Facile synthesis of stable lipid analogues possessing a range of alkyl groups: Application to artificial glycolipids

Article abstract of DOI:10.1016/S0968-0896(02)00266-3

Efficient preparation of lipid analogues is described in which various long alkoxy chains and 2-hydroxyethyl group were covalently linked with benzoic acid derivatives. An α-mannopyranosyl group was stereoselectively introduced by the conventional imidate

Full text of DOI:10.1016/S0968-0896(02)00266-3

Bioorganic & Medicinal Chemistry 10 (2002) 4013–4022
Facile Synthesis of Stable Lipid Analogues Possessing a Range of
Alkyl Groups: Application to Artificial Glycolipids
Yasuo Azefu,a Hitoshi Tamiaki,a,* Reiko Satob and Kazunori Tomab
aDepartment of Bioscience and Biotechnology, Faculty of Science and Engineering, Ritsumeikan University, Kusatsu,
Shiga 525-8577, Japan
bThe Noguchi Institute, Itabashi, Tokyo 173-0003, Japan
Received 6 June 2002;accepted 3 July 2002
Abstract—Efficient preparation of lipid analogues is described in which various long alkoxy chains and 2-hydroxyethyl group were
covalently linked with benzoic acid derivatives. An a-mannopyranosyl group was stereoselectively introduced by the conventional
imidate method into the terminal hydroxy group without any alternation of other moieties in a molecule. The resulting new gly-
coconjugates acted as models of natural glycolipids for protein-carbohydrate interactions.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
and regiocontrolled compounds;(b) instability of the
compounds for reduction, isomerization, racemization
and/or hydrolysis;and (c) difficulty in detection of the
compounds usually lacking any useful chromophores.
For example, sphingolipid-type compounds as the lipid
segment in a designed glycolipid could be prepared only
with great difficulty because of complex synthetic
routes.5 Several groups have already reported synthetic
lipids/glycolipids,6 but to our knowledge there have
been limited systematic preparation of lipid models and
further development into synthetic glycolipids.7 Com-
plicated syntheses of glycolipids that possess a variety of
lengths or numbers at hydrophobic chains prevent us
from systematically investigating their functions.
Carbohydrates exist on a cell surface as a part of the
glycoconjugates, such as glycolipids and glycoproteins.
Numerous cellular recognition processes are triggered
by specific interactions of carbohydrates with proteins
including lectins. Such carbohydrates play important
roles in many biological communications including fer-
tilization, cell signaling, pathogen identification, and
inflammatory response.1,2 The great affinity and specific-
ity are ascribable to clustering of binding events into
multivalent arrays, while the attractive force at the con-
stitutive monovalent interactions is relatively weak.3,4
Both molecular structures of hydrophilic and hydro-
phobic moieties affect the clustering of the glycoconju-
gates in a cell membrane. To elucidate the biological
events at the molecular level, many types of structurally
defined glycoconjugates are necessary and should be
systematically investigated. The synthetic glycoconju-
gates have to be prepared in large quantities as chemi-
cally pure materials, because the naturally occurring
glycoconjugates are scarce and often structurally
heterogeneous.
Here we report on synthesis of lipid and glycolipid
models possessing long alkoxy groups as hydrophobic
tails and benzoic acid derivatives as the linker. Multi-
hydroxylated benzoic acids allow various numbers and
lengths of alkyl groups to be adopted as the tails of
lipids, which resemble fatty acid portions of natural
ones. A linker module with the aromatic ring could be
successively and selectively introduced without any
alternation of alkoxy chains. In the solution phase, we
can employ well-established reactions for the glycosyl
bond formation, which is capable of controlling stereo-
chemistry of anomeric configurations without any
difficulty. The synthetic conjugates are easily separated
from reaction mixtures and conventionally purified due
to their chemical stabilities, appropriate hydrophobicities
There are many problems in preparing naturally occur-
ring glycolipids: (a) difficulty in synthesizing the stereo-
*Corresponding author. Tel.: +81-77-566-1111;fax: +81-77-561-
2659;e-mail: tamiaki@se.ritsumei.ac.jp
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00266-3

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Y. Azefu et al. / Bioorg. Med. Chem. 10 (2002) 4013–4022
and aromatic chromophores possessing a relatively large
absorption at 254 nm. Moreover, we report specific
recognition of lectin with the carbohydrate moiety of
the synthetic glycolipids on a membrane, indicating that
the artificial glycolipids can be good functional models
of natural glycolipids.
methyl esters 7–10 before the reaction with aminoetha-
nol, because the long alkyl ester was unreactive due to
their high steric hindrance around the carbonyl group
and the low solubility in aminoethanol and DMF as the
mixed solvents. NMR spectra of the resulting amides
11–17 in CDCl3 at 293 K show that primary amides 11–
16 were exclusively trans conformers around the CO–
NH bond, but that secondary amide 17 was a mixture of
cis/trans conformers (1:5) in the solution.
Results and Discussion
The lipid components that can link a carbohydrate
residue were prepared as follows. Long alkyl chains
were introduced into phenolic hydroxy groups of com-
mercially available benzoic acid derivatives by slight
modification of previously reported procedures,8 in
which the free carboxyl group in a molecule was ester-
ified simultaneously (Scheme 1). The etherification and
esterification with a series of 1-bromoalkane in the pres-
ence of potassium carbonate in dimethylformamide
(DMF) smoothly proceeded to give alkoxybeonzoates
1–6 possessing various numbers and lengths of alkyl
chains in high yields.
Acid catalyzed alcoholysis of 10 by 1,2-dihydroxyethane
in 1,2-dimethoxyethane gave the corresponding 2-
hydroxyethyl benzoate (18) in a high yield. The ester 18
is applicable for a lipid analogue but not suitable as a
precursor of glycolipid in the present study because of
its instability in removing benzoyl groups as a protect-
ing group of reactive hydroxyl moieties on a carbohy-
drate (see below). Treatment of ester 18 with 1 M
.
solution of borane-tetrahydrofuran (BH3 THF) com-
plex in THF did not afford the reductive product 21, but
predominantly gave benzyl alcohol (19). The desired 21,
having a benzyl ether bond as a spacer tolerant of the
above ester-cleavage, was efficiently prepared from 10
by a series of reactions: reduction of the methyl ester
with LiAlH4 to 19, bromination of the benzyl alcohol
with triphenylphosphine and carbon tetrabromide to 20
and Williamson ether synthesis of the bromide with 1,2-
dihydroxyethane to 21. These three linkages, amide,
methylamide and ether bonds, in the synthetic lipid
analogues were resistant to the following glycosylation
using a conventional imidate method and the successive
cleavage of benzoyl protecting groups on the carbohy-
drate moiety. All the new lipids 11–18 and 21 were
easily purified by flash column chromatography (FCC)
with silica gel and recrystallization, and fully character-
ized by their 1H NMR, FAB-MS and/or FT-IR spectra.
Aminolysis of esters 1 and 6–10 by aminoethanol in
DMF and of ester 5 by neat N-methylaminoethanol
efficiently afforded the corresponding primary amides
11–16 and secondary amide 17, respectively (Scheme 2).
Treatment of 1 and 6–10 with neat aminoethanol gave
none of the desired amide products, but recovered the
starting esters. Use of DMF as a polar solvent was
necessary for the aminolysis by aminoethanol. The
amino group in neat aminoethanol is less reactive to the
carbonyl group because of intra- and intermolecular
hydrogen bonding with amino and hydroxyl groups.
DMF molecules would break the hydrogen bonding
and then the amino group would increase the nucleo-
philicity to react with the benzoates. Enhanced solubil-
ity of the benzoates in DMF rather than in
aminoethanol would also accelerate the aminolysis. In
addition, double chain benzoates 2–5 possessing a long
alkyl ester had to be changed to the corresponding
The introduction of a carbohydrate moiety to the
hydroxyl group of the above synthetic lipids was
achieved as shown in Scheme 3. Glycosylation of lipid
analogues 11–17 and 21 with 2,3,4,6-tetra-O-benzoyl-a-
Scheme 1. Synthesis of precursors for lipid analogues: a. CnH2n+1Br, K2CO3, DMF, 74–88%; b. MeONa/MeOH, CH2Cl2, >96%.

Y. Azefu et al. / Bioorg. Med. Chem. 10 (2002) 4013–4022
4015
Scheme 2. Synthesis of lipid analogues: c. HOCH2CH2NH2, DMF, 77–94%; d. HOCH2CH2NMeH, 90%; e. HOCH2CH2OH, MeOCH2CH2OMe,
.
concd H2SO4, 88%; f. BH3 THF, THF, 82%; g. LiAlH4, THF, 97%; h. CBr4, PPh3, CH2Cl2, 90%; i. HOCH2CH2OH, NaH, THF, 73%.
d-mannopyranosyl trichloroacetimidate, prepared from
a commercially available d-mannopyranoside by reported
procedures,9 in the presence of a catalytic amount of
trimethylsilyl trifluoromethanesulfonate (TMSOTf)
gave protected mannolipids 22–29 moderately after
purification by silica gel column chromatography with
ethyl acetate–hexane. Irrespective of the number and
length of alkyl chains at the hydrophobic moiety, the
glycosylation occurred stereoselectively to give the a-
Interaction of the synthesized glycolipid analogues with
lectins was examined by a commercially available sur-
face plasmon resonance (SPR) instrument (BIACORE
3000) using a fluid ligand-layer formed on the surface of
the sensor chip, HPA chip. A gold surface of HPA is
modified with octadecyl groups attached through thio-
ethers. An additional self-assembled monolayer (SAM)
composed of 20% synthetic glycolipid and phosphati-
dylcholine from egg yolk (EPC) was generated upon the
hydrophobic chip surface.11
1
anomer exclusively from their H NMR analyses. This
glycosylation could be applicable to other saccharides;
for example, use of b-d-galacto- and a/b-d-glucopyra-
nosides instead of the above mannopyranoside gave the
corresponding glycoconjugates.10
The binding amounts of proteins including lectins on
the above surface modified with mannolipid 34 were
evaluated by SPR analysis as shown in Figure 1. Injec-
tion of proteins in an aqueous buffer solution increased
the SPR response by binding the proteins on the surface
and changing the bulk refractive index.4 The response
shows that concanavalin A (Con A), which is one of the
lectins that binds specifically to an a-mannosyl resi-
due,12 had considerably higher affinity for the artificial
Deprotection of all four benzoate groups in 22–29 using
sodium methoxide cleanly afforded the free-OH prod-
ucts 30–37. After silica gel column chromatography
with methanol–CHCl3, these glycolipid analogues were
recrystallized from methanol to yield pure materials as a
white solid. The solids 30–37 were so stable that they
could be stored at room temperature for a long period
(>1 year) without any alteration. All the new glycoli-
pids (22–37) in both protected and unprotected forms
monolayer than did b-galactose specific-binding ricin
12
(RCA120
)
and bovine serum albumin (BSA) lacking
specific affinity with any carbohydrates. The responses
by ricin and BSA were mainly ascribable to bulk
refractive index changes. These results indicate that the
binding events of Con A specifically occurred at the
interface of the aqueous solution with the hydrophilic
surface and that the a-mannosyl residues were present
1
were fully characterized by H NMR, H–H COSY and
FAB-MS spectra. Compounds 28 and 36, having methyl
amide linkage, were a mixture of cis/trans conformers
(1:3 and 1:5, respectively) in CDCl3 at 293 K.

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Y. Azefu et al. / Bioorg. Med. Chem. 10 (2002) 4013–4022
Scheme 3. Synthesis of glycolipid analogues possessing the a-d-mannopyranosyl residue: j. TMSOTf, CH2Cl2; k. MeONa/MeOH. The overall yields
of 30–37 based on consumed 11–17 and 21 were 26–78%.
on the artificial membrane surface. The other glycolipid
analogues showed a similar behavior to 34. Therefore,
the present artificial glycoconjugates (30–37) are prom-
ising as good models of natural glycolipids.
gave a series of glycolipid analogues in an adequate
yield. Moreover, these compounds can be stored at the
solid state for one year or longer without any alteration.
The glycolipid analogues behaved as a membrane com-
ponent with EPC on the SPR surface. The increased
response to Con A over the other proteins demonstrated
that the lipid-like membrane containing the analogues
functioned similarly as a biomembrane. These analo-
gues will contribute elucidating the biological functions
of glycolipids on a cell surface.
Conclusion
In the synthetic lipid molecules, the 2-hydroxyethyl
group and a series of alkyl chains were connected with
hydroxylated benzoic acid derivatives at both ends.
Number and length of the alkyl chains could control
hydrophobicity of the lipid analogues. The number of
alkyl chains actually depended on the starting material:
4-hydroxybenzoic acid, 3,5-dihydroxybenzoic acid and
methyl gallate (methyl 3,4,5-trihydroxybenzoate) were
converted into lipid analogues possessing a single alkyl
chain, double chains and triple chains, respectively.
Carbon number of bromoalkane can define the length
of alkyl chains: using a series of bromoalkane afforded
the corresponding analogues, which have dodecyl, tet-
radecyl, hexadecyl or octadecyl groups at the hydro-
phobic chains. Structurally determined pure lipid
analogues can be systematically and easily synthesized
in this way. A successive conventional imidate method
Experimental
General
All materials were obtained from commercial suppliers
and used as provided. Solvents were distilled by stan-
dard protocols. N2 or Ar gas was used after drying by
CaCl2. All reactions were monitored by TLC on
0.25 mm precoated Merck silica gel 60 F254 and visual-
ized with ultraviolet irradiation (254 nm) or 5%
H2SO4–ethanol stain. All melting points were measured
with a Yanagimoto micro melting point apparatus and

Y. Azefu et al. / Bioorg. Med. Chem. 10 (2002) 4013–4022
4017
Figure 1. Titration of proteins on mannolipid-EPC surface: aqueous buffer solutions of proteins (20 mM in HBS-N buffer) were injected for 60 s
(inset arrow) over a mannolipid 34 and EPC (1:4) monolayer on an octadecyl-coated Au chip. Solid, dashed and dotted lines are responses for Con
A, RCA120 and BSA, respectively.
1
were uncorrected. H NMR spectra were recorded on a
Bruker AC-300 spectrometer (300 MHz) at 293 K
(unless another specific description is given); d-values
are expressed in parts per million relative to CHCl3
(7.26 ppm) or CD2HCOCD3 (2.04 ppm) as an internal
reference. Infrared absorption spectra were measured
with a Shimadzu FTIR-8600 spectrophotometer. FAB-
MS spectrum data were collected on a JEOL JMS-
GCmateIIR using m-nitrobenzyl alcohol as a matrix.
SPR measurements were performed on a BIACORE
3000 operated using the version 3.1.1 software.
vigorous stirring, a small portion of CH2Cl2 was added
until complete dissolution. The solution was neutralized
with Amberlite IR-120 (plus) resin, filtered, and con-
centrated in vacuo. Recrystallization of the residue from
ethanol gave pure methyl ester as a white needle-like
powder.
Aminolysis (ester to amide) for 11–16. Alkyl alkoxy-
benzoate was added to a mixture of 2-aminoethanol
(45 mL) and freshly distilled DMF (58 mL) under N2,
and the mixture was heated to 110 ꢀC. After the ester
was completely consumed, aq 2 M HCl was added to
the ice-cooled solution. The aqueous solution was
extracted with CHCl3 and the combined extracts were
dried over Na2SO4 and concentrated in vacuo. Recrys-
tallization from methanol for 11–14, methanol–ethanol
for 15 or ethyl acetate for 16 afforded N-(2-hydroxy-
ethyl)amide, lipid analogue, as a white powder.
General synthetic procedures
Ether synthesis for 1–6. A mixture of 1-bromoalkane
and K2CO3 in distilled DMF was heated to 65 ꢀC with
stirring under N2 and then hydroxybenzoic acid or
methyl gallate was added. After heating for several hours,
aq 2 M HCl and CH2Cl2 were added to the solution
with stirring at room temperature. The aqueous phase
was separated from the CH2Cl2 phase and successively
extracted with CH2Cl2. The combined CH2Cl2 phases
were dried over Na2SO4 and concentrated in vacuo.
FCC (Merck silica gel 60, 230–400 mesh) with CH2Cl2–
hexane (ca. 1:1) and recrystallization from ethanol gave
alkyl alkoxybenzoate as a white powder for 1, 3–6 or
colorless oil for 2.
Glycosylation for 22–29. Lipid analogue and 2,3,4,6-
tetra - O - benzoyl - a - d - mannopyranosyl trichloro-
acetimidate9 (Bz-Man-imidate) were dissolved in dis-
tilled CH2Cl2 under Ar. The solution was cooled to 0 ꢀC
with stirring and then a diluted CH2Cl2 solution of
TMSOTf was added dropwise for a few min. After stir-
ring for several hours at room temperature, the reaction
mixture was quenched with a small amount of Et3N and
concentrated in vacuo. Purification by silica gel column
chromatography (Wakogel FC-40FM) with 50% ethyl
acetate–hexane yielded benzoyl-protected glycolipid as a
white solid. Unchanged lipid analogue was recovered by
collecting some fractions after eluting the glycolipid on
the column chromatography. The total amount of the
Alcoholysis (long alkyl ester to methyl ester) for 7–10.
The above long alkyl ester and 0.1 M sodium methox-
ide in methanol (10 mL) were dissolved in CH2Cl2, fol-
lowed by stirring at room temperature for 14 h under
N2;if the solid of the starting ester was observed after

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Y. Azefu et al. / Bioorg. Med. Chem. 10 (2002) 4013–4022
glycoside was used in the following deprotection reac-
tion without recrystallization.
C12H24), 0.88 (9H, t, J=7 Hz, O–C15–CH3). MS (FAB)
found: m/z 828. Calcd for C55H103O4: [M+H]+, 828.
Deprotection for 30–37. The above benzoyl-protected
glycolipid and 0.1 M sodium methoxide in methanol
were dissolved in CH2Cl2, and the solution was stirred
at room temperature (30–34, 36–37) or under moderate
heating (35;ca. 40 ꢀC) for several hours under N2. The
solution was neutralized with Amberlite IR-120 (plus)
resin, filtered, and concentrated in vacuo. The residue
was purified by silica gel column chromatography
(Wakogel FC-40FM) with 10% methanol–CHCl3 and
recrystallized from methanol to give glycolipid analogue
as a white solid. The melting points of 30–37 are not
presented here, because the solids were amorphous to
provide broad and indefinite melting points. Yields in
parenthesis (see below) are overall yields for glycosyla-
tion and deprotection based on consumed lipid ana-
logues.
Octadecyl 3,5-bis(octadecyloxy)benzoate (5). 1-Bromo-
octadecane (10.0 g, 30 mmol), K2CO3 (21.1 g,
0.15 mol) and 3,5-dihydroxybenzoic acid (1.54 g,
10 mmol) in DMF (150 mL) for 27 h gave 5 (7.63 g,
84%);mp 56 ꢀC;IR (KBr) 1713 (C ¼O), 1597 cmꢁ1
(C¼C); 1H NMR (CDCl3) d=7.15 (2H, d, J=2 Hz, 2,6-
H), 6.63 (1H, t, J=2 Hz, 4-H), 4.28 (2H, t, J=7 Hz,
COO–CH2), 3.96 (4H, t, J=6 Hz, O–CH2), 1.76 (6H, m,
O–C–CH2), 1.43 (6H, br-m, O–C2–CH2), 1.25 (84H, br-
m, O–C3–C14H28), 0.88 (9H, t, J=7 Hz, O–C17–CH3).
MS (FAB) found: m/z 912. Calcd for C61H115O4:
[M+H]+, 912.
Methyl 3,4,5-tris(octadecyloxy)benzoate (6). Triether 6
was prepared by previously reported procedures;8 see
ref 8 for data of 6.
Octadecyl 4-octadecyloxybenzoate (1). 1-Bromooctadec-
ane (10.0 g, 30 mmol), K2CO3 (20.7 g, 0.15 mol) and 4-
hydroxybenzoic acid (2.07 g, 15 mmol) in ꢀDMF
(200 mL) for 30 h gave 1 (8.44 g, 88%);mp 76–77 C; 1H
NMR (CDCl3) d=7.98 (2H, d, J=9 Hz, 2,6-H), 6.90
(2H, d, J=9 Hz, 3,5-H), 4.27 (2H, t, J=7 Hz, COO–
CH2), 4.00 (2H, t, J=7 Hz, O–CH2), 1.77 (4H, m, O–C–
CH2), 1.43 (4H, br-m, O–C2–CH2), 1.26 (56H, br-m, O–
C3–C14H28), 0.88 (6H, t, J=7 Hz, O–C17–CH3). MS
(FAB) found: m/z 644. Calcd for C43H79O3: [M+H]+,
644.
Methyl 3,5-bis(dodecyloxy)benzoate (7). Dodecyl ester 2
(659 mg, 1.0 mmol) yielded 7 (484 mg, 96%);alternative
preparation and characterization of benzoate 7 was
reported in the literature.13 See ref 13 for data of 7.
Methyl 3,5-bis(tetradecyloxy)benzoate (8). Tetradecyl
ester 3 (743 mg, 1.0 mmol) yielded 8 (541 mg, 96%);mp
71–72 ꢀC; H NMR (CDCl3) d=7.15 (2H, d, J=2 Hz,
1
2,6-H), 6.63 (1H, t, J=2 Hz, 4-H), 3.96 (4H, t, J=7 Hz,
O–CH2), 3.89 (3H, s, COOCH3), 1.77 (4H, m, O–C–
CH2), 1.44 (4H, br-m, O–C2–CH2), 1.26 (40H, br-m, O–
C3–C10H20), 0.88 (6H, t, J=7 Hz, O–C13–CH3). MS
(FAB) found: m/z 561. Calcd for C36H65O4: [M+H]+,
561.
Dodecyl 3,5-bis(dodecyloxy)benzoate (2). 1-Bromodode-
cane (6.02 g, 24 mmol), K2CO3 (5.53 g, 40 mmol) and
3,5-dihydroxybenzoic acid (1.23 g, 8.0 mmol) in DMF
ꢀ
1
(60 mL) for 23 h gave 2 (3.92 g, 74%);mp 32 C; H
NMR (CDCl3) d=7.15 (2H, d, J=2 Hz, 2,6-H), 6.63
(1H, t, J=2 Hz, 4-H), 4.28 (2H, t, J=7 Hz, COO–CH2),
3.96 (4H, t, J=6 Hz, O–CH2), 1.76 (6H, m, O–C–CH2),
1.43 (6H, br-m, O–C2–CH2), 1.26 (48H, br-m, O–C3–
C8H16), 0.88 (9H, t, J=6 Hz, O–C11–CH3). MS (FAB)
found: m/z 660. Calcd for C43H79O4: [M+H]+, 660.
Methyl 3,5-bis(hexadecyloxy)benzoate (9). Hexadecyl
ester 4 (827 mg, 1.0 mmol) yielded 9 (615 mg, 100%);mp
77 ꢀC; H NMR (CDCl3) d=7.15 (2H, d, J=2 Hz, 2,6-
1
H), 6.63 (1H, t, J=2 Hz, 4-H), 3.96 (4H, t, J=6 Hz, O–
CH2), 3.89 (3H, s, COOCH3), 1.77 (4H, m, O–C–CH2),
1.44 (4H, br-m, O–C2–CH2), 1.26 (48H, br-m, O–C3–
C12H24), 0.88 (6H, t, J=7 Hz, O–C15–CH3). MS (FAB)
found: m/z 618. Calcd for C40H73O4: [M+H]+, 618.
Tetradecyl 3,5-bis(tetradecyloxy)benzoate (3). 1-Bromo-
tetradecane (7.01 g, 24 mmol), K2CO3 (3.32 g, 24 mmol)
and 3,5-dihydroxybenzoic acid (1.23 g, 8.0 mmol) in
DMF (50 mL) for 23 h gave 3 (4.86 g, 82%);mp 42–
Methyl 3,5-bis(octadecyloxy)benzoate (10). Octadecyl
ester 5 (911 mg, 1.0 mmol) yielded 10 (675 mg, 100%);
mp 82–83 ꢀC;IR (KBr) 1722 (C ¼O), 1601 cmꢁ1 (C¼C);
1H NMR (CDCl3) d=7.15 (2H, d, J=2 Hz, 2,6-H), 6.63
(1H, t, J=2 Hz, 4-H), 3.96 (4H, t, J=6 Hz, O-CH2),
3.89 (3H, s, COOCH3), 1.77 (4H, m, O–C–CH2), 1.44
(4H, br-m, O–C2–CH2), 1.25 (56H, br-m, O–C3–
C14H28), 0.88 (6H, t, J=7 Hz, O–C17–CH3). MS (FAB)
found: m/z 674. Calcd for C44H81O4: [M+H]+, 674.
43 ꢀC; H NMR (CDCl3) d=7.15 (2H, d, J=2 Hz, 2,6-
1
H), 6.63 (1H, t, J=2 Hz, 4-H), 4.28 (2H, t, J=7 Hz,
COO–CH2), 3.96 (4H, t, J=6 Hz, O–CH2), 1.76 (6H, m,
O–C–CH2), 1.43 (6H, br-m, O–C2–CH2), 1.26 (60H, br-
m, O–C3–C10H20), 0.88 (9H, t, J=7 Hz, O–C13–CH3).
MS (FAB) found: m/z 744. Calcd for C49H91O4:
[M+H]+, 744.
N-(2-Hydroxyethyl)-4-octadecyloxybenzamide (11). Ami-
nolysis of 1 (322 mg, 0.50 mmol) for 64 h afforded 11
(175 mg, 81%) as a white needle-like powder;mp 109–
110 ꢀC; 1H NMR (CDCl3) d=7.74 (2H, d, J=9 Hz, 2,6-
H), 6.91 (2H, d, J=9 Hz, 3,5-H), 6.49 (1H, br-t, NH),
3.99 (2H, t, J=7 Hz, 4–O–CH2), 3.84 (2H, q, J=5 Hz,
N–C–CH2), 3.62 (2H, q, J=5 Hz, N–CH2), 2.66 (1H, t,
J=5 Hz, OH), 1.79 (2H, m, O–C–CH2), 1.45 (2H, br-m,
O–C2–CH2), 1.25 (28H, br-m, O–C3–C14H28), 0.88 (3H,
Hexadecyl 3,5-bis(hexadecyloxy)benzoate (4). 1-Bromo-
hexadecane (7.37 g, 24 mmol), K2CO3 (3.33 g, 24 mmol)
and 3,5-dihydroxybenzoic acid (1.24 g, 8.0 mmol) in
DMF (50 mL) for 27 h gave 4 (4.89 g, 74%);mp 51 ꢀC;
1H NMR (CDCl3) d=7.15 (2H, d, J=2 Hz, 2,6-H), 6.63
(1H, t, J=2 Hz, 4-H), 4.29 (2H, t, J=7 Hz, COO–CH2),
3.96 (4H, t, J=7 Hz, O–CH2), 1.76 (6H, m, O–C–CH2),
1.43 (6H, br-m, O–C2–CH2), 1.26 (72H, br-m, O–C3–

Y. Azefu et al. / Bioorg. Med. Chem. 10 (2002) 4013–4022
4019
t, J=6 Hz, O–C17–CH3). MS (FAB) found: m/z 434.
Calcd for C27H48NO3: [M+H]+, 434.
N - (2 - Hydroxyethyl) - N - methyl - 3,5 - bis(octadecyloxy)
benzamide (17). Ester 5 (1.74 g, 1.9 mmol) was added to
neat 2-(methylamino)ethanol (76 mL) under N2, and
the mixture was heated to 110 ꢀC for 16 h. Allowing the
reaction mixture to stand at room temperature for 4
days gave a crude product as a solid. The resulting pre-
cipitates were filtered and washed with methanol.
Recrystallization from methanol affordꢀed pure 17
(1.23 g, 90%) as a white powder;mp 83–85 C;IR (KBr)
N-(2-Hydroxyethyl)-3,5-bis(dodecyloxy)benzamide (12).
Aminolysis of 7 (251 mg, 0.50 mmol) for 21 h afforded
12 (233 mg, 88%);mp 82–83 ꢀC; 1H NMR (CDCl3)
d=6.87 (2H, d, J=2 Hz, 2,6-H), 6.57 (1H, br-t, 4-H),
6.52 (1H, br-t, NH), 3.96 (4H, t, J=6 Hz, 3,5-O–CH2),
3.84 (2H, q, J=5Hz, N–C–CH2), 3.62 (2H, q, J=5 Hz,
N–CH2), 2.48 (1H, t, J=5 Hz, OH), 1.77 (4H, m, O–C–
CH2), 1.43 (4H, br-m, O–C2–CH2), 1.26 (32H, br-m, O–
C3–C8H16), 0.88 (6H, t, J=6 Hz, O–C11–CH3). MS
(FAB) found: m/z 534. Calcd for C33H60NO4:
[M+H]+, 534.
1
3341 (O–H), 1616 (C¼O), 1587 cmꢁ1 (C¼C); H NMR
(CDCl3 at 323 K14) d=6.53 (2H, d, J=2 Hz, 2,6-H),
6.49 (1H, br-t, 4-H), 3.94 (4H, t, J=7 Hz, 3,5-O–CH2),
3.87 (2H, br-s, N–C–CH2), 3.67 (2H, br-s, N–CH2), 3.06
(3H, s, N–CH3), 1.76 (4H, m, O–C–CH2), 1.44 (4H, br-
m, O–C2–CH2), 1.27 (56H, br-m, O–C3–C14H28), 0.89
(6H, t, J=7 Hz, O–C17–CH3). MS (FAB) found: m/z
717. Calcd for C46H86NO4: [M+H]+, 717.
N-(2-Hydroxyethyl)-3,5-bis(tetradecyloxy)benzamide
(13). Aminolysis of 8 (280 mg, 0.50 mmol) for 21 h
afforded 13 (266 mg, 90%);mp 90–91 ꢀC; 1H NMR
(CDCl3) d=6.87 (2H, d, J=2 Hz, 2,6-H), 6.57 (1H, br-t,
4-H), 6.53 (1H, br-t, NH), 3.96 (4H, t, J=7 Hz, 3,5-O–
CH2), 3.83 (2H, q, J=5 Hz, N–C–CH2), 3.61 (2H, q,
J=5 Hz, N–CH2), 2.50 (1H, t, J=5 Hz, OH), 1.77 (4H,
m, O–C–CH2), 1.44 (4H, br-m, O–C2–CH2), 1.26 (40H,
br-m, O–C3–C10H20), 0.87 (6H, t, J=7 Hz, O–C13–
CH3). MS (FAB) found: m/z 591. Calcd for C37H68
NO4: [M+H]+, 591.
2-Hydroxyethyl 3,5-bis(octadecyloxy)benzoate (18).
Methyl ester 10 (67.4 mg, 100 mmol) was added to a
mixture of 1,2-dihdroxyethane (135mL), 1,2-dimethoxy-
ethane (134 mL) and concd H2SO4 (17 mL). After
refluxing for 31 h, the reaction mixture was cooled to
room temperature and extracted with hexane. The
combined extracts were washed with aq satd NaHCO3
and aq satd NaCl, dried over Na2SO4 and concentrated
in vacuo. Purification by FCC with 0–10% ether-
CH2Cl2 and recrystallization from methanol–eꢀthanol
gave 18 (62.2 mg, 88%) as a white powder;mp 71 C;IR
N-(2-Hydroxyethyl)-3,5-bis(hexadecyloxy)benzamide
(14). Aminolysis of 9 (307 mg, 0.50 mmol) for 21 h
afforded 14 (293 mg, 91%);mp 95 ꢀC; 1H NMR
(CDCl3) d=6.87 (2H, d, J=2 Hz, 2,6-H), 6.57 (1H, br-t,
4-H), 6.53 (1H, br-t, NH), 3.96 (4H, t, J=7 Hz, 3,5-O–
CH2), 3.83 (2H, q, J=5 Hz, N–C–CH2), 3.61 (2H, q,
J=5 Hz, N–CH2), 2.50 (1H, t, J=5 Hz, OH), 1.77 (4H,
m, O–C–CH2), 1.44 (4H, br-m, O–C2–CH2), 1.25 (48H,
br-m, O–C3–C12H24), 0.87 (6H, t, J=6 Hz, O–C15–
CH3). MS (FAB) found: m/z 647. Calcd for C41H76
NO4: [M+H]+, 647.
1
(KBr) 3537 (O–H), 1701 (C¼O), 1605 cmꢁ1 (C¼C); H
NMR (CDCl3) d=7.16 (2H, d, J=2 Hz, 2,6-H), 6.64
(1H, t, J=2 Hz, 4-H), 4.45 (2H, t, J=5 Hz, COO-CH2),
3.96 (6H, m, 3,5-O–CH2, COO–C–CH2), 2.01 (1H, t,
J=6 Hz, OH), 1.77 (4H, m, O–C–CH2), 1.44 (4H, br-m,
O–C2–CH2), 1.25 (56H, br-m, O–C3–C14H28), 0.87 (6H,
t, J=7 Hz, O–C17–CH3). MS (FAB) found: m/z 704.
Calcd for C45H83O5: [M+H]+, 704.
3,5-Bis(octadecyloxy)benzyl alcohol (19). To a dry THF
solution (22 mL) of the above pure 10 (448 mg,
665 mmol) was added LiAlH4 (403 mg, 10.6 mmol) under
N2 and the mixture was then heated to 40 ꢀC for 12 h.
After water (100 mL) was added dropwise, the solution
was extracted with CHCl3. The combined extracts were
washed with aq 2 M HCl and aq 4% NaHCO3, dried
over Na2SO4 and concentrated in vacuo. Purification by
recrystallization from methanol gave 19 (414 mg, 97%)
N-(2-Hydroxyethyl)-3,5-bis(octadecyloxy)benzamide (15).
Aminolysis of 10 (336 mg, 0.50 mmol) for 21 h afforded
15 (330 mg, 94%);mp 95–96 ꢀC;IR (KBr) 3398 (O–H),
1
3298 (N–H), 1628 (C¼O), 1597 cmꢁ1 (C¼C); H NMR
(CDCl3) d=6.87 (2H, d, J=2 Hz, 2,6-H), 6.57 (1H, br-t,
4-H), 6.53 (1H, br-t, NH), 3.96 (4H, t, J=7 Hz, 3,5-O–
CH2), 3.83 (2H, q, J=5 Hz, N–C–CH2), 3.61 (2H, q,
J=5 Hz, N–CH2), 2.49 (1H, t, J=5 Hz, OH), 1.77 (4H,
m, O–C–CH2), 1.44 (4H, br-m, O–C2–CH2), 1.25 (56H,
br-m, O–C3–C14H28), 0.87 (6H, t, J=7 Hz, O–C17–
CH3). MS (FAB) found: m/z 703. Calcd for
C45H84NO4: [M+H]+, 703.
ꢀ
as a white powder;mp 68–70 C;IR (KBr) 3518 (O–H),
1
1589 cmꢁ1 (C¼C); H NMR (CDCl3) d=6.50 (2H, d,
J=2 Hz, 2,6-H), 6.37 (1H, br-t, 4-H), 4.62 (2H, d,
J=6 Hz, 1-CH2), 3.93 (4H, t, J=7 Hz, O–CH2), 1.76
(4H, m, O–C–CH2), 1.43 (4H, br-m, O–C2–CH2), 1.25
(56H, br-m, O–C3–C14H28), 0.88 (6H, t, J=7 Hz, O–
C17–CH3). MS (FAB) found: m/z 645. Calcd for
C43H80O3: M+, 645.
N-(2-Hydroxyethyl)-3,4,5-tris(octadecyloxy)benzamide
(16). Aminolysis of 6 (1.13 g, 1.2 mmol) with 2-amino-
ethanol (109 mL) in DMF (163 mL) for 14 h afforded
16 (894 mg, 77%);mp 83–85 ꢀC; 1H NMR (CDCl3)
d=6.96 (2H, s, 2,6-H), 6.48 (1H, br-t, NH), 4.00 (4H, t,
J=6 Hz, 3,5-O–CH2), 3.98 (2H, t, J=6 Hz, 4-O–CH2),
3.84 (2H, t, J=5 Hz, N–C–CH2), 3.62 (2H, q, J=5 Hz,
N–CH2), 1.77 (6H, m, O–C–CH2), 1.46 (6H, br-m, O–
C2–CH2), 1.25 (84H, br-m, O–C3–C14H28), 0.87 (9H, t,
J=7 Hz, O–C17–CH3). MS (FAB) found: m/z 971.
Calcd for C63H120NO5: [M+H]+, 971.
3,5-Bis(octadecyloxy)benzyl bromide (20). A solution of
alcohol 19 (322 mg, 499 mmol), triphenylphosphine
(160 mg, 609 mmol), and carbon tetrabromide (415 mg,
1.25 mmol) in dry CH2Cl2 (16 mL) was stirred at room
temperature for 4 h under Ar. The solution was washed
with aq satd NaHCO3 and aq satd NaCl, dried over
Na2SO4 and concentrated in vacuo. After purification

4020
Y. Azefu et al. / Bioorg. Med. Chem. 10 (2002) 4013–4022
by FCC with CH2Cl2, the eluate was evaporated to give
20 (318 mg, 90%) as a white solid;mp 70–73 ꢀC;IR
(KBr) 1595 (C¼C), 550 cmꢁ1 (C–Br); 1H NMR (CDCl3)
d=6.51 (2H, d, J=2 Hz, 2,6-H), 6.37 (1H, br-t, 4-H),
4.40 (2H, s, 1-CH2), 3.92 (4H, t, J=6 Hz, O–CH2), 1.76
(4H, m, O–C–CH2), 1.43 (4H, br-m, O–C2–CH2), 1.25
(56H, br-m, O–C3–C14H28), 0.88 (6H, t, J=7 Hz, O–
C17–CH3). MS (FAB) found: m/z 708. Calcd for
C43H80BrO2: [M+H]+, 708.
N-[O-(2,3,4,6-Tetra-O-benzoyl-ꢀ-D-mannopyranosyl)oxy-
ethyl] - 3,5 - bis(tetradecyloxy)benzamide (24). Glycosyla-
tion of 13 (107mg, 0.18mmol) with Bz-Man-imidate (ca.
0.12mmol) in CH2Cl2 (11 mL) and 11mM TMSOTf in
CH2Cl2 (0.54 mL) for 5 h gave 24, and 57.0 mg of 13 was
recovered; 1H NMR (CDCl3) d=8.1–7.2 (20H, m, C6H5),
6.95 (2H, d, J=2 Hz, 2,6-H), 6.71 (1H, br-t, NH), 6.59
(1H, br-t, 4-H), 6.12 (1H, t, J=10 Hz, Man-4-H), 5.91
(1H, dd, J=3, 10Hz, Man-3-H), 5.74 (1H, br-t, Man-2-
H), 5.16 (1H, br-d, Man-1-H), 4.68 (1H, d, J=11 Hz,
Man-6-H), 4.48 (1H, m, Man-6-H), 4.44 (1H, m, Man-
5-H), 4.07 (1H, m, N–C–CH), 3.95 (4H, t, J=6Hz, O–
CH2), 3.78 (3H, m, N–C–CH, N–CH2), 1.72 (4H, m, O–
C–CH2), 1.39 (4H, br-m, O–C2–CH2), 1.25 (40H, br-m,
O–C3–C10H20), 0.88 (6H, t, J=6 Hz, O–C13–CH3).
2-Hydroxyethyl 3,5-bis(octadecyloxy)benzyl ether (21).
Sodium hydride (60% in mineral oil;6.0 mg,
0.15 mmol) was added to a solution of 20 (31.2 mg,
44 mmol) and ethylene glycol (16.1 mg, 0.26 mmol) in
THF (4 mL) under Ar. The mixture was refluxed for 3
days, and aq 2% HCl was added to the ice-chilled mix-
ture. The resulting mixture was extracted with CH2Cl2,
washed aq satd NaCl, dried over Na2SO4, and evapo-
rated to dryness. FCC with 1% methanol–CH2Cl2
afforded 21 (22.1 mg, 73%);mp 51–54 ꢀC;IR (KBr)
3504 (O–H), 1601 (C¼C), 1177 cmꢁ1 (C–O–C); 1H
NMR (CDCl3) d=6.47 (2H, d, J=2 Hz, 2,6-H), 6.38
(1H, br-t, 4-H), 4.48 (2H, s, 1-CH2), 3.92 (4H, t,
J=7 Hz, 3,5-O–CH2), 3.75 (2H, br-q, 1-C–O–C–CH2),
3.59 (2H, t, J=5 Hz, 1-C–O–CH2), 1.98 (1H, br-t, OH),
1.76 (4H, m, O–C–CH2), 1.43 (4H, br-m, O–C2–CH2),
1.25 (56H, br-m, O–C3–C14H28), 0.87 (6H, t, J=7 Hz,
O–C17–CH3). MS (FAB) found: m/z 689. Calcd for
C45H84O4: M+, 689.
N-[O-(2,3,4,6-Tetra-O-benzoyl-ꢀ-D-mannopyranosyl)oxy-
ethyl] - 3,5 - bis(hexadecyloxy)benzamide (25). Glycosyla-
tion of 14 (77.1 mg, 0.12 mmol) with Bz-Man-imidate
(ca. 80 mmol) in CH2Cl2 (16 mL) and 11 mM TMSOTf
in CH2Cl2 (0.36 mL) for 5 h gave 25, and 44.0 mg of 14
1
was recovered; H NMR (CDCl3) d=8.1–7.2 (20H, m,
C6H5), 6.95 (2H, d, J=2 Hz, 2,6-H), 6.68 (1H, t,
J=5 Hz, NH), 6.58 (1H, br-t, 4-H), 6.11 (1H, t,
J=10 Hz, Man-4-H), 5.91 (1H, dd, J=3, 10 Hz, Man-3-
H), 5.74 (1H, dd, J=1, 2 Hz, Man-2-H), 5.16 (1H, d,
J=1 Hz, Man-1-H), 4.68 (1H, d, J=10 Hz, Man-6-H),
4.48 (1H, m, Man-6-H), 4.44 (1H, m, Man-5-H), 4.07
(1H, m, N–C–CH), 3.96 (4H, t, J=6 Hz, O–CH2), 3.78
(3H, m, N–C–CH, N–CH2), 1.73 (4H, m, O–C–CH2),
1.39 (4H, br-m, O–C2–CH2), 1.25 (48H, br-m, O–C3–
C12H24), 0.88 (6H, t, J=7 Hz, O–C15–CH3).
N-[O-(2,3,4,6-Tetra-O-benzoyl-ꢀ-D-mannopyranosyl)oxy-
ethyl]-4-octadecyloxybenzamide (22). Glycosylation of 11
(69.0 mg, 0.16 mmol) with Bz-Man-imidate (ca.
0.16 mmol) in CH2Cl2 (21 mL) and 50 mM TMSOTf in
CH2Cl2 (0.32 mL) for 3 h gave 22, and all of 11 was
consumed; 1H NMR (CDCl3) d=8.1–7.3 (20H, m,
C6H5), 7.82 (2H, d, J=12 Hz, 2,6-H), 6.92 (2H, d,
J=12 Hz, 3,5-H), 6.67 (1H, br-t, NH), 6.10 (1H, t,
J=10 Hz, Man-4-H), 5.92 (1H, dd, J=3, 10 Hz, Man-3-
H), 5.75 (1H, br-t, Man-2-H), 5.15 (1H, br-d, Man-1-
H), 4.66 (1H, dd, J=2, 10 Hz, Man-6-H), 4.46 (1H, m,
Man-6-H), 4.43 (1H, m, Man-5-H), 4.07 (1H, m, N–C–
CH), 3.95 (2H, t, J=7 Hz, O–CH2), 3.79 (3H, m, N–C–
CH, N–CH2), 1.76 (2H, m, O–C–CH2), 1.45 (2H, br-m,
O–C2–CH2), 1.25 (28H, br-m, O–C3–C14H28), 0.87 (3H,
t, J=7 Hz, O–C17–CH3).
N-[O-(2,3,4,6-Tetra-O-benzoyl-ꢀ-D-mannopyranosyl)oxy-
ethyl] - 3,5 - bis(octadecyloxy)benzamide (26). Glycosyla-
tion of 15 (56.3 mg, 80 mmol) with Bz-Man-imidate (ca.
80 mmol) in CH2Cl2 (5.6 mL) and 5.5 mM TMSOTf in
CH2Cl2 (0.36 mL) for 17 h gave 26, and 14.7 mg of 15
1
was recovered; H NMR (CDCl3) d=8.1–7.2 (20H, m,
C6H5), 6.95 (2H, d, J=2 Hz, 2,6-H), 6.71 (1H, t,
J=5 Hz, NH), 6.58 (1H, br-t, 4-H), 6.11 (1H, t,
J=10 Hz, Man-4-H), 5.91 (1H, dd, J=3, 10 Hz, Man-3-
H), 5.74 (1H, dd, J=1, 2 Hz, Man-2-H), 5.15 (1H, d,
J=1 Hz, Man-1-H), 4.67 (1H, d, J=10 Hz, Man-6-H),
4.47 (1H, m, Man-6-H), 4.44 (1H, m, Man-5-H), 4.06
(1H, m, N–C–CH), 3.95 (4H, t, J=6 Hz, O–CH2), 3.78
(3H, m, N–C–CH, N–CH2), 1.72 (4H, m, O–C–CH2),
1.39 (4H, br-m, O–C2–CH2), 1.25 (56H, br-m, O–C3–
C14H28), 0.87 (6H, t, J=6 Hz, O–C17–CH3).
N-[O-(2,3,4,6-Tetra-O-benzoyl-ꢀ-D-mannopyranosyl)-
oxyethyl]-3,5-bis(dodecyloxy)benzamide (23). Glycosyla-
tion of 12 (126 mg, 0.24 mmol) with Bz-Man-imidate
(ca. 0.16 mmol) in CH2Cl2 (6.4 mL) and 11 mM
TMSOTf in CH2Cl2 (0.72 mL) for 5 h gave 23, and
63.9 mg of 12 was recovered; 1H NMR (CDCl3) d=8.1–
7.2 (20H, m, C6H5), 6.95 (2H, d, J=2 Hz, 2,6-H), 6.71
(1H, br-t, NH), 6.58 (1H, br-t, 4-H), 6.12 (1H, t,
J=10 Hz, Man-4-H), 5.91 (1H, dd, J=3, 10 Hz, Man-3-
H), 5.74 (1H, br-t, Man-2-H), 5.15 (1H, br-d, Man-1-
H), 4.68 (1H, d, J=10 Hz, Man-6-H), 4.48 (1H, m,
Man-6-H), 4.43 (1H, m, Man-5-H), 4.07 (1H, m, N–C–
CH), 3.95 (4H, t, J=6 Hz, O–CH2), 3.78 (3H, m, N–C–
CH, N–CH2), 1.72 (4H, m, O–C–CH2), 1.39 (4H, br-m,
O–C2–CH2), 1.25 (32H, br-m, O–C3–C8H16), 0.88 (6H,
t, J=6 Hz, O–C11–CH3).
N-[O-(2,3,4,6-Tetra-O-benzoyl-ꢀ-D-mannopyranosyl)oxy-
ethyl]-3,4,5-tris(octadecyloxy)benzamide (27). Glycosyla-
tion of 16 (156 mg, 0.16 mmol) with Bz-Man-imidate
(ca. 0.16 mmol) in CH2Cl2 (16 mL) and 50 mM
TMSOTf in CH2Cl2 (0.80 mL) for 1.5 h gave 27, and
1
5.9 mg of 16 was recovered; H NMR (CDCl3) d=8.1–
7.3 (20H, m, C6H5), 7.04 (2H, s, 2,6-H), 6.66 (1H, br-t,
NH), 6.11 (1H, t, J=10 Hz, Man-4-H), 5.90 (1H, dd,
J=3, 10 Hz, Man-3-H), 5.73 (1H, br-dd, Man-2-H),
5.15 (1H, br-d, Man-1-H), 4.68 (1H, d, J=10 Hz, Man-
6-H), 4.47 (1H, m, Man-6-H), 4.43 (1H, m, Man-5-H),
4.02 (7H, m, N–C–CH, O–CH2), 3.78 (3H, m, N–C–
CH, N–CH2), 1.76 (6H, m, O–C–CH2), 1.41 (6H, br-m,

Y. Azefu et al. / Bioorg. Med. Chem. 10 (2002) 4013–4022
4021
O–C2–CH2), 1.25 (84H, br-m, O–C3–C14H28), 0.87 (9H,
t, J=6 Hz, O–C17–CH3).
N-[O-(ꢀ-D-Mannopyranosyl)oxyethyl]-3,5-bis(tetradecyl-
oxy)benzamide (32). Deprotection of 24 with 0.1 M
MeONa/MeOH (6 mL) in CH2Cl2 (2 mL) for 8 h gave
1
N-Methyl-N-[O-(2,3,4,6-tetra-O-benzoyl-ꢀ-D-manno-
pyranosyl)oxyethyl]-3,5-bis(octadecyloxy) benzamide (28).
Glycosylation of 17 (57.5 mg, 80 mmol) with Bz-Man-
imidate (59.2 mg, 80 mmol) in CH2Cl2 (5.7 mL) and
50 mM TMSOTf in CH2Cl2 (0.40 mL) for 4 h gave 28,
32 (34.7 mg, 55%); H NMR (CDCl3 at 323 K) d=6.87
(2H, d, J=1 Hz, 2,6-H), 6.70 (1H, br-t, NH), 6.55 (1H, br-
t, 4-H), 4.87 (1H, br-d, Man-1-H), 3.93, 3.86, 3.64, 3.57
(18H, m, 3,5-O–CH2, N–C–CH2, N–CH2, Man-
2,3,4,5,6,6-H, Man-2,3,4,6-OH), 1.74 (4H, m, O–C–CH2),
1.43 (4H, br-m, O–C2–CH2), 1.28 (40H, br-m, O–C3–
C10H20), 0.89 (6H, t, J=7 Hz, O–C13–CH3). MS (FAB)
found: m/z 775. Calcd for C43H77NNaO9: [M+Na]+, 775.
1
and 10.5 mg of 17 was recovered; H NMR (CDCl3 at
323 K14) d=8.1–7.2 (20H, m, C6H5), 6.56 (2H, d,
J=2 Hz, 2,6-H), 6.49 (1H, t, J=2 Hz, 4-H), 6.10 (1H, t,
J=10 Hz, Man-4-H), 5.88 (1H, dd, J=3, 10 Hz, Man-3-
H), 5.70 (1H, br-t, Man-2-H), 5.14 (1H, br-s, Man-1-H),
4.69 (1H, dd, J=2, 12 Hz, Man-6-H), 4.53 (1H, dd,
J=5, 12 Hz, Man-6-H), 4.44 (1H, br-d, Man-5-H), 4.08
(1H, br-m, N–C–CH), 3.94 (4H, t, J=7 Hz, O–CH2),
3.82 (3H, br-m, N–C–CH, N–CH2), 3.18 (3H, s, N–
CH3), 1.72 (4H, m, O–C–CH2), 1.45 (4H, br-m, O–C2–
CH2), 1.28 (56H, br-m, O–C3–C14H28), 0.89 (6H, t,
J=6 Hz, O–C17–CH3).
N-[O-(ꢀ-D-Mannopyranosyl)oxyethyl]-3,5-bis(hexadecyl-
oxy)benzamide (33). Deprotection of 25 with 0.1 M
MeONa/MeOH (4 mL) in CH2Cl2 (2 mL) for 8 h gave
1
33 (26.5 mg, 64%); H NMR (CDCl3 at 323 K) d=6.87
(2H, d, J=2 Hz, 2,6-H), 6.58 (1H, br-t, 4-H), 6.38 (1H,
br-t, NH), 4.90 (1H, br-d, Man-1-H), 3.98 (5H, t,
J=7 Hz, 3,5-O–CH2, Man-2-H), 3.83 (5H, m, N–C–
CH2, Man-3,4,6-H), 3.68 (4H, m, N–CH2, Man-5,6-H),
2.41 (1H, d, J=5 Hz, Man-3-OH), 2.36 (1H, br-d, Man-
4-OH), 2.25 (1H, d, J=4 Hz, Man-2-OH), 1.99 (1H, br-
t, Man-6-OH), 1.78 (4H, m, O–C–CH2), 1.47 (4H, br-m,
O–C2–CH2), 1.28 (48H, br-m, O–C3–C12H24), 0.89 (6H,
t, J=7 Hz, O-C15-CH3). MS (FAB) found: m/z 831.
Calcd for C47H85NNaO9: [M+Na]+, 831.
O-(2,3,4,6-Tetra-O-benzoyl-ꢀ-D-mannopyranosyl)oxy-
ethyl 3,5-bis(octadecyloxy)benzyl ether (29). Glycosyla-
tion of 21 (13.8 mg, 20 mmol) with Bz-Man-imidate (ca.
30 mmol) in CH2Cl2 (2 mL) and 5.5 mM TMSOTf in
CH2Cl2 (0.14 mL) for 10 h gave 29, and 1.5 mg of 21 was
recovered; 1H NMR (CDCl3) d=8.1–7.2 (20H, m,
C6H5), 6.52 (2H, d, J=2 Hz, 2,6-H), 6.37 (1H, br-t, 4-
H), 6.14 (1H, t, J=10 Hz, Man-4-H), 5.94 (1H, dd,
J=3, 10 Hz, Man-3-H), 5.75 (1H, br-dd, Man-2-H),
5.17 (1H, br-d, Man-1-H), 4.69 (1H, dd, J=2, 12 Hz,
Man-6-H), 4.56 (2H, s, 1-CH2), 4.52 (1H, m, Man-5-H),
4.46 (1H, dd, J=4, 12 Hz, Man-6-H), 3.99 (1H, m, 1-C–
O–C–CH), 3.91 (4H, t, J=7 Hz, 3,5-O–CH2), 3.84(1H,
m, 1-C–O–C–CH), 3.75 (2H, t, J=4 Hz,1-C–O–CH2),
1.71 (4H, m, O–C–CH2), 1.39 (4H, br-m, O–C2–CH2),
1.26 (56H, br-m, O–C3–C14H28), 0.88 (6H, t, J=6 Hz,
O–C17–CH3).
N-[O-(ꢀ-D-Mannopyranosyl)oxyethyl]-3,5-bis(octadecyl-
oxy)benzamide (34). Deprotection of 26 with 0.1 M
MeONa/MeOH (5 mL) in CH2Cl2 (3 mL) for 26 h gave
1
34 (39.0 mg, 76%); H NMR (CDCl3 at 323 K) d=6.87
(2H, d, J=2 Hz, 2,6-H), 6.58 (1H, br-t, 4-H), 6.37 (1H, br-
t, NH), 4.90 (1H, br-d, Man-1-H), 3.98 (5H, t, J=7Hz,
3,5-O–CH2, Man-2-H), 3.85 (5H, m, N–C–CH2, Man-
3,4,6-H), 3.68 (4H, m, N–CH2, Man-5,6-H), 2.37 (1H, d,
J=6 Hz, Man-3-OH), 2.30 (1H, d, J=3 Hz, Man-4-OH),
2.19 (1H, d, J=4 Hz, Man-2-OH), 1.94 (1H, t, J=6 Hz,
Man-6-OH), 1.78 (4H, m, O–C–CH2), 1.46 (4H, br-m,
O–C2–CH2), 1.28 (56H, br-m, O–C3–C14H28), 0.89 (6H,
t, J=7 Hz, O–C17–CH3). MS (FAB) found: m/z 887.
Calcd for C51H93NNaO9: [M+Na]+, 887.
N-[O-(ꢀ-D-Mannopyranosyl)oxyethyl]-4-octadecyloxy-
benzamide (30). Deprotection of 22 with 0.1 M MeONa/
MeOH (8 mL) in CH2Cl2 (2 mL) for 17 h gave 30
1
(39.8 mg, 42%); H NMR ((CD3)2CO) d=7.85 (2H, d,
N-[O-(ꢀ-D -Mannopyranosyl)oxyethyl]-3,4,5-tris(octa-
decyloxy)benzamide (35). Deprotection of 27 with
0.1 M MeONa/MeOH (8 mL) in CH2Cl2 (8 mL) for 8 h
gave 35 (107 mg, 61%); 1H NMR (CDCl3) d=6.98 (2H,
s, 2,6-H), 6.34 (1H, br-t, NH), 4.90 (1H, br-d, Man-1-
H), 4.02 (7H, m, 3,4,5-O–CH2, Man-2-H), 3.83 (5H, m,
N–C–CH2, Man-3,4,6-H), 3.66 (4H, m, N–CH2, Man-
5,6-H), 2.37 (1H, d, J=6 Hz, Man-3-OH), 2.30 (1H, br-
d, Man-4-OH), 2.19 (1H, d, J=4 Hz, Man-2-OH), 1.93
(1H, t, J=6 Hz, Man-6-OH), 1.81 (6H, m, O–C–CH2),
1.53 (6H, br-m, O–C2–CH2), 1.28 (84H, br-m, O–C3–
C14H28), 0.89 (9H, t, J=7 Hz, O–C17–CH3). MS (FAB)
found: m/z 1132. Calcd for C69H129NO10: M+, 1132.
J=9 Hz, 2,6-H), 7.77 (1H, br-t, NH), 6.95 (2H, d,
J=9 Hz, 3,5-H), 4.78 (1H, br-d, Man-1-H), 4.03 (2H, t,
J=6 Hz, 4-O–CH2), 3.98 (1H, m, Man-2-H), 3.78 (5H,
m, N–C–CH2, Man-3,4,6-H), 3.60 (8H, m, N–CH2,
Man-5,6-H, Man-2,3,4,6-OH), 1.77 (2H, m, O–C–CH2),
1.46 (2H, br-m, O–C2–CH2), 1.27 (28H, br-m, O–C3–
C14H28), 0.86 (3H, br-t, O–C17–CH3). MS (FAB) found:
m/z 596. Calcd for C33H58NO8: [M+H]+, 596.
N-[O-(ꢀ-D -Mannopyranosyl)oxyethyl]-3,5-bis(dodecyl-
oxy)benzamide (31). Deprotection of 23 with 0.1 M
MeONa/MeOH (8 mL) in CH2Cl2 (2 mL) for 8 h gave
1
31 (45.3 mg, 56%); H NMR (CDCl3 at 323 K) d=6.86
(2H, d, J=2 Hz, 2,6-H), 6.56 (2H, br-m, 4-H, NH), 4.87
(1H, br-d, Man-1-H), 3.95, 3.84, 3.8–3.5 (18H, m, 3,5-
O–CH2, N–C–CH2, N–CH2, Man-2,3,4,5,6,6-H, Man-
2,3,4,6-OH), 1.76 (4H, m, O–C–CH2), 1.44 (4H, br-m,
O–C2–CH2), 1.28 (32H, br-m, O–C3–C8H16), 0.89 (6H,
t, J=7 Hz, O–C11–CH3). MS (FAB) found: m/z 696.
Calcd for C39H69NO9: M+, 696.
N-[O-(ꢀ-D-Mannopyranosyl)oxyethyl]-N-methyl-3,5-bis-
(octadecyloxy)benzamide (36). Deprotection of 28 with
0.05 M MeONa/MeOH (1 mL) in CH2Cl2 (1 mL) for
20 h gave 36 (15.2 mg, 26%); 1H NMR (CDCl3 at
323 K14) d=6.49 (3H, br-s, 2,4,6-H), 4.85 (1H, br-s,
Man-1-H), 3.95 (5H, t, J=6 Hz, 3,5-O–CH2, Man-2-H),
3.84 (5H, br-m, N–C–CH2, Man-3,4,6-H), 3.64 (4H, br-

4022
Y. Azefu et al. / Bioorg. Med. Chem. 10 (2002) 4013–4022
m, N–CH2, Man-5,6-H), 3.06 (3H, br-s, N–CH3), 1.77
(4H, br-m, O–C–CH2), 1.45 (4H, br-m, O–C2–CH2),
1.28 (56H, br-m, O–C3–C14H28), 0.92 (6H, t, J=7 Hz,
O–C17–CH3). MS (FAB) found: m/z 901. Calcd for
C52H95NNaO9: [M+Na]+, 901.
An aq 10 mM NaOH solution (10 mL, 10 mL/min)
removed the binding protein to regenerate the lipid sur-
face. Aq 20 mM BSA (SIGMA, Albumin, Bovine), ricin
tetramer (WAKO, Ricin 120 solution) and Con A
(Nacalai Tesque, Concanavalin A from Jack Beans)
buffer solutions were used for the present measurement.
The used HPA chip was finally recycled by washing
with an aqueous 40 mM solution of octyl glucoside
(25 mL, 10 mL/min).
O-(ꢀ-D-Mannopyranosyl)oxyethyl 3,5-bis(octadecyloxy)-
benzyl ether (37). Deprotection of 29 with 0.1 M
MeONa/MeOH (0.5 mL) in CH2Cl2 (3.5 mL) for 15 h
1
gave 37 (11.9 mg, 78%); H NMR (CDCl3 at 323 K)
d=6.46 (2H, d, J=2 Hz, 2,6-H), 6.38 (1H, br-t, 4-H),
4.89 (1H, br-d, Man-1-H), 4.47 (2H, s, 1-CH2), 3.94
(5H, t, J=7 Hz, 3,5-O–CH2, Man-2-H), 3.87 (2H, br-s,
1-C–O–C–CH2), 3.82 (3H, m, Man-3,4,6-H), 3.63 (4H,
m, 1-C–O–CH2, Man-5,6-H), 2.56 (4H, br-s, Man-
2,3,4,6-OH), 1.76 (4H, m, O–C–CH2), 1.45 (4H, br-m,
O–C2–CH2), 1.28 (56H, br-m, O–C3–C14H28), 0.89 (6H,
t, J=7 Hz, O–C17–CH3). MS (FAB) found: m/z 874.
Calcd for C51H94NaO9: [M+Na]+, 874.
Acknowledgements
We thank Dr. Tomohiro Miyatake of Ryukoku Uni-
versity for measurement of FAB-MS spectra and Pro-
fessor Dr. Masakazu Kikuchi of Ritsumeikan
University for measurement of SPR sensorgrams.
References and Notes
Preparation of liposomes
1. Drickamer, K.;Taylor, M. E. Annu. Rev. Cell Biol. 1993, 9,
237.
2. Varki, A. Glycobiology 1993, 3, 97.
3. (a) Mortell, K. H.;Weatherman, R. V.;Kiessling, L. L. J.
Am. Chem. Soc. 1996, 118, 2297. (b) Mortell, K. H.;Gingras,
M.;Kiessling, L. L. J. Am. Chem. Soc. 1994, 116, 12053.
4. Lundquist, J. J.;Toone, E. J. Chem. Rev. 2002, 102, 555.
5. A review of syntheses of sphingosine: Koskinen, P. M.;
Koskinen, A. M. P. Synthesis 1998, 1075.
6. (a) Kunitake, T. Angew. Chem., Int. Ed. Engl. 1992, 31,
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An aqueous suspension of liposome was prepared by
either of the following two procedures: (a) a 5 mM etha-
nol solution (500 mL) of glycolipid was mixed with a
20 mM ethanol solution (500 mL) of EPC (SIGMA, l-a-
phosphatidylcholine from frozen egg yolk). The mixed
ethanol solution (400 mL) was injected into a magneti-
cally stirred HBS-N buffer (4.6 mL;Biacore AB)
through a Hamilton syringe (Hamilton, Reno) with
20 mL/min at 313 K. The resulting suspension was par-
tially evaporated to afford an almost ethanol-free lipo-
somal suspension;or (b) a 2 mM chloroform solution
(100 mL) of glycolipid, an 8 mM chloroform solution
(100 mL) of EPC, chloroform (600 mL) and methanol
(200 mL) was mixed in a vial. The mixture was dried
with a rotary evaporator and kept under high vacuum
for at least 2 h. The lipid mixture was hydrated in a
HBS-N buffer (1 mL) and shaken with a tube mixer.
One-half of the suspension of multilamellar vesicles was
extruded 11 times to give small unilamellar vesicles
through two polycarbonate membrane filters (AVES-
TIN, pore diameter 100 nm) mounted in the mini-
extruder (AVESTIN, LiposoFast) fitted with two
500 mL Hamilton syringes.15
8. Tamiaki, H.;Obata, T.;Azefu, Y.;Toma, K.
Soc. Jpn. 2001, 74, 733.
Bull. Chem.
9. Schmidt, R. R.;Kinzy, W. Adv. Carbohydr. Chem. Bio-
chem. 1994, 50, 21.
10. (a) Toma, K.;Sato, R.;Tamiaki, H. JP Patent 2001–
122889, 2001. (b) Tamiaki, H.;Azefu, Y.;Toma, K.;Sato, R.
JP Patent 2001–253896, 2001. (c) Sato, R.;Toma, K.;
Tamiaki, H. JP Patent 2002–30091, 2002.
11. (a) Kalb, E.;Frey, S.;Tamm, L. K. Biochim. Biophys.
Acta 1992, 1103, 307. (b) Kuziemko, G. M.;Stroh, M.;Ste-
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12. Sharon, N.;Lis, H. LECTINS;Chapman and Hall: Lon-
don, 1989.
SPR experiments
The hydrophobic surface of an HPA chip (Biacore AB)
was washed with 20 mM CHAPS solution (25 mL, 5 mL/
min;PIERCE). The above suspension of liposomes was
injected (60 mL, 1 mL/min) to generate an artificial lipid
monolayer. An aq 10 mM NaOH solution (5 mL, 5 mL/
min) washed away the weakly attached lipid vesicles.
After changing the solvents to a running buffer (HBS-N
buffer), binding assays were carried out by injection of
the aqueous solution of a protein (10 mL, 10 mL/min).
13. Metzger, R. M.;Wiser, D. C.;Laidlaw, R. K.;Takassi,
M. A. Langmuir 1990, 6, 350.
1
14. At 293 K, the H NMR spectrum was complex due to the
cis/trans mixture (see text). At 323 K, the cis and trans con-
formers exchanged so rapidly in the NMR measurement time
scale that the spectrum was simple.
15. MacDonald, R. C.;MacDonald, R. I.;Menco, B.P.M.;
Takeshita, K.;Subbarao, N. K.;Hu, L. R. Biochim. Biophys.
Acta 1991, 1061, 297.