),
99.4 (C-1), 70.0, 67.9, 67.4 (CH2Fmoc, CH2Ph, b-CH2Ser),
68.0, 67.6, 67.3 (C-3, C-4, C-5), 61.8 (C-6), 57.5 (C-2),
54.6 (a-CHSer), 47.1 (C-9Fmoc), 20.7, 20.6 (3CH3COO).
1
CHCl3); Rf 0.30; H NMR (CDCl3): d 7.77 (d, 2H, J
7.5 Hz, aromatic HFmoc), 7.62 (d, 2H, J 7.3 Hz, aromatic
HFmoc), 7.41 (t, 2H, J 7.3 Hz, aromatic HFmoc), 7.33 (t,
2H, J 7.5 Hz, aromatic HFmoc), 7.18 (t, 1H, J 5.4 Hz,
CONH), 6.24 (d, 1H, JNH,2 9.6 Hz, NHCOCH3), 6.00
(d, 1H, J 7.4 Hz, NHCOOFmoc), 5.36 (br d, 1H, J3,4
3.3, J4,5 <1 Hz, H-4), 5.10 (dd, 1H, J2,3 11.0 Hz, H-3),
4.83 (d, 1H, J1,2 3.5 Hz, H-1), 4.58 (ddd, 1H, H-2),
4.41 (m, 3H, a-CHSer, NHCH2CH2O), 4.23 (t, 1H, J
6.9 Hz, H-9Fmoc), 4.13 (br t, 1H, J5,6a, J5,6b 6.6 Hz, H-
5), 4.08 (dd, 1H, J6a,6b 11.0 Hz, H-6a), 4.02 (dd, 1H,
H-6b), 3.95 (dd, 1H, J 3.5 Hz, J 10.1 Hz, b-CHSer),
3.78 (dd, 1H, b-CHSer), 3.72–3.66 (m, 2H, CH2Fmoc),
3.64 (quint, 1H, J 5.4 Hz, CH(CH2OC11H23)), 3.55–
3.49 (m, 2H, NHCH2CH2O), 3.43 (m, 8H, 2CH2O-
CH2C10H21), 2.15, 2.04, 2.02, 1.96 (4s, 12H, 3CH3COO,
1CH3CON), 1.62–1.48 (m, 4H, 2OCH2CH2C9H19),
1.34–1.18 (m, 32H, 16CH2 alkyl chains), 0.87 (t, 6H, J
6.9 Hz, 2CH3 alkyl chains); 13C NMR (CDCl3): d
171.1, 170.8, 170.7, 169.4 (4CH3CO, CH2CON), 156.3
(NHCOOFmoc), 144.1, 141.7 (aromatic CFmoc), 128.2,
127.5, 125.4, 120.5 (aromatic CHFmoc), 99.5 (C-1), 78.8
(CH(CH2OC11H23)2), 72.2, 71.3 (CH(CH2OCH2C10-
H21)2), 69.9 (b-CH2Ser), 69.1 (CH2Fmoc, C-3), 67.7,
67.6 (C-4, C-5) 67.6 (NHCH2CH2O), 62.2 (C-6), 55.0

N. Laurent et al. / Carbohydrate Research 341 (2006) 823–835
833
3.25. Benzyl O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
a-D-galactopyranosyl)-N-[(9H-fluoren-9-ylmethoxy)-
carbonyl]-L-serinate (20b)
3.27. 8-(Cholest-5-en-3b-yloxy)-3,6-dioxaoctyl 3-O-
(2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl)-2-azido-
4,6-O-benzylidene-2-deoxy-a-D-galactopyranoside (23)
Prepared from 20a (0.146 g, 0.20 mmol) as described
above for 16b. After purification by column chromato-
graphy (5:1 EtOAc–petroleum ether), 20b (0.106 g,
71%) was obtained as an amorphous solid; [a]D +47.5
(c 1.0, CHCl3) [lit.29 [a]D +60.0 (c 1.0, CHCl3)]; Rf 0.50;
1H NMR (CDCl3): selected values: d 7.65 (d, 2H, J
7.5 Hz, aromatic HFmoc), 7.61 (d, 2H, J 6.8 Hz, aromatic
HFmoc), 7.51–7.30 (m, 9H, 4 aromatic HFmoc, 5 aromatic
HBn), 6.01 (d, 1H, JNH,2 8.4 Hz, NHCOCH3), 5.80 (d,
1H, J 9.0 Hz, NHCOOFmoc), 5.33 (dd, 1H, J3,4 2.3, J4,5
<1 Hz, H-4), 5.20 (s, 2H, CH2Ph), 5.07 (dd, 1H, J2,3
11.4 Hz, H-3), 4.78 (d, 1H, J1,2 3.2 Hz, H-1); 13C NMR
(CDCl3): d 170.9, 170.4, 170.3, 170.3, 170.0 (4CH3CO,
COOBn), 155.9 (NHCOOFmoc), 143.7, 141.4 (aromatic
CFmoc), 135.0 (aromatic CBn), 128.9, 128.4, 127.9,
127.2, 125.0, 120.1 (aromatic CHFmoc), 99.1 (C-1), 69.8,
67.7, 67.3 (CH2Fmoc, CH2Ph, b-CH2Ser), 68.2, 67.3, 67.2
(C-3, C-4, C-5), 62.0 (C-6), 54.6 (a-CHSer), 47.6, 47.1
(C-2, C-9Fmoc), 23.2 (CH3CON), 20.7, 20.6 (3CH3COO).
A suspension of glycoside 18d (0.396 g, 0.500 mmol) and
2,3,4,6-tetra-O-acetyl-a-D-galactopyranosyl trichloro-
acetimidate30 (0.542 g, 1.10 mmol) in CH2Cl2 (8.0 mL)
was stirred for 1 h at rt in the presence of crushed acti-
˚
vated 4 A molecular sieves (0.370 g). After cooling to
ꢀ25 ꢁC, TMSOTf (23 lL, 0.119 mmol) was added; stir-
ring was maintained for 0.5 h at ꢀ25 ꢁC and the mixture
was then allowed to reach rt. After 1 h stirring at rt, the
mixture was neutralized by addition of i-Pr2NEt
(25 lL), filtrated, concentrated and purified by two suc-
cessive column chromatographies (1:1 EtOAc–petro-
leum ether, then 2:1 EtOAc–petroleum ether) to afford
compound 23 (0.356 g, 71%) as a colourless oil; [a]D
+50.3 (c 1.0, CHCl3); Rf 0.38 (2:1 EtOAc–petroleum
1
ether); H NMR (CDCl3): d 7.56–7.35 (m, 5H, C6H5),
0
0
0
0
5.57 (s, 1H, CHPh), 5.43 (br d, 1H, J3 ;4 3.5, J4 ;5
1.3 Hz, H-40), 5.37–5.35 (m, 1H, H-6chol), 5.31 (dd,
1H, J1 ;2 7.9, J2 ;3 10.4 Hz, H-20), 5.11 (d, 1H, J1,2
3.5 Hz, H-1), 5.05 (dd, 1H, H-30), 4.81 (d, 1H, H-10),
4.39 (dd, 1H, J3,4 3.1, J4,5 0.5 Hz, H-4), 4.27 (dd, 1H,
0
0
0
0
0
0
3.26. 4-Undecyloxymethyl-3,6-dioxaheptadecyl
O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-D-galacto-
pyranosyl)-L-serinamide (22b)
J5,6a 1.6 Hz, J6a,6b 12.3 Hz, H-6a), 4.22 (dd, 1H, J5 ;6 a
6.7 Hz, J6 a;6 b 10.9 Hz, H-60a), 4.18 (dd, 1H, J2,3
0
0
10.8 Hz, H-3), 4.17 (dd, 1H, J5 ;6 b 6.7 Hz, H-60b), 4.07
(dd, 1H, J5,6b 1.6 Hz, H-6b), 3.97 (ddd, 1H, H-50),
3.89 (ddd, 1H, 1/2 CH2OC-1), 3.85 (m, 1H, H-5), 3.84
(dd, 1H, H-2), 3.78 (ddd, 1H, 1/2 CH2OC-1), 3.76–
3.68 (m, 10H, 5OCH2), 3.23–3.19 (m, 1H, H-3chol),
2.17, 2.07, 2.06, 1.99 (4s, 12H, 4CH3COO), 2.41–0.67
(m, 43H, H cholesterol); 13C NMR (CDCl3): d 170.3,
170.2, 170.0, 169.4 (4CH3CO), 140.8 (C-5chol), 137.8,
128.8, 128.1, 126.2 (C6H5), 121.6 (C-6chol), 102.4 (C-
10), 100.6 (CHC6H5), 98.9 (C-1), 79.5 (C-3chol), 76.2
(C-3, C-4), 71.5 (C-30), 71.2 (C-50), 71.4, 71.1, 71.0,
70.7 (OCH2), 69.4 (C-6), 68.0 (CH2OC-1), 67.7 (CH2O-
Chol), 69.1 (C-20), 67.4 (C-40), 63.5 (C-5) 61.8 (C-60),
59.30 (C-2), 56.8 (C-14chol), 56.1 (C-17chol), 50.2 (C-
0
0
Compound 19b (0.060 g, 0.055 mmol) was reacted for
45 min with freshly distilled morpholine (0.8 mL). After
evaporation and coevaporation from Et2O, the residue
was diluted with CH2Cl2 and the organic layer was
washed with brine, before drying (Na2SO4). After con-
centration under diminished pressure and purification
by column chromatography (20:1 CHCl3–MeOH),
product 22b (0.036 g, 76%) was recovered as an amor-
phous solid; [a]D +47.6 (c 1.0, CHCl3); Rf 0.35; 1H
NMR (CDCl3): d 7.75–7.71 (m, 1H, NHCO), 6.35 (d,
1H, JNH,2 9.6 Hz, NH), 5.37 (dd, 1H, J3,4 2.5, J4,5
<1 Hz, H-4), 5.09 (dd, 1H, J2,3 11.3 Hz, H-3), 4.88 (d,
1H, J1,2 3.5 Hz, H-1), 4.61 (ddd, 1H, H-2), 4.25–4.20
(m, 1H, a-CHSer), 4.15–4.08 (m, 2H, OCH2CH2N),
9
chol), 42.4 (C-13chol), 39.8 (C-12chol), 39.1 (C-24chol),
39.1 (C-4chol), 37.2 (C-1chol), 36.9 (C-10chol), 36.2 (C-
22chol), 35.8 (C-20chol), 32.0 (C-7chol), 31.9 (C-8chol),
28.4 (C-2chol), 28.3 (C-16chol), 28.0 (C-25chol), 24.3 (C-
15chol), 23.9 (C-23chol), 22.9 (C-27chol), 22.6 (C-26chol),
21.1 (C-11chol), 20.7, 20.5 (4CH3COO), 19.4 (C-19chol),
18.7 (C-21chol), 11.9 (C-18chol). Anal. Calcd for
C60H89N3O17Æ1.5H2O (1151.354): C, 62.58; H, 8.05; N,
3.64. Found: C, 62.42; H, 7.72; N, 3.30.
3.93–3.40 (m, 16H, H-5, H-6a, H-6b, b-CH2Ser
CH(CH2OC11H23)2, NHCH2CH2O, 2CH2OCH2-
,
C10H21), 2.16, 2.06, 2.05, 1.98 (4s, 12H, 3CH3COO,
CH3CON), 1.84 (2s, 2H, NH2), 1.60–1.48 (m, 4H,
2OCH2CH2C9H19), 1.35–1.18 (m, 32H, 16CH2 alkyl
chains), 0.88 (t, 6H, J 6.0 Hz, 2CH3 alkyl chains); 13C
NMR (CDCl3): d 170.8, 170.5, 170.4 (4CH3CO, NHCO-
Ser), 98.9 (C-1), 78.2 (CH(CH2OC11H23)2), 71.9, 71.8,
70.9, 68.9 (CH(CH2OCH2C10H21)2, NHCH2CH2O,
b-CH2Ser), 68.6, 67.3, 67.2 (C-3, C-4, C-5), 61.6 (C-6),
55.0 (a-CHSer), 47.7 (C-2), 39.4 (NHCH2CH2O), 32.3,
30.0, 29.9, 29.7, 29.6, 26.4, 22.7 (CH2 alkyl chains),
23.1 (CH3CON), 20.8 (3CH3COO), 14.1 (CH3 alkyl
chains).
3.28. 8-(Cholest-5-en-3b-yloxy)-3,6-dioxaoctyl 3-O-
(2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl)-2-azido-2-
deoxy-a-D-galactopyranoside (24)
Ethanethiol (0.72 mL, 3.63 mmol) and borontrifluoride
etherate (17 lL, 0.134 mmol) were successively added

834
N. Laurent et al. / Carbohydrate Research 341 (2006) 823–835
0
0
0
0
at 0 ꢁC to
a
soln of disaccharide 23 (0.400 g,
<1.0 Hz, H-4), 5.35 (br d, 1H, J3 ;4 3.2, J4 ;5 0.9 Hz,
H-40), 5.345–5.33 (m, 1H, H-6chol), 5.10 (dd, 1H, J1 ;2
0
0
0.356 mmol) in dry CH2Cl2 (5.6 mL). The mixture was
stirred for 3 h at 0 ꢁC and neutralized by addition of
satd aq NaHCO3 (0.1 mL). The organic phase was dried
(Na2SO4) and concentrated. The residue was then puri-
fied by column chromatography (EtOAc) to afford diol
24 (0.274 g, 74%) as a colourless oil; [a]D +33.8 (c 1.0,
CHCl3); Rf 0.37 (5:1 EtOAc–petroleum ether); 1H
7.9, J2 ;3 10.4 Hz, H-20), 4.92 (dd, 1H, H-30), 4.85 (d,
0
0
1H, J1,2 3.5 Hz, H-1), 4.59 (d, 1H, H-10), 4.57 (ddd,
0
0
0
0
1H, J2,3 10.6 Hz, H-2), 4.17 (dd, 1H, J5 ;6 a 6.0, J6 a;6 b
11.0 Hz, H-60a), 4.16–4.13 (m, 2H, H-6a,60b), 4.11 (br
dd, 1H, J5,6a 6.0, J5,6b 6.5 Hz, H-5), 4.00 (dd, 1H,
J6a,6b 9.8 Hz, H-6b), 3.98 (dd, 1H, H-3), 3.87 (br dd,
1H, J5 ;6 b 6.5 Hz, H-50), 3.81–3.77 (m, 1H, 1/2
CH2OC-1), 3.70–3.61 (m, 11H, 1/2 CH2OC-1, OCH2-
CH2(OCH2CH2)2), 3.21–3.19 (m, 1H, H-3chol), 2.16,
2.13, 2.05, 2.04, 1.99, 1.96, 1.95 (7s, 21H, 6CH3COO,
CH3CON), 2.42–0.67 (m, 43H, H cholesterol); 13C
NMR (CDCl3): d 170.6, 170.3, 170.2, 170.0, 169.6
(7CH3CO), 140.6 (C-5chol), 121.9 (C-6chol), 101.5 (C-
10), 98.4 (C-1), 79.6 (C-3chol), 72.9 (C-3), 70.8 (C-30),
70.6 (C-50), 70.7, 70.5, 70.4, 70.1, 67.1, 66.8
((OCH2CH2)3), 69.2 (C-4), 68.7 (C-20), 67.5 (C-5), 67.1
(C-40), 62.9 (C-6), 61.0 (C-60), 56.8 (C-14chol), 56.2 (C-
17chol), 50.2 (C-9chol), 48.7 (C-2), 42.4 (C-13chol), 39.8
(C-12chol), 39.5 (C-24chol), 39.0 (C-4chol), 37.2 (C-1chol),
36.9 (C-10chol), 36.2 (C-22chol), 35.8 (C-20chol), 31.9 (C-
0
0
0
0
0
0
NMR (CDCl3): d 5.41 (br d, 1H, J3 ;4 3.2, J4 ;5 0.6 Hz,
H-40), 5.35–5.33 (m, 1H, H-6chol), 5.30 (dd, 1H, J1 ;2
0
0
7.8, J2 ;3 10.4 Hz, H-20), 5.04 (dd, 1H, H-30), 5.02 (d,
1H, J1,2 3.2 Hz, H-1), 4.74 (d, 1H, H-10), 4.18–3.64 (m,
21H, H-2, H-3, H-4, H-5, H-6a, H-6b, H-50, H-60a,
H-60b, (OCH2CH2)3), 3.20–3.18 (m, 1H, H-3chol), 2.17,
2.09, 2.07, 2.00 (4s, 12H, 4CH3COO), 2.41–0.67 (m,
43H, H cholesterol); 13C NMR (CDCl3): d 170.4,
170.1, 170.0, 169.60 (4CH3CO), 140.8 (C-5chol), 121.6
(C-6chol), 101.9 (C-10), 98.9 (C-1), 79.5 (C-3chol), 78.5
(C-3), 71.2, 70.7 (C-30, C-50), 70.8, 70.7, 70.4, 67.4,
67.2 ((OCH2CH2)3), 69.7, 68.9, 68.5 (C-4, C-5, C-20),
67.0 (C-40), 62.2 (C-6), 61.5 (C-60), 58.7 (C-2), 56.8 (C-
14chol), 56.2 (C-17chol), 50.2 (C-9chol), 42.3 (C-13chol),
39.8 (C-12chol), 39.5 (C-24chol), 39.0 (C-4chol), 37.2 (C-
0
0
7
chol), 31.8 (C-8chol), 28.4 (C-2chol), 28.2 (C-16chol), 28.0
1
chol), 36.9 (C-10chol), 36.2 (C-22chol), 35.8 (C-20chol),
(C-25chol), 24.3 (C-15chol), 23.8 (C-23chol), 23.2
(CH3CON), 22.8 (C-27chol), 22.6 (C-26chol), 21.1 (C-
11chol), 20.8, 20.7, 20.5 (6CH3COO), 19.4 (C-19chol),
18.7 (C-21chol), 11.9 (C-18chol). Anal. Calcd for
C59H93NO20Æ1.5H2O (1163.364): C, 60.91; H, 8.32; N,
1.19. Found: C, 60.77; H, 8.32; N, 1.10.
31.9 (C-7chol), 31.9 (C-8chol), 28.3 (C-2chol), 28.2 (C-
16chol), 28.0 (C-25chol), 24.3 (C-15chol), 23.8 (C-23chol),
22.8 (C-27chol), 22.6 (C-26chol), 21.1 (C-11chol), 20.6,
20.5 (4CH3COO), 19.4 (C-19chol), 18.7 (C-21chol), 11.9
(C-18chol). Anal. Calcd for C53H85N3O17Æ1.5H2O
(1063.255): C, 59.87; H, 8.34; N, 3.96. Found: C,
59.92; H, 8.23; N, 3.76.
References
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