Synthesis of new galactosyl and lactosyl carbamate-containing glycolipids

Article abstract of DOI:10.1081/CAR-100108661

An efficient synthesis of mono-, di- and tetrasaccharides linked to a lipid has been developed. Galactose or lactose was covalently coupled to a glycyldioctadecylamide, via well-defined chemical steps, in both an α and β anomeric configuration. The multia

Full text of DOI:10.1081/CAR-100108661

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SYNTHESIS OF NEW GALACTOSYL AND LACTOSYL
CARBAMATE-CONTAINING GLYCOLIPIDS
Michel Azoulay a , Virginie Escriou b , Jean-Claude Florent a & Claude Monneret a
a Section de Recherche , UMR 176 CNRS-Institut Curie , 26 rue d'Ulm, Paris, Cedex 05,
F-75248, France
b CRVA, UMR 7001 CNRS-Aventis-Gencell , 13 Quai Jules Guesde, B.P. 14, Vitry-sur-Seine,
F-94403, France
Published online: 19 Aug 2006.
To cite this article: Michel Azoulay , Virginie Escriou , Jean-Claude Florent & Claude Monneret (2001) SYNTHESIS OF NEW
GALACTOSYL AND LACTOSYL CARBAMATE-CONTAINING GLYCOLIPIDS, Journal of Carbohydrate Chemistry, 20:9, 841-853
To link to this article: http://dx.doi.org/10.1081/CAR-100108661
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J. CARBOHYDRATE CHEMISTRY, 20(9), 841–853 (2001)
SYNTHESIS OF NEW GALACTOSYL AND
LACTOSYL CARBAMATE-CONTAINING
GLYCOLIPIDS
Michel Azoulay,1 Virginie Escriou,2 Jean-Claude Florent,1
and Claude Monneret1
1UMR 176 CNRS-Institut Curie, Section de Recherche, 26 rue d’Ulm,
F-75248 Paris Cedex 05, France
2UMR 7001 CNRS-Aventis-Gencell, CRVA, 13 Quai Jules Guesde,
B.P. 14, F-94403 Vitry-sur-Seine, France
ABSTRACT
An efficient synthesis of mono-, di- and tetrasaccharides linked to a lipid has
been developed. Galactose or lactose was covalently coupled to a glycyldioc-
tadecylamide, via well-defined chemical steps, in both an ꢀ and ꢁ anomeric
configuration. The multiantennary galactosyl ligands were obtained using 1,3-
diamino-2-propanol as a scaffold.
INTRODUCTION
Gene therapy is a promising and rapidly growing field of medical research di-
rected towards correcting genetic or somatic disorders.1 One of the major obstacles
in gene therapy consists in finding methods which allow for specific and efficient
delivery of the therapeutic genes. Besides the viruses, cationic liposomes are at-
tractive systems for use in gene delivery. Non-viral vectors represent safe and ef-
ficient gene transfer agents2 which, unlike viral vectors, do not elicit immune re-
sponses.3 However, the non-viral gene carriers lack cell specificity, thus limiting
their in vivo application. To solve this problem, cell targeting ligands were intro-
duced to synthetic vectors4 based on the concept of receptor-mediated endocyto-
sis.5 Hepatocytes exclusively express large numbers of high affinity cell-surface
841
Copyright © 2002 by Marcel Dekker, Inc.
www.dekker.com

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AZOULAY ET AL.
receptors that bind asialoglycoproteins (ASGP-R) and subsequently internalize
them to the cell interior.6 Selective transfection of hepatocytes via ASGP-R has
been accomplished with a ligand-possessing galactose residue such as asialooro-
somucoid,7 galactosylated proteins or polymer8 and galactosylated synthetic lig-
ands.9
A study of available data agrees with the fact that human hepatocytes have a
membrane lectin recognizing glycoproteins terminated by a ꢁ-D-galactose
residue.10 Moreover, recent results11 indicate that the substitution of this ꢁ-D-
galactosyl residue, coupled to polyethylenimine (PEI) to give a linear tetragalac-
tose Galꢀ3-Galꢁ4-Galꢀ3-Galꢁ-PEI, improves the gene transfer into hepatocytes.
In the same report, it was also observed that the terminal ꢀ-galactosyl residue is
easily accessible and recognized by using a galactose-binding RCA120 lectin-me-
diated agglutination.
RESULTS AND DISCUSSION
The aim of the work reported here was to determine if the ꢀ or ꢁ anomeric
configuration of the galactose moiety plays an essential role in the binding with the
lectin-mediated gene transfer into hepatocytes. To address this question, we un-
dertook the synthesis of six new glycolipids (1 a, 1 b, 2 a, 2 b, 3, 4) and subse-
quently investigated their potentiality for a specific and efficient targeting of hep-
atocytes.
In this paper, we describe the synthesis of glycolipids having a single ꢀ or ꢁ
galactosyl unit 1 a or 1 b (for lactose 2 a, 2 b) linked to a glycine nitrogen via a car-
bamate function, with the glycine carboxyl group protected as an N,N-dialky-
lamide. We were interested in the development of a carbamate-linked galactose to
insure better stability towards glycosidases.
The first two glycolipids were prepared according to reaction steps depicted
in Scheme 1.
Compound 6 was prepared by reaction between benzyloxylcarbonyl-glycyl-
p-nitrophenyl ester 5 and dioctadecylamine as reported.12 Then, 6 was hy-
drogenolysed for 48 h at atmospheric pressure to remove the benzyloxycarbonyl
protective group (Z), giving the glycyldioctadecylamide 712 in good yield.
In regard to preparation of the galactosyl unit of the target lipids, galactose 8
was first peracetylated13 and the pentaacetyl galactose 9 was selectively depro-
tected at the anomeric position using hydrazine acetate14 in order to give the
2,3,4,6-tetra-O-acetyl-D-galactopyranose 10, as inseparable mixture of anomers.

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MULTIANTENNARY GALACTOSYL LIGANDS
843
Scheme 1. Reagents and conditions: a) CH2Cl2, dioctadecylamine, Et3N, 40°C, 45%; b)
CH2Cl2/EtOH (1:1), 10% Pd/C, H2, 85%; c) CH2Cl2, 4-nitrophenylchloroformate, pyridine, 0°C,
80% (anomeric ratio ꢀ/ꢁ, 4/1); d) DME, 4-nitrophenylchloroformate, NaH, 65% (anomeric ratio ꢀ/ꢁ,
1/2); e) CH2Cl2, glycyldioctadecylamide 7, Et3N, 75% from 11 a, 70% from 11 b; f) CH3OH,
NaOMe 1M, 75% from 12 a, 69% from 12 b. Z ꢂ benzyloxycarbonyl, pNP ꢂ para-nitrophenyl.
Crude 10 was then treated with 4-nitrophenylchloroformate under two different
conditions. First, with dimethylformamide (DMF) at 0°C in the presence of pyri-
dine, the ꢀ-anomer was afforded with a good diastereoselectivity (molar ratio of
ꢀ/ꢁ, 4/1). In order to enhance ꢁ-anomer formation, we used the conditions as de-
scribed by Klotz and Schmidt15—1,2-dimethoxyethane (DME) in the presence of
sodium hydride at room temperature. As the anomeric diastereocontrol is temper-
ature-dependent when carried out at room temperature, the ꢁ-anomer was prefer-
entially obtained (molar ratio of ꢀ/ꢁ, 1/2). At this stage, the ꢀ and ꢁ anomers 11 a,
b were separated by flash chromatography. The carbamate 12 a was obtained by
reaction between 4-nitrophenyl 2,3,4,6-tetra-O-acetyl-ꢀ-D-galactopyranosyl car-
bonate 11 a and compound 7. Transesterification of 12 a with sodium methoxide
in methanol led to the desired compound 1 a. The carbamate 1 b was prepared by
the same procedure from the ꢁ-activated glycoside 11b.
The synthesis of the next two glycolipids 2 a, b was also accomplished by a
similar reactional sequence described in Scheme 2, using lactose 13 as starting ma-
terial.
Preparations of the corresponding carbonate-activated lactose 16 a, b were
performed with low diastereoselectivity using the same conditions as previously

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AZOULAY ET AL.
described (DMF, 0°C, pyridine or DME, rt, NaH) since the molar ratios of ꢁ/ꢀ
were 1/2 and 1/1, respectively.
Numerous studies have established that individual protein-sugar interactions
are weak (Kd, mM). To overcome this restriction, many processes mediated by
oligosaccharide-lectin interactions involve multivalent binding.16 For example,17
the specificity for terminal galactose residue, binding to the ASGP-receptor
strongly depends on the oligosaccharide structure: mono-, bi- and tetraantennary
galactose-terminal oligosaccharides bind with increasing affinity (Kd, from mM to
nM). In agreement with these data, indicating that oligomeric structures are re-
quired for efficient binding to a galactose receptor, we decided to prepare the bi-
and tetraantennary galactosyl lipids 3, 4 as described in Scheme 3.
The biantennary glycolipid 3 was obtained by linking the glycosyl carbonate
11 a with the commercially available 1,3-diamino-2-propanol to give the bianten-
nary galactosylated compound 18. The free alcohol of this molecule was activated
with 4- nitrophenylchloroformate before coupling with glycyldioctadecylamide 7
to yield 20. The last step to give 3 was the removal of the acetyl groups by brief
treatment with the Zemplén method. The tetraantennary galactosyl lipid 4 was ob-
tained following a similar reactional sequence involving condensation of a second
molecule of 1,3-diamino-2-propanol with the bis-galactosyled carbonate deriva-
tive 19 to give first the tetraantennary compound 21 which was converted to the
carbonate 22. Coupling of 22 with compound 7 yielded the corresponding glycol-
ipid 23, which was then deacetylated under Zemplén conditions to give the final
product.
Scheme 2. Reagents and conditions: a) CH2Cl2, 4-nitrophenylchloroformate, pyridine, 0°C, 72%
(anomeric ratio ꢀ/ꢁ, 2/1); b) DME, 4-nitrophenylchloroformate, NaH, 55% (anomeric ratio ꢀ/ꢁ,
1/1); c) CH2Cl2, 7, Et3N, 65% from 16 a, 60% from 16 b; d) CH3OH, NaOMe 1M, 70% from 17 a,
60% from 17 b.

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MULTIANTENNARY GALACTOSYL LIGANDS
845
Scheme 3.
CONCLUSION
The synthetic procedure described here exploited converting the free
anomeric hydroxyl group of an otherwise protected glycopyranosyl residue into
the corresponding activated ꢀ or ꢁ carbonate for coupling with diaminopropanol
as a scaffold to provide an entry for the synthesis of a multiantennary compound.
In summary, we succeeded in the preparation of artificial glycolipids involving a
single ꢀ or ꢁ galactosyl or lactosyl unit, as well as the synthesis of bi- or tetraan-
tennary-ꢀ-galactosyl lipids. These compounds offer the advantage of being fully
synthetic, rapidly prepared and easily purified. The lectin recognition and trans-
fection potential of these new galactosyl lipids are under investigation and results
regarding this aspect will be forthcoming.
EXPERIMENTAL
General Methods. Thin-layer chromatography (TLC) was performed on
silica gel 60F254 (Merck) and visualized first with light, and second by heating af-
ter alcoholic sulfuric or phosphomolybdic acid treatment. Column chromatogra-
phy was performed on SiO2 (Merck, particle size 0.004–0.063 nm) using the flash

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AZOULAY ET AL.
chromatography technique. Optical rotations were determined with a Perkin-Elmer
241 polarimeter (589 nm) at 20°C with a concentration expressed in g/100 mL. For
mass spectra, CI (NH3) were recorded with a Nermag R10-10C, FAB with a Jeol
MS-700 and MALDI/Tof with a Voyager (Applied Biosystems) using reflector
mode and 2,5-dihydroxybenzoic acid (DHB) as the matrix. 1H NMR spectra were
recorded using a Bruker AC-300 (300 MHz). Chemical shifts are expressed in ppm
downfield from internal Me4Si with notation indicating the multiplicity of the sig-
nal. For the NMR assignments, galactose atoms are noted 1–6, those of glucose 1ꢃ-
6ꢃ. Compounds 6, 7;12 9, 14;13 1014,18 and 1519 were prepared as described in the
literature.
4-Nitrophenyl 2,3,4,6-tetra-O-acetyl-ꢀ-D-galactopyranosyl carbonate
(11 a) and 4-Nitrophenyl 2,3,4,6-tetra-O-acetyl-ꢁ-D-galactopyranosyl carbon-
ate (11 b).
First procedure: To a cooled solution of 10 (1.26 g, 3.33 mmol) in CH2Cl2
(50 mL) were successively added 4-nitrophenyl chloroformate (1 g, 5 mmol) and
pyridine (0.44 mL, 5 mmol). After stirring for 4 h at 0°C, the crude mixture was
extracted with additional CH2Cl2 (ꢀ150 mL) and the organic layer was washed
with water and brine. Evaporation of solvent, followed by flash chromatography
(cyclohexane-EtOAc, 2:1) afforded 11 a as a crystalline compound (1.13 g, 66%);
mp 67°C; [ꢀ]D ꢄ103 (c 1, CHCl3); 1H NMR (CDCl3) ꢅ 2.01, 2.05, 2.06, 2.16 (4s,
4 ꢆ 3H, OAc), 4.14 (d, 2H, J ꢂ 6.6 Hz, H-6a, H-6b), 4.45 (m, 1H, H-5), 5.40 (m,
2H, H-2, H-3), 5.55 (m, 1H, H-4), 6.32 (d, 1H, J1,2 ꢂ 3 Hz, H-1), 7.40 (d, 2H,
J ꢂ 10 Hz, Ar), 8.30 (d, 2H, J ꢂ 10 Hz, Ar), and 11 b as a colourless oil (0.28 g,
1
16%); [ꢀ]D ꢄ55 (c 1.3, CHCl3), H NMR (CDCl3) ꢅ 1.99, 2.01, 2.05, 2.16 (4s,
4 ꢆ 3H, OAc), 4.15 (m, 3H, H-5, H-6a, H-6b), 5.10 (dd, 1H, J3,2 ꢂ 4 Hz, J3,4 ꢂ
10 Hz, H-3), 5.40 (m, 2H, H-2, H-4), 5.60 (d, 1H, J1,2 ꢂ 8.1 Hz, H-1), 7.44 (d, 2H,
J ꢂ 10 Hz, Ar), 8.33 (d, 2H, J ꢂ 10 Hz, Ar).
Second procedure: To a solution of 10 (0.52 g, 1.5 mmol) in dry DME (20
mL) at room temperature, NaH (42 mg, 1.65 mmol, 96% suspension in paraffin oil)
was added. After 15 min, the 4-nitrophenyl chloroformate (450 mg, 2.25 mmol)
was added and stirring was continued for 15 h. The reaction mixture was filtered
through silica and the silica was washed with ethyl acetate (50 mL). The clear so-
lution was washed with saturated NaCl (3 ꢆ 75 mL), dried with MgSO4, concen-
trated and then purified by flash chromatography (cyclohexane-ethyl acetate, 2:1);
11 a (160 mg, 20%) and 11 b (340 mg, 44%) were obtained, respectively.
N-(2,3,4,6-Tetra-O-acetyl-ꢀ-D-galactopyranosyloxycarbonyl)-L-glycyl-
dioctadecylamide (12 a). To a solution of 11 a (980 mg, 1.9 mmol) in CH2Cl2
(50 mL) were successively added glycyldioctadecylamide (650 mg, 2.8 mmol) and
Et3N (400 ꢇL, 2.8 mmol). The mixture was stirred at room temperature for 8 h,
then concentrated under reduced pressure. Water was added, and the aqueous
phase was extracted with ethyl acetate. The combined organic layers were dried,
filtered, concentated and purified by flash chromatography (cyclohexane-ethyl ac-
etate, 6:4) to give 12 a (1360 mg, 75%) as an oil; [ꢀ]D ꢄ 2 (c 0.8, CHCl3), 1H NMR

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MULTIANTENNARY GALACTOSYL LIGANDS
847
(CDCl3) ꢅ 0.88 (t, 2 ꢆ 3H, CH3), 1.20 (br s, 60H, MCH2M), 1.60 (br s, 4H,
CH2MCH3), 2.00, 2.04, 2.05, 2.16 (4s, 4 ꢆ 3H, OAc), 3.25 (t, 2H, CH2MN), 3.35
(t, 2H, CH2MN), 4.00 (m, 2H, NHMCH2MCO), 4.10 (m, 2H, H-6a, H-6b), 4.35
(t, 1H, H-5), 5.33 (m, 2H, H-2, H-3), 5.50 (m, 1H, H-4), 6.00 (t, 1H, NH), 6.30 (d,
1H, J ꢂ 3 Hz, H-1); Positive FAB-MS: m/z 953 (M ꢄ H)ꢄ, 975 (M ꢄ Na)ꢄ.
N-(ꢀ-D-Galactopyranosyloxycarbonyl)-L-glycyldioctadecylamide (1 a).
To a cooled solution of 12 a (320 mg, 0.34 mmol) in 10 mL of a CH2Cl2MCH3OH
mixture (2:1), 30 mg of sodium methoxide powder were added. The solution
was stirred for 4 h, then neutralized with IRC 50-S Amberlite and filtered. The
filtrate was concentrated to an oily residue and purified by chromatography
(CH2Cl2MMeOH, 9:1) to give 1 a (200 mg, 75%) as a white foam; [ꢀ]D ꢄ 55
(c 0.2, CHCl3), 1H NMR (CDCl3MCD3OD) ꢅ 0.88 (t, 2 ꢆ 3H, CH3), 1.20 (br s,
60H, MCH2M), 1.50 (br s, 4H, CH2MCH3), 2.50 (br s, 2H, OH), 3.15 (br s, 2H,
CH2MN), 3.30 (br s, 2H, CH2MN), 4.10 (m, 6H, H-2, H-3, H,4, H-5, H-6a, H-6b),
4.95 (br s, 1H, OH), 5.24 (br s, 1H, OH), 6.10 (br s, 1H, H-1); Positive FAB-MS:
m/z 785 (M ꢄ H)ꢄ.
Anal. Calcd for C45H88N2O8: C, 68.83; H, 11.38; N, 3.57. Found: C, 68.99;
H, 11.76; N, 3.25.
N-(2,3,4,6-Tetra-O-acetyl-ꢁ-D-galactopyranosyloxycarbonyl)-L-glycyl-
dioctadecylamide (12 b). Compound 12b was obtained from 11 b (870 mg, 1.7
mmol) as described for the preparation of 12 a and isolated as an oil in 70% yield
after flash chromatography (cyclohexane-ethyl acetate, 1:1); [ꢀ]D ꢄ32 (c 1,
1
CHCl3), H NMR (CDCl3) ꢅ 0.86 (t, 2 ꢆ 3H, CH3), 1.22 (br s, 60H, MCH2M),
1.43 (br s, 4H, CH2MCH3), 1.99, 2.04, 2.05, 2.17 (4s, 4 ꢆ 3H, OAc), 3.12 (br t,
2H, CH2MN), 3.30 (br t, 2H, CH2MN), 4.00 (d, 2H, J ꢂ 4 Hz, CH2MCO), 4.15
(m, 3H, H-5, H-6a, H-6b), 5.09 (dd, 1H, J3,2 ꢂ 3.5 Hz, J3,4 ꢂ 10.3 Hz, H-3), 5.30
(m, 2H, H-2, H-4), 5.60 (d, 1H, J ꢂ 8.2 Hz, H-1), 6.05 (m, 1H, NH); Positive FAB-
MS: m/z 953 (M ꢄ H)ꢄ.
N-(ꢁ-D-Galactopyranosyloxycarbonyl)-L-glycyldioctadecylamide (1 b).
Compound 1b was prepared in 69% yield by deprotection of 12 b (950 mg, 1
mmol) as described for 1 a; [ꢀ]D ꢄ 3 (c 2.2, CHCl3), 1H NMR (CDCl3MCD3OD)
ꢅ 0.88 (t, 2 ꢆ 3H, CH3), 1.26 (br s, 60H, MCH2M), 1.50 (br s, 4H, CH2MCH3),
2.42 (br s, 2H, OH), 3.15 (br s, 2H, CH2MN), 3.25 (br s, 2H, CH2MN), 3.80 (m,
6H, H-2, H-3, H-4, H-5, H-6a, H-6b), 4.80 (br s, 1H, OH), 5.14 (br s, 2H, OH, NH),
5.42 (br d, 1H, J ꢂ7 Hz, H-1), 7.10 (br s, 1H, NH); Positive FAB-MS: m/z 785 (M
ꢄ H)ꢄ, 807 (M ꢄ Na)ꢄ.
Anal. Calcd for C45H88N2O8: C, 68.83; H, 11.38; N, 3.57. Found: C, 69.23;
H, 11.67; N, 3.52.
4-Nitrophenyl-[(2,3,4,6-tetra-O-acetyl-ꢁ-D-galactopyranosyl)-(1→4)-
(2,3,6-tri-O-acetyl-ꢀ-D-glucopyranosyl)] carbonate (16 a) and 4-Nitrophenyl-
[(2,3,4,6-tetra-O-acetyl-ꢁ-D-galactopyranosyl)-(1→4)-(2,3,6-tri-O-acetyl-ꢁ-D-

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AZOULAY ET AL.
glucopyranosyl)] carbonate (16 b). Both compounds were obtained from 15 as
described for the preparation of 11 a and 11 b.
By the first procedure: 16 a was obtained in 48% yield after purification as
white solid; mp 143–144°C; [ꢀ]D ꢄ16 (c 3.5, CHCl3), 1H NMR (CDCl3) ꢅ 1.98,
2.02, 2.06, 2.07, 2.13, 2.14 (7s, 7 ꢆ 3H, OAc), 3.90 (m, 2H, H-5, H-4ꢃ), 4.15 (m,
4H, H-6ꢃa, H-5ꢃ, H-6a, H-6b), 4.51 (m, 2H, H-1, H-6ꢃb), 5.00 (dd, 1H, J3ꢈ2 ꢂ 10.25
Hz, J3–4 ꢂ 3.1 Hz, H-3), 5.10 (m, 2H, H-2ꢃ, H-2), 5.35 (br d, 1H, H-4ꢃ), 5.50 (t, 1H,
J3ꢃ-4ꢃ ꢂ 9.2 Hz, H-3ꢃ), 6.22 (d, 1H, J ꢂ 3.6 Hz, H-1ꢃ), 7.45 (d, 2H, J ꢂ 8 Hz, Ar),
8.30 (d, 2H, J ꢂ 8 Hz, Ar); CI (NH3)-MS: m/z 802 (M ꢄ H)ꢄ, 819 (M ꢄ NH4)ꢄ.
16 b was obtained in 24% yield as an oil after chromatography; [ꢀ]D ꢄ52 (c 0.4,
CHCl3), 1H NMR (CDCl3) ꢅ 1.98, 2.06, 2.08, 2.09, 2.10, 2.14, 2.17 (7s, 7 ꢆ 3H,
OAc), 3.90 (m, 3 H, H-5, H-5ꢃ, H-4ꢃ), 4.10 (m, 3H, H-6a, H-6b, H-6ꢃa), 4.50 (m,
2H, H-1, H-6ꢃb), 5.00 (dd, 1H, J3–4 ꢂ 3.5 Hz, J3ꢈ2 ꢂ 10.4 Hz, H-3), 5.15 (m, 2H,
H-2, H-2ꢃ), 5.30 (t, 1H, J ꢂ 8 Hz, H-3), 5.38 (br d, 1H, J ꢂ 3 Hz, H-4), 5.65 (d, 1H,
J ꢂ 8 Hz, H-1ꢃ), 7.45 (d, 2H, J ꢂ 8 Hz, Ar), 8.30 (d, 2H, J ꢂ 8 Hz, Ar).
By the second procedure: Both 16 a and 16 b were obtained in 24% yield.
N-[(2,3,4,6-Tetra-O-acetyl-ꢁ-D-galactopyranosyl)-(1→4)-(2,3,6-tri-O-
acetyl-ꢀ-D-glucopyranosyloxycarbonyl)]-L-glycyldioctadecylamide (17 a).
To a solution of 16 a (300 mg, 0.37 mmol) in dry dichloromethane (20 mL) were
added glycyldioctadecylamide (130 mg, 0.55 mmol) and Et3N (80 ꢇL, 0.56
mmol). The mixture was stirred at rt for 6 h, then concentrated under reduced
pressure. Water was added and the aqueous phase was extracted with CH2Cl2.
The combined organic phases were dried over MgSO4, filtered and concentrated.
Purification by flash chromatography (cyclohexane-ethyl acetate, 2:1) afforded
1
17 a (300 mg, 65%); [ꢀ]D ꢄ31 (c 1.5, CHCl3), H NMR (CDCl3) ꢅ 0.88 (t, 2
ꢆ 3H, CH3), 1.22 (br s, 60H, MCH2M), 1.96, 2.03, 2.04, 2.05, 2.06, 2.12, 2.15
(7s, 7 ꢆ 3H, OAc), 3.12 (br t, 2H, CH2MN), 3.30 (br t, 2H, CH2MN), 3.85 (m,
3H, H-5, H-5ꢃ, H-4ꢃ), 4.10 (m, 5H, NHMCH2MCOM, H-6a, H-6b, H-6ꢃa), 4.50
(m, 2H, H-1, H-6ꢃb), 5.00 (m, 2H, H-2, H-3), 5.10 (dd, 1H, J2ꢃꢈ1ꢃ ꢂ 3 Hz, J2ꢃ–3ꢃ
ꢂ 10 Hz, H-2ꢃ), 5.35 (m, 1H, H-4), 5.40 (t, 1H, J ꢂ 9.8 Hz, H-3ꢃ), 6.05 (br t,
1H, NH), 6.17 (d, 1H, J ꢂ 3.5 Hz, H-1ꢃ); Positive FAB-MS: m/z 1241 (M
ꢄ H)ꢄ, 1263 (M ꢄ Na)ꢄ.
N-[(ꢁ-D-Galactopyranosyl)-(1→4)-(ꢀ-D-glucopyranosyloxy-carbonyl)]-
L-glycyldioctadecylamide (2 a). To a cold solution of 17 a (190 mg, 0.15 mmol)
in 30 mL of a CH2Cl2MCH3OH mixture (2:1), 30 mg of sodium methoxide pow-
der were added. The solution was stirred for 4 h and neutralized with Amberlite
IRC-50S. Filtration, followed by solvent evaporation from the filtrate under re-
duced pressure, afforded a crude residue which was purified by flash chromatog-
raphy (CH2Cl2MMeOH, 8:2), giving 2 a (100 mg, 70%) as a white solid; mp
166–167°C; [ꢀ]D ꢄ 9 (c 1, CHCl3MMeOH, 1:1), 1H NMR (CDCl3MCD3OD) ꢅ
0.88 (t, 2 ꢆ 3H, CH3), 1.20 (m, 60H, MCH2M), 1.50 (br s, 4H, CH2MCH3), 2.50
(br s, 4H, OH), 3.15 (br s, 2H, CH2MN), 3.30 (br s, 2H, CH2MN), 3.90 (m, 9H,
NHMCH2MCBO, H-4ꢃ, H-5ꢃ, H-5, H-6ꢃa, H-6ꢃb, H-6a, H-1), 4.50 (m, 5H, H-2,

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849
H-2ꢃ, H-3, H-3ꢃ, H-4), 4.90 (br s, 2H, OH), 5.22 (br s, 3H, OH, NH), 5.90 (br s, 1H,
H-1); Positive FAB-MS: m/z 947 (M ꢄ H)ꢄ, 969 (M ꢄ Na)ꢄ.
Anal. Calcd for C51H98N2O13: C, 64.66; H, 10.43; N, 2.96. Found: C, 64.60;
H, 10.54; N, 3.09.
N-[(2,3,4,6-Tetra-O-acetyl-ꢁ-D-galactopyranosyl)-(1→4)-(2,3,6-tri-O-
acetyl-ꢁ-D-glucopyranosyloxycarbonyl)]-L-glycyldioctadecylamide (17 b).
Compound 17b was obtained from 16 b as described for the preparation of 17 a
and isolated in 60% yield as an oil after flash chromatography (cyclohexane-ethyl
acetate, 6:4); [ꢀ]D–3 (c 1, CHCl3), 1H NMR (CDCl3) ꢅ 0.88 (t, 2 ꢆ 3H, CH3), 1.20
(br s, 60H, MCH2M), 1.97, 2.03, 2.04, 2.05, 2.07, 2.12, 2.16 (7s, 7 ꢆ 3H, OAc),
3.10 (br t, 2H, CH2MN), 3.30 (br t, 2H, CH2MN), 3.90 (m, 3H, H-5, H-5ꢃ, H-4),
4.00 (d, 2H, J ꢂ 3 Hz, NHMCH2MCBO), 4.10 (m, 3H, H-6a, H-6b, H-6ꢃa), 4.50
(m, 2H, H-1, H-6ꢃb), 4.95 (dd, 1H, J3–4 ꢂ 3 Hz, J3ꢈ2 ꢂ 11 Hz, H-3), 5.10 (m, 2H,
H-2, H-2ꢃ), 5.25 (t, 1H, J ꢂ 9 Hz, H-3ꢃ), 5.35 (m, 1H, H-4), 5.64 (d, 1H, J ꢂ 9 Hz,
H-1ꢃ), 6.05 (br t, 1H, NH); Positive FAB-MS: m/z 1241 (M ꢄ H)ꢄ.
N-[(ꢁ-D-Galactopyranosyl)-(1→4)-(ꢁ-D-glucopyranosyloxy-carbonyl)]-
L-glycyldioctadecylamide (2 b). Compound 2 b was prepared in 60% yield by
deprotection of 17 b as described for 2 a; [ꢀ]D–35 (c 1, CHCl3MMeOH, 1:1), 1H
NMR (CDCl3MCD3OD) ꢅ 0.88 (t, 2 ꢆ 3H, CH3), 1.60 (br s, 60H, MCH2M), 2.50
(br s, 4H, OH), 3.12 (br s, 2H, CH2MN), 3.28 (br s, 2H, CH2MN), 3.80 (m, 8H, H-
6ꢃa, H-6ꢃb, H-6a, H-6b, H-5, H-5ꢃ, H-4ꢃ, H-1), 4.50 (m, 5H, H-3, H-3ꢃ, H-2, H-2ꢃ,
H-4), 4.90 (br s, 2H, OH), 5.22 (br s, 3H, OH, NH), 5.40 (br d, 1H, J ꢂ 9 Hz, H-
1ꢃ), 6.05 (br t, 1H, NH); Positive FAB-MS: m/z 947 (M ꢄ H)ꢄ.
Anal. Calcd for C51H98N2O13: C, 64.66; H, 10.43; N, 2.96. Found: C, 64.89;
H, 10.64; N, 3.11.
N,Nꢃ-(2,3,4,6-Tetra-O-acetyl-ꢀ-D-glucopyranosyloxycarbonyl)-1,3-di-
aminopropan-2-ol (18). To a solution of 11 a (3.6 g, 7 mmol) in dry CH2Cl2 (75
mL) were added 1,3-diaminopropanol (315 mg, 3.5 mmol) and triethylamine
(1 mL, 7 mmol). After stirring for 1 h at rt, the reaction mixture was diluted with
water and extracted with CH2Cl2 (2 ꢆ 50 mL). The combined organic layers
were washed with a saturated NaCl solution, dried over MgSO4 and concentrated
under reduced pressure. The residue was purified by flash chromatography
(CH2Cl2MCH3OH, 95:5) to afford 18 (4.7 g, 80%); [ꢀ]D ꢄ 36 (c 2.8, CHCl3), 1H
NMR (CDCl3) ꢅ 1.97, 1.99, 2.03, 2.15 (4s, 24H, OAc), 3.30 (m, 4H, CH2MCH-
MOH), 3.87 (m, 1H, CH2MCHMOH), 4.08 (d, 4H, J ꢂ 6 Hz, H-6a, H-6b), 4.34
(t, 2H, J ꢂ 7 Hz, H-5), 5.30 (m, 4H, H-2, H-3), 5.47 (m, 2H, H-4), 5.60 (m, 2H,
NH), 6.26 (br s, 2H, H-1); Positive FAB-MS: m/z 839 (M ꢄ H)ꢄ, 861 (M ꢄ Na)ꢄ.
4-Nitrophenyl-[N,Nꢃ-(2,3,4,6-tetra-O-acetyl-ꢀ-D-galactopyranosyloxy-
carbonyl)-1,3-diaminopropyl]-carbonate (19). To a solution of 4-nitrophenyl
chloroformate (300 mg, 1.5 mmol) in CH2Cl2 (25 mL) and pyridine (120 ꢇL, 1.5
mmol), a solution of 18 (650 mg, 0.77 mmol) in CH2Cl2 (25 mL) was gradually

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AZOULAY ET AL.
added. The mixture was stirred over night, then concentrated under reduced pres-
sure. After purification by flash chromatography (ethyl acetate-cyclohexane, 2:1),
1
19 was obtained as an oil (680 mg, 88%); [ꢀ]D ꢄ 30 (c 1, CHCl3), H NMR
(CDCl3) ꢅ 1.98, 2.00, 2.03, 2.05, 2.16 (4s, 2(4 ꢆ 3H), OAc), 3.45 (m, 2H,
CH2MCHMOMCO), 3.65 (m, 2H, CH2MCHMOMCO), 4.10 (d, 4H, J ꢂ 6 Hz,
H-6a, H-6b), 4.30 (m, 2H, H-5), 4.85 (m, 1H, CH2MCHMOMCO), 5.30 (m, 4H,
H-2, H-3), 5.50 (m, 4H, H-5, NH), 6.30 (d, 1H, J ꢂ 2.5 Hz, H-1), 6.35 (d, 1H,
J ꢂ 2.5 Hz, H-1), 7.44 (d, 2H, J ꢂ 10 Hz, Ar), 8.30 (d, 2H, J ꢂ 10 Hz, Ar); Posi-
tive FAB-MS: m/z 1004 (M ꢄ H)ꢄ.
N,Nꢃ-[(2,3,4,6-Tetra-O-acetyl-ꢀ-D-galactopyranosyloxycarbonyl)-1,3-di-
aminopropyloxycarbonyl]-L-glycyldioctadecylamide (20). To a solution of 19
(435 mg, 0.43 mmol) in dry CH2Cl2 (30 mL) were successively added glycyldioc-
tadecylamide (112 mg, 0.47 mmol) and Et3N (70 ꢇL, 0.49 mmol). The mixture was
stirred overnight at rt, then extracted following the classical procedure. Purification
by flash chromatography (cyclohexane-ethyl acetate, 1:1) led to compound 20 (495
mg, 70%) as a white foam; [ꢀ]D ꢄ 11 (c 1.5, CHCl3), 1H NMR (CDCl3) ꢅ 0.88 (t,
2 ꢆ 3H, CH3), 1.26 (br s, 60H, MCH2M), 1.60 (m, 4H, CH2MCH3), 2.00, 2.04,
2.05, 2.16 (4s, 24H, OAc), 3.15 (m, 2H, CH2MCHMOMCO), 3.30 (m, 2H,
CH2MN), 3.40 (m, 2H, CH2MCHMOMCO), 3.50 (m, 2H, CH2MN), 4.00 (d, 2H,
J ꢂ 3 Hz, NHMCH2MCBO), 4.15 (br d, 4H, H-6a, H-6b), 4.33 (m, 2H, H-5), 4.82
(m, 1H, CH2MCHMOMCO), 5.30 (m, 4H, H-2, H-3), 5.50 (m, 4H, NH, H-4),
5.71 (br t, 1H, NH), 6.30 (br s, 2H, H-1); Positive FAB-MS: m/z 1443 (M ꢄ H)ꢄ,
1465 (M ꢄ Na)ꢄ.
N,Nꢃ-[(ꢀ-D-galactopyranosyloxycarbonyl)-1,3-diaminopropyloxycar-
bonyl]-L-glycyldioctadecylamide (3). To a cold solution of 20 (470 mg, 0.33
mmol) in 50 mL of CH2Cl2ꢈMMeOH (2:1) were added 140 mg of MeONa. The
reaction mixture was stirred for 3 h and neutralized with Amberlite IRC-50S. Af-
ter filtration, the filtrate was concentrated under reduced pressure to give a solid.
Flash chromatography (CH2Cl2MMeOH, 7:3) gave 3 (208 mg, 57%) as white
1
solid; mp 162–164°C; [ꢀ]D ꢄ 21 (c 1, CHCl3MCH3OH, 1:1); H NMR
(CDCl3MCD3OD) ꢅ 0.88 (t, 2 ꢆ 3H, CH3), 1.20 (br s, 60H, MCH2M), 1.50 (br s,
4H, CH2MCH3), 2.54 (br s, 4H, OH), 3.15 (br s, 4H, CH2MN, CH2MCH-
MOMCO), 3.30 (br s, 4H, CH2MN, CH2MCHMOMCO), 3.80 (m, 12H, H-2, H-
3, H-4, H-5, H-6a, H-6b), 4.80 (br s, 2H, OH), 5.14 (br s, 2H, OH), 6.10 (br s, 2H,
H-1); Positive FAB-MS: m/z 1107 (M ꢄ H)ꢄ.
Anal. Calcd for C56H106N4O17: C, 60.73; H, 9.65; N, 5.06. Found: C, 61.05;
H, 9.69; N, 5.17.
N,Nꢃ-[Bis-N,Nꢃ-(2,3,4,6-tetra-O-acetyl-ꢀ-D-galactopyranosyloxycar-
bonyl)-1,3-diaminopropyloxycarbonyl]-1,3-diaminopropan-2-ol (21). Com-
pound 21 was obtained in 78% yield from 19 by the same protocol used for the
preparation of compound 18; [ꢀ]D ꢄ 49 (c 2, CHCl3), 1H NMR (CDCl3) ꢅ 2.05 (m,
36H, OAc), 2.20 (br s, 12H, OAc), 3.32 (m, 12H, NHMCH2M), 3.80 (m, 1H,

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MULTIANTENNARY GALACTOSYL LIGANDS
851
CH2MCHMOH), 4.10 (m, 8H, H-6a, H-6b), 4.30 (m, 4H, H-5), 4.80 (m, 1H,
CH2MCHMOMCBO), 5.30 (m, 8H, H-2, H-3), 5.65 (m, 4H, H-4), 5.90 (m, 4H,
NH), 6.25 (m, 4H, H-1); Positive FAB-MS: m/z 1841 (M ꢄ Na)ꢄ, 1857 (M ꢄ K)ꢄ.
4-Nitrophenyl-[N,Nꢃ-(bis-N,Nꢃ-(2,3,4,6-tetra-O-acetyl-ꢀ-D-galactopyra-
nosyloxycarbonyl)-1,3-diaminopropyloxycarbonyl)-1,3-diaminopropyl]-car-
bonate (22). Compound 22 was prepared in 78% yield as described for 19; [ꢀ]D
ꢄ74 (c 2, CHCl3), 1H NMR (CDCl3) ꢅ 2.01 (m, 36H, OAc), 2.20 (br s, 12H, OAc),
3.40 (m, 12H, NHMCH2M), 4.10 (m, 8H, H-6a, H-6b), 4.20 (m, 4H, H-5), 4.80
(m, 2H, CHMOMCOMNH), 5.07 (m, 1H, CHMOMCOMO), 5.30 (m, 8H, H-2,
H-3), 5.48 (m, 4H, H-4), 5.90 (m, 6H, NH), 6.15 (m, 4H, H-1); Positive FAB-MS:
m/z 1984 (M ꢄ H)ꢄ, 2006 (M ꢄ Na)ꢄ.
N,Nꢃ-[Bis-N,Nꢃ-((2,3,4,6-tetra-O-acetyl-ꢀ-D-galactopyranosyloxycar-
bonyl)-1,3-diaminopropyloxycarbonyl)-1,3-diaminopropyl]-L-glycyldioc-
tadecylamide (23). Compound 23 was obtained from 22 as described for the
preparation of 20 and isolated in 55% yield as a white foam; [ꢀ]D ꢄ 42 (c 3.4,
CHCl3), 1H NMR (CDCl3) ꢅ 0.88 (t, 6H, CH3), 1.20 (br s, 60H, MCH2M), 1.60
(br s, 4H, CH2MCH3), 2.05 (m, 36H, OAc), 2.15 (br s, 12H, OAc), 3.40 (m, 14H,
NHMCH2MCHM, NHMCH2MCO), 4.10 (m, 8H, H-6a, H-6b), 4.35 (m, 4H, H-
5), 4.80 (m, 2H, CHMOMCOMNH), 5.01 (m, 1H, CHMOMCOMO), 5.30 (m,
8H, H-2, H-3), 5.47 (m, 4H, H-4), 6.28 (m, 4H, H-1); MALDI/Tof-MS (DHB):
m/z 2424 (M ꢄ H)ꢄ, 2446 (M ꢄ Na)ꢄ.
Anal. Calcd for C110H174N8O51: C, 54.49; H, 7.23; N, 4.62. Found: C, 54.85;
H, 7.56; N, 4.84.
N,Nꢃ-[Bis-N,Nꢃ-((ꢀ-D-galactopyranosyloxycarbonyl)-1,3-diaminopropy-
loxycarbonyl)-1,3-diaminopropyl]-L-glycyldioctadecylamide (4). Compound
4 was prepared by deprotection of compound 23 (450 mg, 0.18 mmol) as described
for 3, after purification by flash chromatography (CH2Cl2MMeOH, 7:3). Com-
pound 4 (146 mg, 45%) was isolated as a white solid; mp 196–198°C; [ꢀ]D ꢄ 66
(c 0.5, CHCl3MCH3OH, 1:1); MALDI/Tof-MS (DHB): m/z 1773 (M ꢄ Na)ꢄ,
1789 (M ꢄ K)ꢄ.
Anal. Calcd for C78H142N8O35: C, 53.47; H, 8.17; N, 6.40. Found: C, 53.37;
H, 8.04; N, 6.33.
ACKNOWLEDGMENTS
We are grateful to Dr. J.-P. Le Caer (UMR 7637) for performing the
MALDI/Tof mass spectra.
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Received May 18, 2001
Accepted November 6, 2001

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