Synthesis of the sialyl Lewis X epitope attached to glycolipids with different core structures and their selectin-binding characteristics in a dynamic test system

Article abstract of DOI:10.1002/(SICI)1521-3765(20000103)6:1<111::AID-CHEM111>3.0.CO;2-X

Sialyl Lewis X (sLe(X))/selectin-mediated leukocyte rolling along endothelial cells has recently gained wide interest. In this paper the influence of the spacer length of laterally clustered neoglycolipids 1a-d on cell rolling in a dynamic test system is

Full text of DOI:10.1002/(SICI)1521-3765(20000103)6:1<111::AID-CHEM111>3.0.CO;2-X

FULL PAPER
Synthesis of the Sialyl Lewis X Epitope Attached to Glycolipids
with Different Core Structures and their Selectin-Binding Characteristics
in a Dynamic Test System
Christian Gege,[a] Jan Vogel,[b] Gerd Bendas,[b] Ulrich Rothe,[c] and Richard R. Schmidt*[a]
Abstract: Sialyl Lewis X (sLeX)/selec-
tin-mediated leukocyte rolling along
endothelial cells has recently gained
wide interest. In this paper the influence
of the spacer length of laterally clustered
neoglycolipids 1a ± d on cell rolling in a
dynamic test system is investigated. The
required di-O-hexadecyl glycerols with
none, and with three, six, or nine ethyl-
ene glycol units as spacer groups (com-
pounds 4a ± d) could be readily ob-
tained. The synthesis of 1-O-thexyldi-
methylsilyl-protected sLeX 24 was based
on sialylation of 2,3,4-O-unprotected
galactose derivative 11 with sialyl phos-
phite 8 as donor; this afforded the
desired disaccharide 12, which was
transformed into trichloroacetimidate
14 as disaccharide donor. Reaction of
3-O-unprotected glucosamine derivative
18 with fucosyl donor 20 afforded dis-
accharide 21, which was transformed
into the 4-O-unprotected derivative 23.
Reaction of 14 with 23 furnished the
desired tetrasaccharide 24 in good yield.
Transformation of 24 into the trichlor-
oacetimidate 26 as donor, followed by
the reaction with 4a ± d as acceptor gave,
after deprotection, the target molecules
1a ± d. For comparison, 4d was also
connected with a sialyl residue (!31)
and with an N-acetylglucosamine resi-
due (!34). Compounds 1c and 1d with
a hexaethylene glycol and a nonaethy-
lene glycol spacer, respectively, were
much more efficient in mediating selec-
tin-dependent cell rolling in the dynamic
test system than compounds 1a and 1b,
which had no spacer (1a), or only a
triethylene glycol spacer (1b).
Keywords: cell adhesion ´ glycoli-
pids ´ membranes ´ molecular rec-
ognition ´ selectin
amines as binding epitopes; these are glycosidically linked to
the peptide backbone.[3±5] The tetrasaccharide sialyl Lewis X
(sLeX) (Neu5Aca2-3Galb1-4[Fuca1-3]GlcNAc) is of key
importance. The selectins bind these carbohydrates at their
N-terminal lectin domain. Although numerous studies per-
formed under static conditions have confirmed fundamental
findings about selectin-binding characteristics and the struc-
ture ± activity relationships of their binding epitopes,[6±9] they
do not provide information on how ligands interact with
selectins under dynamic conditions. Additional binding stud-
ies under the simulated shear force conditions of the
vasculature confirmed that tethering, rolling velocity, and
shear force resistance of neutrophils are regulated by the
kinetics of bond formation and dissociation, and are different
for each of the individual selectins.[10±12]
However, several questions about the essential structure of
the carbohydrate ligands for the mediation of rolling remain
unanswered. For instance, the molecular basis of the high
affinity of the natural sialomucins to the selectins could not be
fully elucidated, since single binding epitopes like sLeX exhibit
only low affinity.[13] Multiple protein ± carbohydrate interac-
tions are therefore thought to be the reason for a higher
binding efficiency mediated by a special molecular arrange-
ment of binding epitopes.[14] Therefore, various research
groups have prepared synthetic sLeX clusters, but these
Introduction
The recruitment of leukocytes to sites of injury or infection is
a receptor-mediated process essential for the immune re-
sponse. Leukocyte rolling along endothelial cells under the
shear force in postcapillary venules represents the first step in
a sequence of adhesive interactions that lead to firm attach-
ment and subsequent emigration through the venular wall.[1]
The selectins, a family of three adhesion molecules on both
the leukocyte and endothelial surfaces, are thought to mediate
rolling by binding carbohydrate-presenting ligands with rapid
association and dissociation rate constants.[2] The identified
natural selectin ligands are extended mucin-like glycoproteins
that present a series of sialylated and fucosylated polylactos-
[a] Prof. Dr. R. R. Schmidt, Dipl.-Chem. C. Gege
Fakultät für Chemie M726, Universität Konstanz
D-78457 Konstanz, (Germany)
Fax : (‡ 49)7531-88-3135
E-mail: rrs@dg3.chemie.uni-konstanz.de
[b] Dipl.-Pharm. J. Vogel, Dr. G. Bendas
Fachbereich Pharmazie, Martin-Luther-Universität Halle
Wolfgang-Langenbeck-Strasse 4, D-06120 Halle, (Germany)
[c] Priv. Doz. Dr. U. Rothe
Institut für Physiologische Chemie, Medizinische Fakultät
Martin-Luther-Universität Halle, Hollystrasse 1
D-06097 Halle, (Germany)
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R. R. Schmidt et al.
FULL PAPER
studies could only in part support this hypothesis of increased
affinity.[15, 16]
nature (Scheme 1, A), an oligoethylene glycol spacer was
selected instead. Its length was chosen in order to mimic b(1-
3)-linked lactose and/or lactosamine residues, that is, tri-
ethylene glycol units were combined. Thus, 1a (m ˆ n ˆ 0)
corresponds to a compound in which the sLeX moiety is
directly linked to the ceramide moiety, which has not been
found in nature, 1b (m ˆ 0, n ˆ 1) corresponds to natural sLeX,
1c (m ˆ n ˆ 1) to dimer sLeX, and 1d (m ˆ 2, n ˆ 1) to trimer
sLeX. In addition, in 1a ± d the ceramide residue is replaced by
the 1,2-di-O-hexadecyl glycerol moiety to avoid physical
demixing.[21]
Furthermore, the extended flexible structure of the sialo-
mucins is thought to be essential for the mediation of cell
rolling. However, lipid based sLeX molecules (glycosphingo-
lipids) at the leukocyte surface, as well as selectin ligands,
have been postulated to act in the rolling process.[17, 18]
In our previous study we introduced a dynamic model
system for the investigation of ligand characteristics that are
fundamental for the rolling process.[19] The adhesion and
rolling of selectin-presenting cells along a ligand-containing
supported membrane were analyzed; this reflects molecular
recognition requirements like ligand structure, concentration,
and lateral distribution. It was shown that sLeX-glycolipids
mediate a selectin-dependent cell rolling when they are
arranged in lateral clusters in the model membrane.[19] This
study supports the hypothesis of multivalent binding and
extends its applicability to glycolipids.
The present study focuses on the molecular features of
glycolipid ligands that are important for the mediation of
rolling. To this end, four sLeX glycolipids were synthesized
that differ in their spacer length between the hydrophobic
moiety and the carbohydrate headgroup. The resulting differ-
ences in flexibility and accessibility of the sLeX in these
compounds, when incorporated into the model membrane of
the flow chamber system, should give new insights into the
cell-rolling process.
Results and Discussion
Synthesis of sLeX neoglycolipids 1a ± 1d: The starting materi-
al for the molecules with 1,2-di-O-hexadecylglycerol was
the readily available 1,2-di-O-isopropylidene-sn-glycerol
(Scheme 2). Its transformation into known 1-O-benzyl-sn-
glycerol (2a) followed standard procedures.[22] Transforma-
tion of 2a into the di-O-hexadecyl intermediate 3a and then
hydrogenolytic O-debenzylation (!4a) was performed with
some modification of known procedures.[23]
For the attachment of one, two, or three triethylene glycol
units to the glycerol residue, commercially available triethyl-
ene glycol monochlorohydrin 5 was chosen; this was treated
with benzyl bromide in the presence of NaH as base to afford
O-benzyl-protected derivative 6. Reaction of 1,2-di-O-isopro-
pylidene-sn-glycerol with 6 in the presence of NaH as base
and tetrabutylammonium iodide (TBAI) as activator gave
alkylation product 7. Acid catalyzed de-O-isopropylidenation
(!2b), then O-alkylation with hexadecyl bromide under the
above described conditions (!3b), and finally hydrogenolytic
O-debenzylation afforded glycerol derivative 4b[24] with one
For the synthesis of the sLeX tetrasaccharide moiety a
highly convergent strategy was designed, which differed in the
protective-group pattern of the required glucosamine building
block and in the sequence of the connection of the four sugar
residues from previous strategies.[20]
In order to minimize the influence of the carbohydrate
backbone, to which the sLeX moiety is generally attached in
Scheme 1.
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Laterally Clustered Neoglycolipids
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yield (gated proton-decoupled
13C NMR:[28] JC-1,H-3ax ˆ 6.0 Hz).
Removal of the 1a-O-TDS
group by treatment with tetra-
butylammonium
fluoride
(TBAF) in THF/HOAc afford-
ed 1a-O-unprotected 13, which
on reaction with trichloroace-
tonitrile in the presence of 1,8-
diazabicyclo[5.4.0]undec-7-ene
(DBU) as base afforded tri-
chloroacetimidate 14 in almost
quantitative yield.
For the glucosamine-derived
acceptor, N-trichloroethoxycar-
bonyl (N-Teoc) protection was
selected because it is compat-
ible with standard protecting
group manipulations required
in oligosaccharide synthesis; as
a neighboring group it supports
b-glycoside bond formation,
and convenient replacement
by the N-acetyl group has
also been reported.[29] Thus,
the known compound 15
(Scheme 4) was transformed
into TDS-protected derivative
16, which on O-deacetylation
Scheme 2.
(!17) and ensuing 4,6-O-ben-
zylidenation gave 3-O-unpro-
O-unprotected triethylene glycol unit. Compound 4b served
also for the chain extension in the synthesis of 4c and 4d.
Reaction of 4b with alkylating agent 6 gave 3c under the
above described conditions, which afforded on hydrogenolytic
O-debenzylation compound 4c. Repetition of the same two
steps with 4c furnished via 3d compound 4d. Thus, all four
compounds 4a ± d are available that are required for the
synthesis of 1a ± d.
tected derivative 18. Additionally, known trichloroacetimi-
date 19 was obtained from 15; this was required at a later
stage of the synthetic studies. As fucosyl donor the trichloro-
acetimidate 20 of 2-O-benzyl-3,4-di-O-acetylfucose was
chosen as it has been shown to give a-fucopyranosides in
high yield.[30]
Fucosylation of 18 with donor 20 in the presence
of TMSOTf as catalyst afforded a-linked disaccharide 21 in
almost quantitative yield (1H NMR: J1b,2bˆ3.6 Hz). Acid-
catalyzed cleavage of the 4a,6a-O-benzylidene group (!22)
and then regioselective 6a-O-benzoylation afforded 4a-O-
unprotected 23. All yields could be improved over previously
published results.[31]
Glycosylation of acceptor 23 with disaccharide donor 14
(Scheme 5) was performed in dichloromethane, and TMSOTf
again served as catalyst, to afford the desired sLeX inter-
mediate 24 in 66% yield (1H NMR: J1c,2cꢀ8.3 Hz). 1a-O-
Desilylation with TBAF in THF/HOAc afforded 1a-O-
unprotected 25; treatment with trichloroacetonitrile in the
presence of DBU as base furnished trichloroacetimidate
26, which served as tetrasaccharide donor for the acceptors
4a ± d.
The glycosylation reactions of 4a ± d with 26 were per-
formed in dichloromethane as solvent and with TMSOTf as
the catalyst; they afforded the desired b-glycosides 27a ± d in
56 ± 88% yields (27d: 1H NMR: J1a,2a ꢀ 8.0 Hz, 13C NMR:
C-1a d ˆ 100.22). Treatment of 27a ± d with Zn in acetic
anhydride[29] led to replacement of the N-Teoc group by the N-
acetyl group; immediate hydrogenolytic O-debenzylation
For the synthesis of the sLeX epitope, proper ligation of N-
acetylneuraminic acid, d-galactose, l-fucose, and N-acetyl-d-
glucosamine is required. Several successful approaches to
solving this problem have already been reported.[9, 20] We
selected here a convergent strategy that seemed to be
particularly appropriate; we employed sialylation of a gal-
actose-derived acceptor and fucosylation of a glucosamine-
derived acceptor and finally ligation of the two disaccharides
to the tetrasaccharide. As the sialyl donor the known
phosphite derivative 8[25] (Scheme 3) was chosen. The high
acceptor reactivity of 2,3,4-O-unprotected galactose residues
in sialylation reactions[20, 25, 26] was reason to transform known
6-O-benzyl derivative 9 into thexyldimethylsilyl (TDS)-pro-
tected compound 10, which on O-deacetylation at low
temperatures afforded acceptor 11 without silyl group migra-
tion.[27] Sialylation of 11 with 8 in acetonitrile at 408C and in
the presence of trimethylsilyl trifluoromethanesulfonate
(TMSOTf) as the catalyst and then immediate O-acetylation
of the product mixture afforded the desired a-linked di-
saccharide 12, as derived from the NMR data, in 51% overall
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R. R. Schmidt et al.
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Scheme 3.
Scheme 4.
afforded partly O-acylated intermediates 28a ± d, which on
treatment with NaOMe in MeOH and then with KOH (0.2n)
gave target molecules 1a ± d; they were isolated as triethyl-
ammonium salts after chromatography with chloroform/
MeOH/H2O/NEt3 as eluents.
Cell-rolling investigations: In order to investigate the ability
of the compounds 1a ± d, 31a, 31b, and 34 to mediate selectin-
induced cell rolling, the previously described dynamic model
system was employed.[19] By means of the Langmuir± Blodg-
ett technique, the glycolipids were incorporated into a well
defined support-fixed model membrane and assembled into a
flow chamber. In contrast to the established static binding
assays, which solely focus on quantifying cell-binding events,[9]
this flow chamber assay considers the physiological function
of selectins and selectin ligands in the mediation of rolling of
fluorescently labeled selectin-presenting cells under physio-
logical shear force conditions; this process can be directly
followed by microscopic means. It was shown that selectin-
mediated cell rolling, as a special form of cell adhesion with a
much lower velocity than free flowing cells, is sensitively
balanced between firm adhesion and detachment by the
ligand densities in the model membrane. Whereas in static
In order to differentiate molecular recognition between
selectins on CHO-E cells and the sLeX epitope from
unspecified interactions, ligation of 4d directly to the sialyl
residue and to the N-acetylglucosamine residue was per-
formed for comparison. To this end, glycosylation of 4d with 8
was carried out; this gave a 1:1 a/b-mixture of glycosides
29a,b (Scheme 6). De-O-acetylation (!30a,b) and then
saponification of the ester moiety gave the desired 31a and
also 31b. Similarly, reaction of 4d with donor 19 led to b-
linked glycoside 32 (1H NMR: J1,2ˆ8.6 Hz) in high yield.
Treatment of 32 with Zn in acetic anhydride led to the N-
acetyl derivative 33, which furnished on treatment with
NaOMe in MeOH the desired N-acetylglucosamine deriva-
tive 34.
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Laterally Clustered Neoglycolipids
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arrangement was proven (data not shown). These membranes
were mounted into the flow chamber, and Chinese hamster
ovarian cells that stably express E-selectin (CHO-E) were
allowed to interact with the model membrane under static
conditions for five minutes. After that time, the cell adhesion
1
and rolling were analyzed at a shear rate of 200 s , as shown
in Figure 1. At concentrations above 0.1% of each compound
in the membranes, all cells in the analyzed area stay adhered
and did not show any mobility. Reduction of the ligand
concentrations led to great differences in cell-membrane
interactions. Whereas the spacerless compound 1a was no
longer able to mediate cell binding and all cells were detached
under the flow conditions, lower concentrations of the spacer-
containing compounds 1b ± d induced a cell movement that
could be defined as rolling (rolling velocity of about 5 ±
1
25 mms versus 1 ± 2 mms 1 of freely flowing cells).
Furthermore, spacer elongation, which corresponds to an
increased headgroup flexibility, is of crucial importance in
supporting effective cell rolling. The compounds 1c and 1d
with long spacers mediated a rolling at lower concentrations
down to 0.0025% in the lipid matrix compared with com-
pound 1b, which has a shorter spacer. To focus on the
influence of spacer length and structure on rolling, a
glycolipid with a naturally occurring, relatively stiff lactose
spacer between the sLeX headgroup and a ceramide moiety
[i.e. sLeX glycosphingolipid A (m ˆ 0, n ˆ 1)[20, 35], Scheme 1]
was used. Membranes containing this substance were nearly
as effective as those with compound 1b; this should be
attributed to the similar spacer length of these two com-
pounds, illustrated in Scheme 1. Because of the higher
flexibility of the ethylene glycol spacer in 1b, this substance
seems to be slightly more efficient in cell binding, as reflected
in the reduced cell-rolling velocity. All these cell-binding
events were of a specific nature, since preincubation of the
cells with a blocking E-selectin antibody prevented any cell-
membrane interactions. Compounds 31a, 31b, and 34 were
not able to interact with selectin-containing cells, since nearly
all cells were removed from these model membranes. This
demonstrates that a complete sLeX structure is essential for
selectin binding.
Scheme 5.
Thus, it could be demonstrated that sLeX glycolipids are
able to mediate a selectin-dependent rolling when they are
laterally clustered in a model phospholipid matrix, and
minimal flexibility and accessibility of the sLeX moiety is
sufficient to maintain cell rolling. Therefore, it can be
concluded that the extended silaomucin structures of the
natural ligands, which are extended and not well defined with
respect to structural requirements for mediating cell rolling,
are not ultimately necessary for rolling; however, the
sialomucin structures with their high flexibility offer optimal
conditions for a rolling event.
binding assays relatively high ligand concentrations or pure
ligand layers were used, in the dynamic system cell rolling
could be detected within a relatively low concentration range
(around 0.05% glycolipid incorporated in a phospholipid
matrix), which corresponds better to the physiological con-
ditions.
Furthermore, it could be proven that the phospholipid
matrix is of great importance because of its ligand organizing
effects. A clustering of glycolipid ligands within the matrix
was regarded to be essential for effective selectin recogni-
tion.[19] The compounds 1a ± d, 31a,b, and 34, with spacers of
different lengths, were investigated in order to focus on the
influence of sLeX mobility and accessibility at the membrane
surface on the rolling movement.
Conclusion
In the present study a series of sLeX glycolipids were
successfully synthesized which differed in their ethylene
glycol spacer length. Thus, the influence of the carbohydrate
headgroup mobility in glycolipids on the biological recogni-
Model membranes were prepared by incorporating differ-
ent concentrations of compounds 1a ± d into a 1,2-distearoyl-
sn-glycero-3-phosphocholine (DSPC) matrix and, as a pre-
requisite for the following investigations, their clustered
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Scheme 6.
Experimental Section
General techniques: Solvents were purified according to the standard
procedures. Flash chromatography was performed on J.T. Baker silica
gel 60 (0.040 ± 0.063 mm) at a pressure of 0.4 bar. Thin-layer chromatog-
raphy was performed on Merck silica gel plastic plates, 60F254 or Merck
silica gel glass plates, HPTLC 60F254; compounds were visualized by
treatment with a solution of (NH4)6Mo7O24 ´ 4H2O (20 g) and Ce(SO4)2
(0.4 g) in 10% sulfuric acid (400 mL) and heating at 1508C. Optical
rotations were measured on a Perkin ± Elmer polarimeter 241 in a 1 dm cell
at 228C. NMR measurements were recorded at 228C on a Bruker AC250
Cryospec or a Bruker DRX600. TMS or the resonance of the deuterated
solvent was used as internal standard; solvents: CDCl3, d ˆ 7.24; C6D6, d ˆ
7.15; D2O, d ˆ 4.63. Target molecules 1a ± d (max. 6 mg) were measured in a
320 mmolar solution of [D25]sodiumdodecyl sulfate (SDS) in 0.5 mL D2O.
MALDI-mass spectra were recorded on a Kratos Kompact MALDI I
instrument using a 2,5-dihydroxybenzoic acid matrix.
Figure 1. Selectin-mediated cell rolling along model membranes contain-
ing different ligand structures (see Scheme 1).
3-O-Benzyl-1,2-di-O-hexadecyl-sn-glycerol (3a): Sodium hydride (50 mg,
21 mmol) was added in small portions to a solution of 2a[22] (750 mg,
4.12 mmol), hexadecyl bromide (3.80 mL, 12.4 mmol), and a catalytic
amount of tetrabutylammonium iodide in dry DMF (15 mL) at room
temperature. After stirring for 36 h at room temperature, the mixture was
diluted with ethyl acetate (200 mL) and washed several times with brine.
The organic layer was dried over magnesium sulfate and concentrated
under reduced pressure. Flash chromatography (petroleum ether to
tion phenomena of selectin-dependent cell rolling could be
analyzed for the first time.
It was demonstrated that a minimal headgroup mobility is
an important factor for mediating cell rolling, since sLeX-
lipids with longer spacers were much more effective
than those with shorter spacers. The study gives an insight
into the mechanism of leukocyte rolling along the endothel-
ium and it provides structural information on natural selectin
ligands.
petroleum ether/ethyl acetate 25:1) afforded 3a (1.77 g, 68%) as
a
colorless oil. The physical properties found for 3a are in full accordance
with those described in reference [23].
1,2-Di-O-hexadecyl-sn-glycerol (4a): Compound 3a (1.72 g, 2.73 mmol)
was dissolved in THF/methanol (1:1, 20 mL) and palladium on charcoal
(0.17 g, 10% Pd) was added. The mixture was stirred vigorously under a
hydrogen atmosphere at normal pressure. After 16 h the catalyst was
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Laterally Clustered Neoglycolipids
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filtered off and washed with THF. Evaporation of the solvent and flash
chromatography (petroleum ether/ethyl acetate 19:1 to 9:1) furnished 4a
(1.28 g, 87%) as a colorless solid. The physical properties found for 3b are
in full accordance with those described in reference [23].
stirring for 15 h under reflux, additional 6 (7.20 g, 27.8 mmol) and sodium
hydride (0.67 g, 28 mmol) were added. After a further night under reflux
the mixture was filtered and washed with THF. Evaporation of the solvent
and flash chromatography (toluene/ethyl acetate 4:1 to 1:1) furnished pure
3c (22.2 g, 89%) as a colorless solid. Rf ˆ 0.38 (petroleum ether/ethyl
acetate 1:2); 1H NMR (250 MHz, CDCl3): d ˆ 0.88 (t, 3J ˆ 6.7 Hz, 6H;
2CH3), 1.25 (brs, 52H; 26 CH2), 1.52 ± 1.58 (m, 4H; 2OCH2CH2), 3.40 ±
3.66 (m, 33H; 33HCO), 4.57 (s, 2H; CH2Ph), 7.26 ± 7.34 (m, 5H; C6H5);
C54H102O9 (895.4): calcd C 72.44, H 11.48; found C 72.23, H 11.28%.
8-Benzyloxy-1-chloro-3,6-dioxaoctane (6): Sodium hydride (7.92 g,
330 mmol) was added in small portions to a solution of triethylene glycol
monochlorohydrin 5[32] (43.6 mL, 300 mmol) and benzyl bromide (107 mL,
900 mmol) in dry DMF (100 mL) at 08C. After stirring overnight at room
temperature, the mixture was filtered and concentrated under reduced
pressure. Flash chromatography (toluene to toluene/ethyl acetate 1:1)
afforded 6 (76.2 g, 98%) as a colorless oil. The physical properties found for
6 are in full accordance with those described in reference [33].
1,2-Di-O-hexadecyl-3-O-[17-hydroxy-3,6,9,12,15-pentaoxaheptadecyl]-sn-
glycerol (4c): Compound 3c (21.5 g, 24.0 mmol) was dissolved in THF
(300 mL), and palladium on charcoal (1.9 g, 10% Pd) was added. The
mixture was stirred vigorously under a hydrogen atmosphere at normal
pressure. After 16 h the catalyst was filtered off and washed. Evaporation
of the solvent furnished 4c (19.2 g, 99%) as a colorless solid. Rf ˆ 0.67
(CHCl3/methanol 15:1); 1H NMR (250 MHz, CDCl3): d ˆ 0.88 (t, 6H;
2CH3), 1.25 (brs, 52H; 26CH2), 1.50 ± 1.57 (m, 4H; 2OCH2CH2), 2.28 (brs,
1H; OH), 3.39 ± 3.75 (m, 33H; 33HCO); C47H96O9 (805.3): calcd C 70.10, H
12.02; found C 70.03, H 11.95%.
3-O-[8-Benzyloxy-3,6-dioxaoctyl]-1,2-O-isopropylidene-sn-glycerol
(7):
Sodium hydride (3.22 g, 134 mmol) was added to a solution of 1,2-di-O-
isopropylidene-sn-glycerol[32, 34] (11.8 g, 89.3 mmol), compound 6 (34.6 g,
134 mmol), and tetrabutylammonium iodide (1.7 g, 4.6 mmol) in dry THF
(150 mL). After stirring for 15 h under reflux, additional
6 (11.5 g,
44.5 mmol) and sodium hydride (1.07 g, 44.6 mmol) were added. After
6 h under reflux the solution was concentrated under reduced pressure, and
brine (500 mL) was slowly added. The solution was extracted with ethyl
acetate (3 Â 500 mL), the combined organic layers were dried over
magnesium sulfate and concentrated in vacuo. Flash chromatography
(toluene/ethyl acetate 2:1 to 1:1) yielded 7 (24.2 g, 76%) as a colorless oil.
Rf ˆ 0.64 (CHCl3/methanol 15:1); [a]D ˆ ‡8.9 (c ˆ 1.0 in CHCl3); 1H NMR
(250 MHz, CDCl3): d ˆ 1.36, 1.42 (2s, 6H; 2CH3), 3.49, 3.58 (2dd, 2J ˆ
10.0 Hz, 3J ˆ 5.6 Hz, 2H; CH2 glycerol), 3.61 ± 3.71 (m, 12H; 3OCH2-
3-O-[26-Benzyloxy-3,6,9,12,15,18,21,24-octaoxahexacosyl]-1,2-di-O-hexa-
decyl-sn-glycerol (3d): Sodium hydride (130 mg, 5.3 mmol) was added to a
solution of compound 4c (2.86 g, 3.55 mmol), compound
6 (1.38 g,
5.33 mmol), and tetrabutylammonium iodide (70 mg, 0.19 mmol) in dry
THF (100 mL). After stirring for 15 h under reflux additional 6 (0.92 g,
3.6 mmol) and sodium hydride (85 mg, 3.5 mmol) were added. After a
further night under reflux the mixture was filtered and washed with THF.
Evaporation of the solvent and flash chromatography (toluene/ethyl
acetate 4:1 to 2:1) furnished pure 3d (3.25 g, 89%) as a colorless solid.
Rf ˆ 0.51 (CHCl3/methanol 15:1); 1H NMR (250 MHz, CDCl3): d ˆ 0.88 (t,
6H; 2CH3), 1.25 (brs, 52H; 26CH2), 1.51 ± 1.57 (m, 4H; 2OCH2CH2),
3.36 ± 3.70 (m, 45H; 45HCO), 4.57 (s, 2H; CH2Ph), 7.26 ± 7.34 (m, 5H;
C6H5); C60H114O12 (1027.6): calcd C 70.13, H 11.18; found C 70.03, H
11.00%.
2
3
CH2O), 3.72, 4.04 (2dd, J ˆ 10.0 Hz, J ˆ 5.6 Hz, 2H; CH2 glycerol), 4.28
3
(tt, J ˆ 5.8 Hz and 6.2 Hz, 1H; CH glycerol), 4.57 (s, 2H; CH2Ph), 7.25 ±
7.35 (m, 5H; C6H5); C19H30O6 (354.4): calcd C 64.39, H 8.53; found C 63.94,
H 8.43%.
3-O-[8-Benzyloxy-3,6-dioxaoctyl)-sn-glycerol (2b): A solution of 7 (19.7 g,
55.6 mmol) in methanol (80 mL) was treated with a solution of hydro-
chloric acid (1m, 50 mL). After stirring for 1 h at room temperature, the
solution was concentrated in vacuo and coevaporated with toluene. The
residue was purified by flash chromatography (ethyl acetate/methanol 9:1)
to give 2b (16.5 g, 97%) as a colorless oil. Rf ˆ 0.40 (CHCl3/methanol 15:1);
1,2-Di-O-hexadecyl-3-O-[26-hydroxy-3,6,9,12,15,18,21,24-octaoxahexaco-
syl]-sn-glycerol (4d): Compound 3d (2.35 g, 2.29 mmol) was dissolved in
THF (60 mL), and palladium on charcoal (0.24 g, 10% Pd) was added. The
mixture was stirred vigorously under a hydrogen atmosphere at normal
pressure. After 16 h the catalyst was filtered off and washed. Evaporation
of the solvent and flash chromatography (toluene/acetone 1:1) furnished
4d (2.08 g, 97%) as a colorless solid. Rf ˆ 0.36 (toluene/acetone 1:1);
1H NMR (250 MHz, CDCl3): d ˆ 0.88 (t, 6H; 2CH3), 1.25 (brs, 52H;
26CH2), 1.50 ± 1.57 (m, 4H; 2OCH2CH2), 2.5 (brs, 1H; OH), 3.40 ± 3.75 (m,
45H; 45HCO); C53H108O12 (937.4): calcd C 67.91, H 11.61; found C 67.73, H
11.53%.
[a]D
ˆ
3.2 (c ˆ 1.0 in CHCl3); 1H NMR (250 MHz, CDCl3): d ˆ 2.48 (brs,
2H; 2OH), 3.56 ± 3.67 (m, 16H; 8CH2O), 3.85 (m, 1H; CH of glycerol),
4.57 (s, 2H; CH2Ph), 7.26 ± 7.35 (m, 5H; C6H5); C16H26O6 (314.4): calcd C
61.13, H 8.34; found C 60.92, H 8.35%.
3-O-[8-Benzyloxy-3,6-dioxaoctyl)-1,2-di-O-hexadecyl-sn-glycerol (3b): So-
dium hydride (4.38 g, 183 mmol) was added to a solution of 2b (16.4 g,
52.2 mmol), hexadecyl bromide (64 mL, 0.21 mol), and tetrabutylammo-
nium iodide (0.96 g, 2.6 mmol) in dry THF (250 mL). After stirring for 20 h
under reflux, methanol (50 mL) was added carefully, and the solution was
concentrated in vacuo. The residue was diluted with water (50 mL) and
subsequently extracted with ethyl acetate (2 Â 400 mL). The combined
organic layers were dried over magnesium sulfate, and the solvent was
evaporated. Purification was accomplished by flash chromatography
(toluene/ethyl acetate 9:1), which furnished pure 3b (29.4 g, 74%) as a
colorless solid. Rf ˆ 0.43 (petroleum ether/ethyl acetate 4:1); 1H NMR
(250 MHz, CDCl3): d ˆ 0.88 (t, 3J ˆ 6.6 Hz, 6H; 2CH3), 1.25 (brs, 52H;
26CH2), 1.50 ± 1.59 (m, 4H; 2OCH2CH2), 3.40 ± 3.71 (m, 21H; 21HCO),
4.57 (s, 2H; CH2Ph), 7.26 ± 7.35 (m, 5H; C6H5); C48H90O6 (763.2): calcd C
75.54, H 11.89; found C 75.82, H 11.86%.
Thexyldimethylsilyl 2,3,4-tri-O-acetyl-6-O-benzyl-b-d-galactopyranoside
(10): Compound 9[35] (28.7 g, 72.4 mmol) and imidazole (8.38 g, 123 mmol)
were dissolved in dry DMF (250 mL). Thexyldimethylsilyl chloride
(21.4 mL, 109 mmol) was added at room temperature to the mixture. After
stirring overnight, methanol (50 mL) was added. The solution was
concentrated in vacuo and then diluted with ethyl acetate (1000 mL).
The organic layer was washed with brine (3 Â 300 mL), dried over
magnesium sulfate, and evaporated. Flash chromatography (toluene/ethyl
acetate 2:1) gave compound 10 (37.1 g, 95%) as a colorless oil. Rf ˆ 0.41
(petroleum ether/ethyl acetate 4:1); [a]D
ˆ
15.8 (c ˆ 1.0 in CHCl3);
1H NMR (250 MHz, CDCl3): d ˆ 0.15, 0.18 (2s, 6H; Si(CH3)2), 0.83, 0.83,
0.84, 0.87 (4s, 12H; 4CH3), 1.55 ± 1.66 (m, 1H; CH), 1.98, 2.02, 2.06 (3s, 9H;
3COCH3), 3.46 ± 3.58 (m, 2H; 6-, 6'-H), 3.81 ± 3.87 (m, 1H; 5-H), 4.43, 4.56
(2d, 2J ˆ 12.0 Hz, 2H; CH2Ph), 4.69 (d, J(1,2) ˆ 7.5 Hz, 1H; 1-H), 4.99 (dd,
J(2,3) ˆ 10.5 Hz, J(3,4) ˆ 3.4 Hz, 1H; 3-H), 5.14 (dd, J(1,2) ˆ 7.5 Hz,
J(2,3) ˆ 10.5 Hz, 1H; 2-H), 5.44 (dd, J(3,4) ˆ 3.4 Hz, J(4,5) ˆ 1.1 Hz, 1H;
4-H), 7.26 ± 7.37 (m, 5H; C6H5); C27H42O9Si (538.7): calcd C 60.20, H 7.86;
found C 60.56, H 7.92%.
1,2-Di-O-hexadecyl-3-O-[8-hydroxy-3,6-dioxaoctyl)-sn-glycerol
(4b):
Compound 3b (27.1 g, 35.5 mmol) was dissolved in THF (400 mL), and
palladium on charcoal (1.3 g, 10% Pd) was added. The mixture was stirred
vigorously under a hydrogen atmosphere at normal pressure. After 16 h the
catalyst was filtered off and washed. Evaporation of the solvent and flash
chromatography (toluene/ethyl acetate 4:1) furnished 4b (23.0 g, 96%) as a
colorless solid. Rf ˆ 0.42 (petroleum ether/ethyl acetate 1:2); 1H NMR
(250 MHz, CDCl3): d ˆ 0.88 (t, 3J ˆ 6.6 Hz, 6H; 2CH3), 1.25 (brs, 52H;
26CH2), 1.51 ± 1.59 (m, 4H; 2OCH2CH2), 2.26 ± 2.31 (m, 1H; OH), 3.40 ±
3.75 (m, 21H; 21HCO); C41H84O6 (673.1): calcd C 73.16, H 12.58; found C
72.74, H 12.68%.
Thexyldimethylsilyl 6-O-benzyl-b-d-galactopyranoside (11): A solution of
10 (38.9 g, 72.2 mmol) in dry methanol (220 mL) was treated at 258C with
a solution of sodium methoxide (1m) in methanol (1.5 mL). After stirring
overnight at 158C the solution was neutralized with Amberlite IR120
‡
3-O-[17-Benzyloxy-3,6,9,12,15-pentaoxaheptadecyl]-1,2-di-O-hexadecyl-
sn-glycerol (3c): Sodium hydride (1.0 g, 42 mmol) was added to a solution
of compound 4b (18.8 g, 27.9 mmol), compound 6 (10.8 g, 41.9 mmol), and
tetrabutylammonium iodide (0.52 g, 1.4 mmol) in dry THF (200 mL). After
(H ), filtered, and evaporated. Flash chromatography (ethyl acetate)
yielded 11 (28.6 g, 96%) as a colorless oil. Rf ˆ 0.53 (ethyl acetate); [a]D
ˆ
4.5 (c ˆ 1.0 in CHCl3); 1H NMR (250 MHz, CDCl3): d ˆ 0.18, 0.19 (2s,
6H; Si(CH3)2), 0.87, 0.87, 0.89, 0.90 (4s, 12H; 4CH3), 1.59 ± 1.70 (m, 1H;
Chem. Eur. J. 2000, 6, No. 1
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
0947-6539/00/0601-0117 $ 17.50+.50/0
117

R. R. Schmidt et al.
FULL PAPER
CH), 3.55 ± 3.57 (m, 2H; 2-, 3-H), 3.62 (td, J(4,5) ˆ 1.0 Hz, J(5,6) ˆ J(5,6') ˆ
5.5 Hz, 1H; 5-H), 3.71 (dd, 2J ˆ 10.0 Hz, J(5,6) ˆ 5.4 Hz, 1H; 6-H), 3.78
(dd, 2J ˆ 10.0 Hz, J(5,6') ˆ 5.6 Hz, 1H; 6'-H), 3.98 (dd, J(3,4) ˆ 2.7 Hz,
J(4,5) ˆ 1.1 Hz, 1H; 4-H), 4.45 (d, J(1,2) ˆ 7.3 Hz, 1H; 1-H), 4.57 (s, 2H;
CH2Ph), 7.25 ± 7.39 (m, 5H; C6H5); C21H36O6Si (412.6): calcd C 61.13, H
8.79; found C 60.82, H 8.79%.
3.63 ± 3.65 (m, 1H; 6b-CH3), 3.83 or 3.84 (2s, 3H; COOCH3), 3.85 (dd,
J(5,6) ˆ J(5,6') ˆ 6.3 Hz, 1H; 5a-H), 3.95 ± 3.99 (m, 1H; 9b-H), 4.04 (ddd,
J(4,5) ˆ J(5,6) ˆ J(5,N) ˆ 10.4 Hz, 1H; 5b-H), 4.36 (d, 2J ˆ 12.3 Hz, 1H;
2
9'b-H), 4.41, 4.51 (d, J ˆ 11.8 Hz, 2H; CH2Ph), 4.59 (dd, J(2,3) ˆ 10.1 Hz,
J(3,4) ˆ 3.4 Hz, 1H; 3a-H), 4.82 (d, J(1,2) ˆ 8.0 Hz, 1H; 1a-H), 4.87 (m,
1H; 4b-H), 4.89 (dd, 1H; 2a-H), 5.00 (d, J(3,4) ˆ 3.4 Hz, 1H; 4a-H), 5.13 (d,
J(N,5) ˆ 10.4 Hz, 1H; NH), 5.35 (dd, J(6,7) ˆ 2.7 Hz, J(7,8) ˆ 9.0 Hz, 1H;
7b-H), 5.53 (ddd, J(7,8) ˆ 8.7 Hz, J(8,9) ˆ 2.7 Hz, J(8,9') ˆ 5.9 Hz, 1H; 8b-
Thexyldimethylsilyl O-(methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-di-
deoxy-d-glycero-a-d-galacto-2-nonulopyranosylonate)-(2 !3)-2,4-di-O-
acetyl-6-O-benzyl-b-d-galactopyranoside (12): Acceptor 11 (5.11 g,
12.4 mmol) was dissolved in dry acetonitrile (80 mL) over molecular sieves
(4 Š). After one hour the solution was added to 8[25] (12.7 g, 20.8 mmol) and
cooled to 408C. After addition of trimethylsilyl trifluoromethanesulfo-
nate (500 mL, 0.28 mmol), the solution was stirred for 45 min and then
neutralized with triethylamine (500 mL) and concentrated in vacuo. The
residue was dissolved in chloroform (200 mL) and washed with hydro-
chloric acid (1m). The organic layer was neutralized with sodium
bicarbonate and dried over magnesium sulfate. The remaining foam was
dissolved in pyridine (80 mL), and acetic anhydride (80 mL) was added.
After 2 days the solution was evaporated, and the residue was purified by
flash chromatography (toluene/acetone 3:1) to give 12 (6.16 g, 51%) as a
‡
‡
H), 7.24 ± 7.31 (m, 5H; C6H5); MALDI: m/z: 851 [M‡Na] , 868 [M‡K] ;
C37H49NO20 ´ H2O (845.8): calcd C 52.54, H 6.08, N 1.66; found C 52.59, H
6.24, N 1.59%.
O-(Methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-glycero-a-d-
galacto-2-nonulopyranosylonate)-(2 !3)-2,4-di-O-acetyl-6-O-benzyl-a/b-
d-galactopyranosyl trichloroacetimidate (14): Trichloroacetonitrile
(3.6 mL, 3.6 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (27 mL,
0.18 mmol) were added to a solution of 13 (2.95 g, 3.56 mmol) in dry
dichloromethane (100 mL). After 1 h the mixture was concentrated in
vacuo. Flash chromatography (toluene/acetone 2:1 ‡ 1% triethylamine)
furnished 14 (3.34 g, 97%) as a colorless foam in a ratio of a/b ꢀ 1:9. Rf ˆ
0.59 (toluene/acetone 1:1); 1H NMR (250 MHz, CDCl3): d ˆ 1.74 (dd, 2J ˆ
J(3ax,4) ˆ 12.4 Hz, 1H; 3bax-H), 2.01, 2.02, 2.04, 2.07, 2.07, 2.17, 2.18 (7s,
21H; 7COCH3), 2.59 (dd, 2J ˆ 12.6 Hz, J(3eq,4) ˆ 4.6 Hz, 1H; 3beq-H), 3.46
(dd, 2J ˆ 10.0 Hz, J(5,6) ˆ 6.9 Hz, 1H; 6a-H), 3.57 (dd, 2J ˆ 9.9 Hz, J(5,6') ˆ
5.7 Hz, 1H; 6'a-H), 3.65 (dd, J(5,6) ˆ 10.7 Hz, J(6,7) ˆ 2.7 Hz, 1H; 6b-
CH3), 3.88 (s, 3H; COOCH3), 3.98 (dd, 2J ˆ 12.4 Hz, J(8,9) ˆ 5.9 Hz, 1H;
9b-H), 3.98 ± 4.09 (m, 2H; 5a-, 5b-H), 4.32 ± 4.46 (m, 3H; 3a-, 9'b-H,
CHHPh), 4.55 (d, 2J ˆ 11.9 Hz, 1H; CHHPh), 4.72 (dd, J(2,3) ˆ 10.2 Hz,
J(3,4) ˆ 3.4 Hz, 1H; 3a-H), 4.90 (ddd, J(3ax,4) ˆ 12.1 Hz, J(3eq,4) ˆ 4.6 Hz,
J(4,5) ˆ 10.3 Hz, 1H; 4b-H), 5.04 (d, J(5,N) ˆ 10.1 Hz, 1H; NH), 5.11 (d,
J(3,4) ˆ 2.7 Hz, 1H; 4a-H), 5.30 (dd, J(1,2) ˆ 8.3 Hz, J(2,3) ˆ 10.1 Hz, 1H;
2a-H), 5.37 (dd, J(6,7) ˆ 2.7 Hz, J(7,8) ˆ 8.9 Hz, 1H; 7b-H), 5.58 (ddd,
J(7,8) ꢀ 8.6 Hz, J(8,9) ˆ 5.9 Hz, J(8,9') ˆ 2.5 Hz, 1H; 8b-H), 5.95 (d,
pale yellow foam. Rf ˆ 0.54 (toluene/acetone 1:1); [a]D
ˆ
11.4 (c ˆ 1.0 in
CHCl3); 1H NMR (600 MHz, CDCl3): d ˆ 0.15, 0.16 (2s, 6H; Si(CH3)2),
0.83 ± 0.87 (m, 12H; 2C(CH3)2), 1.53 ± 1.64 (m, 1H; CH), 1.71 (dd, 2J ˆ
J(3ax,4) ˆ 12.4 Hz, 1H; 3bax-H), 1.83, 1.98, 2.01, 2.03, 2.05, 2.13, 2.16 (7s,
21H; 7COCH3), 2.56 (dd, 1H; 3beq-H), 3.45 (dd, 2J ˆ 9.9 Hz, J(5,6) ˆ
6.1 Hz, 1H; 6a-H), 3.47 (dd, 2J ˆ 10.0 Hz, J(5,6') ˆ 6.2 Hz, 1H; 6'a-H),
3.62 (dd, J(5,6) ˆ 10.7 Hz, J(6,7) ˆ 2.8 Hz, 1H; 6b-CH3), 3.78 ± 3.81 (m,
2
1H; 5a-H), 3.82 (s, 3H; COOCH3), 4.00 (dd, J ˆ 12.4 Hz, J(8,9) ˆ 5.8 Hz,
1H; 9b-H), 4.04 (ddd, J(4,5) ˆ J(5,6) ˆ J(5,N) ˆ 10.5 Hz, 1H; 5b-H), 4.33
(dd, 2J ˆ 12.4 Hz, J(8,9') ˆ 2.7 Hz, 1H; 9'b-H), 4.42 (d, 2J ˆ 11.8 Hz, 1H;
CHHPh), 4.47 (dd, J(2,3) ˆ 10.2 Hz, J(3,4) ˆ 3.4 Hz, 1H; 3a-H), 4.51 (d,
2J ˆ 11.8 Hz, 1H; CHHPh), 4.83 (d, J(1,2) ˆ 7.7 Hz, 1H; 1a-H), 4.85 (ddd,
J(3ax,4) ˆ 12.1 Hz, J(3eq,4) ˆ 4.6 Hz, J(4,5) ˆ 10.3 Hz, 1H; 4b-H), 4.93 ±
4.96 (m, 2H; 2a-, 4a-H), 5.05 (d, J(5,N) ˆ 10.3 Hz, 1H; NH), 5.34 (dd,
J(6,7) ˆ 2.8 Hz, J(7,8) ꢀ 8.9 Hz, 1H; 7b-H), 5.53 (ddd, J(7,8) ꢀ 8.9 Hz,
J(8,9) ˆ 5.8 Hz, J(8,9') ˆ 2.7 Hz, 1H; 8b-H), 7.25 ± 7.31 (m, 5H; C6H5);
13C NMR (151 MHz, CDCl3): d ˆ 3.32 (SiCH3), 1.99 (SiCH3), 18.47,
18.51, 19.90, 19.97 (4CH3), 20.73, 20.76, 20.85, 21.07, 21.39, 23.16, 24.82
(7COCH3), 33.91 (CHMe2), 37.54 (3b-C), 49.10 (5b-C), 53.12 (OCH3),
62.36 (9b-C), 67.20 (7b-C), 67.90 (8b-C), 68.43 (4a-C), 68.49 (6a-C), 69.42
(4b-C), 71.76 (3a-C), 71.84 (2a-C), 72.01 (6b-C), 72.09 (5a-C), 73.37
(CH2Ph), 95.72 (2b-C), 96.90 (1a-C), 127.59 ± 138.04 (phenyl-C), 167.87 (1b-
C), 169.61, 169.67, 170.20, 170.33, 170.41, 170.46, 170.89 (7COMe);
C45H67NO20Si (970.1): calcd C 55.71, H 6.96, N 1.44; found C 55.27, H
6.80, N 1.28%.
ˆ
J(1,2) ˆ 8.3 Hz, 1H; 1a-H), 7.23 ± 7.35 (m, 5H; C6H5), 8.67 (s, 1H; NH);
C39H49Cl3N2O20 (972.2): calcd C 48.18, H 5.08, N 2.88; found C 47.99, H 5.24,
N 2.84%.
Thexyldimethylsilyl O-(methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-di-
deoxy-d-glycero-a-d-galacto-2-nonulopyranosylonate)-(2 !3)-(2,4-di-O-
acetyl-6-O-benzyl-b-d-galactopyranosyl)-(1 !4)-[(3,4-di-O-acetyl-2-O-
benzyl-a-l-fucopyranosyl)-(1!3)]-6-O-benzoyl-2-deoxy-2-(2,2,2-trichloro-
ethoxycarbonylamino)-b-d-glucopyranoside (24):
A solution of 14
(860 mg, 885 mmol) and 23[31] (1.51 g, 1.64 mmol) in dry dichloromethane
(10 mL) was treated at room temperature with trimethylsilyl trifluoro-
methanesulfonate (16 mL, 88 mmol). After stirring for 15 min, the mixture
was neutralized with triethylamine and concentrated in vacuo. The residue
was purified by flash chromatography (toluene/acetone 2:1) to give starting
material 23 and 24 (1.01 g, 66%) as a colorless foam. Rf ˆ 0.64 (toluene/
O-(Methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-glycero-a-d-
galacto-2-nonulopyranosylonate)-(2 !3)-2,4-di-O-acetyl-6-O-benzyl-a/b-
d-galactopyranose (13): Compound 12 (766 mg, 790 mmol) was dissolved in
dry THF (10 mL). Acetic acid (90 mL, 1.7 mmol) and then a solution of
tetrabutylammonium fluoride (1m) in THF (1.7 mL, 1.7 mmol) were added
at 258C. The mixture was slowly warmed to room temperature and
stirred for 3 days. After addition of water (100 mL) the solution was washed
with diethyl ether (5 Â 100 mL). The combined organic layers were dried
over magnesium sulfate and concentrated in vacuo. Purification was
accomplished by flash chromatography (toluene/acetone 3:1 to 2:1) to
furnish pure 13 (523 mg, 80%) as a colorless foam in a ratio of a/b ˆ 4:3.
Rf ˆ 0.39 and 0.42 (CHCl3/methanol 15:1); 1H NMR (600 MHz, CDCl3):
13a: d ˆ 1.65 ± 1.75 (m, 1H; 3bax-H), 1.83 (s, 3H; NCOCH3), 1.99 ± 2.22 (m,
21H; 7COCH3), 2.57 (dd, 2J ˆ 12.6 Hz, J(3eq,4) ˆ 4.5 Hz, 1H; 3beq-H), 3.38
acetone 1:1); [a]D
ˆ
24.7 (c ˆ 1.0 in CHCl3); 1H NMR (600 MHz, CDCl3):
d ˆ 0.01, 0.03 (2s, 6H; Si(CH3)2), 0.73 (2s, 6H; SiC(CH3)2), 0.77, 0.78 (2d,
3J ˆ 6.7 Hz, 6H; CH(CH3)2), 1.18 (d, J(5,6) ˆ 6.4 Hz, 3H; 6b-CH3), 1.54 ±
2
1.62 (m, 1H; CH), 1.67 (dd, J ˆ J(3ax,4) ꢀ 12.4 Hz, 1H; 3dax-H), 1.81 (2s,
6H; 2NCOCH3), 1.92, 1.97, 1.98, 2.03, 2.06, 2.10, 2.21 (7s, 21H, 7COCH3),
2
2.54 (dd, J ˆ 12.6 Hz, J(3eq,4) ˆ 4.3 Hz, 1H; 3deq-H), 2.93 (m, 1H; 2a-H),
3.58 (dd, J(5,6) ˆ 10.7 Hz, J(6,7) ˆ 2.8 Hz, 1H; 6d-H), 3.63 ± 3.66 (m, 2H;
5a-, 6c-H), 3.76 (t, J(5,6) ˆ 6.4 Hz, 1H; 5c-H), 3.79 (s, 3H; COOCH3),
3.84 ± 3.86 (m, 2H; 2b-, 6c-H), 3.92 (dd, J(3,4) ˆ J(4,5) ˆ 9.5 Hz, 1H; 4a-H),
4.01 (ddd, J(4,5) ˆ J(5,6) ˆ J(5,N) ˆ 10.4 Hz, 1H; 5d-H), 4.13 (dd, 2J ˆ
12.8 Hz, J(8,9) ˆ 4.0 Hz, 1H; 9d-H), 4.16 (dd, 2J ˆ 11.9 Hz, J(5,6) ˆ
5.8 Hz, 1H; 6a-H), 4.26 (dd, 2J ˆ 12.8 Hz, J(8,9') ˆ 2.1 Hz, 1H; 9'd-H),
4.30 (dd, J(2,3) ˆ J(3,4) ˆ 9.6 Hz, 1H; 3a-H), 4.49 ± 4.53 (m, 3H, 3c-H,
CHHPh, CHHCCl3), 4.62 (d, 2J ˆ 11.7 Hz, 1H; CHHPh), 4.67 (s, 2H;
CH2Ph), 4.72 (d, 2J ˆ 11.9 Hz, 1H; CHHCCl3), 4.83 (ddd, J(3ax,4) ˆ
J(4,5) ˆ 10.7 Hz, J(3eq,4) ˆ 4.6 Hz, 1H; 4d-H), 4.88 (d, J(1,2) ˆ 8.2 Hz,
1H; 1c-H), 4.95 (dd, J(1,2) ꢀ 8.2 Hz, J(2,3) ˆ 9.1 Hz, 1H; 2c-H), 4.99 ± 5.02
(m, 3H; 4c-H, 2NH), 5.04 ± 5.06 (m, 2H; 6a-, 5b-H), 5.12 (d, J(1,2) ˆ 7.7 Hz,
1H; 1a-H), 5.20 (d, J(1,2) ˆ 3.6 Hz, 1H; 1b-H), 5.26 ± 5.28 (m, 2H; 3b-, 4b-
H), 5.38 (dd, J(6,7) ˆ 2.7 Hz, J(7,8) ˆ 9.4 Hz, 1H; 7d-H), 5.56 (ddd, J(7,8) ˆ
9.3 Hz, J(8,9) ˆ J(8,9') ˆ 3.3 Hz, 1H; 8d-H), 7.15 ± 7.97 (m, 15H; 3C6H5);
13C NMR (151 MHz, CDCl3): d ˆ 15.89 (6b-C), 18.41 ± 24.64 (9COCH3,
4CH3), 34.04 (CHMe2), 37.39 (3d-C), 49.08 (5d-C), 53.14 (OCH3), 61.89
(2a-C), 62.01 (9d-C), 62.55 (6a-C), 64.19 (5b-C), 66.65 (7d-C), 67.64 (8d-C),
67.97 (6c-C), 68.11 (4c-C), 69.45 (4d-C), 69.91 (2c-C), 70.26 (3b-C), 71.59
2
2
(dd, J ˆ 9.7 Hz, J(5,6) ˆ 6.7 Hz, 1H; 6a-H), 3.48 (dd, J ˆ 9.7 Hz, J(5,6') ˆ
5.9 Hz, 1H; 6'a-H), 3.63 ± 3.65 (m, 1H; 6b-CH3), 3.83 or 3.84 (s, 3H;
COOCH3), 3.95 ± 3.99 (m, 1H; 9b-H), 4.06 (ddd, J(4,5) ˆ J(5,6) ˆ J(5,N) ˆ
10.4 Hz, 1H; 5b-H), 4.19 (dd, J(5,6) ˆ J(5,6') ˆ 6.3 Hz, 1H; 5a-H), 4.36 (d,
2J ˆ 12.3 Hz, 1H; 9'b-H), 4.41, 4.51 (2d, 2J ˆ 11.8 Hz, 2H; CH2Ph), 4.62 (dd,
J(2,3) ˆ 10.7 Hz, J(3,4) ˆ 3.5 Hz, 1H; 3a-H), 4.87 (m, 1H; 4b-H), 5.04 (d,
J(3,4) ˆ 3.4 Hz, 1H; 4a-H), 5.11 ± 5.15 (m, 2H; 2a-H, NH), 5.28 ± 5.30 (m,
2H; 1a-, 7b-H), 5.62 (ddd, J(7,8) ˆ 9.1 Hz, J(8,9) ˆ 6.7 Hz, J(8,9') ˆ 2.5 Hz,
1H; 8b-H), 7.24 ± 7.31 (m, 5H; C6H5). 13b: d ˆ 1.65 ± 1.75 (m, 1H; 3bax-H),
1.83 (s, 3H; NCOCH3), 1.99 ± 2.22 (m, 21H; 7COCH3), 2.57 (dd, 2J ˆ
12.6 Hz, J(3eq,4) ˆ 4.5 Hz, 1H; 3beq-H), 3.41(dd, 2J ˆ 9.8 Hz, J(5,6) ˆ
6.3 Hz, 1H; 6a-H), 3.51 (dd, 2J ˆ 9.2 Hz, J(5,6') ˆ 6.2 Hz, 1H; 6'a-H),
118
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Chem. Eur. J. 2000, 6, No. 1

Laterally Clustered Neoglycolipids
111±122
(3c-C), 71.94 (6d-C), 72.06 (4b-C), 72.88 (5c-C), 73.13 (PhCH2), 73.27 (5a-
C), 73.33 (PhCH2), 73.58 (3a-C), 74.24 (2b-C), 74.65 (CH2CCl3), 75.09 (4a-
C), 93.86 (1a-C), 95.15 (CH2CCl3), 96.94 (2d-C), 97.22 (1b-C), 99.84 (1c-C),
127.36 ± 138.66 (phenyl-C), 153.39 (COCH2CCl3), 165.62 (COPh), 167.71
(1d-C), 169.41, 169.53, 169.56, 170.01, 170.18, 170.36, 170.58, 170.76, 170.81
(9COMe); C78H103Cl3N2O33Si (1731.1): calcd C 54.12, H 6.00, N 1.62; found
C 54.10, H 6.00, N 2.01%.
di-O-acetyl-2-O-benzyl-a-l-fucopyranosyl)-(1 !3)]-6-O-benzoyl-2-deoxy-
2-(2,2,2-trichloroethoxycarbonylamino)-b-d-glucopyranoside (27a): A sol-
ution of 4a (192 mg, 355 mmol) and 26 (410 mg, 237 mmol) in dry
dichloromethane (4 mL) was treated at room temperature with a solution
of trimethylsilyl trifluoromethanesulfonate (0.25m) in dichloromethane
(95 mL, 24 mmol). After stirring for 30 min, the mixture was neutralized
with triethylamine and concentrated in vacuo. The residue was purified by
flash chromatography (toluene/acetone 4:1) to give 27a (400 mg, 80%) as a
(Methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-glycero-a-d-
galacto-2-nonulopyranosylonate)-(2 !3)-(2,4-di-O-acetyl-6-O-benzyl-b-d-
galactopyranosyl)-(1 !4)-[(3,4-di-O-acetyl-2-O-benzyl-a-l-fucopyrano-
syl)-(1 !3)]-6-O-benzoyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylami-
no)-a-d-glucopyranose (25): Compound 24 (1.42 g, 820 mmol) was dis-
solved in dry THF (10 mL). Acetic acid (320 mL, 5.6 mmol) and then a
solution of tetrabutylammonium fluoride (1m) in THF (1.1 mL, 1.1 mmol)
were added at room temperature, and the mixture was stirred for 5 days.
An additional solution of tetrabutylammonium fluoride (1m) in THF
(0.1 mL, 0.1 mmol) was added, and the mixture was stirred for a further
4 days. After the addition of a saturated sodium bicarbonate solution
(20 mL), the mixture was extracted with ethyl acetate (3 Â 100 mL). The
combined organic layers were dried over magnesium sulfate and concen-
trated in vacuo. Purification was accomplished by flash chromatography
(toluene/acetone 3:2), which furnished pure 25 (1.14 g, 87%) as a colorless
colorless foam. Rf ˆ 0.23 (toluene/acetone 3:1); [a]D
ˆ
20.3 (c ˆ 1.0 in
CHCl3); C105H155Cl3N2O35 (2111.7): calcd C 59.72, H 7.40, N 1.33; found C
59.46, H 7.52, N 1.69%.
[8-(1,2-Di-O-hexadecyl-sn-glycer-3-oxy)-3,6-dioxaoct-1-yl]-O-(methyl-5-
acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-glycero-a-d-galacto-2-non-
ulopyranosylonate)-(2 !3)-(2,4-di-O-acetyl-6-O-benzyl-b-d-galactopyra-
nosyl)-(1 !4)-[(3,4-di-O-acetyl-2-O-benzyl-a-L-fucopyranosyl)-(1 !3)]-
6-O-benzoyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-b-d-gluco-
pyranoside (27b): A solution of 4b (220 mg, 327 mmol) and 26 (330 mg,
190 mmol) in dry dichloromethane (5 mL) was treated at room temperature
with a solution of trimethylsilyl trifluoromethanesulfonate (0.25m) in
dichloromethane (75 mL, 19 mmol). After stirring for 30 min, the reaction
mixture was neutralized with triethylamine and concentrated in vacuo. The
residue was purified by flash chromatography (toluene/acetone 5:1 to 3:1)
to give 27b (375 mg, 88%) as a colorless foam. Rf ˆ 0.64 (toluene/acetone
1
foam. Rf ˆ 0.41 (CHCl3/methanol 15:1); H NMR (600 MHz, CDCl3): d ˆ
2
1.12 (d, J(5,6) ˆ 6.2 Hz, 3H; 6b-CH3), 1.53 (s, 3H; COCH3), 1.71 (dd, J ˆ
1:1); [a]D
ˆ
19.4 (c ˆ 1.0 in CHCl3); C111H167Cl3N2O38 (2243.9): calcd C
J(3ax,4) ꢀ 12.4 Hz, 1H; 3dax-H), 1.79, 1.81, 1.96, 2.00, 2.08, 2.08, 2.10, 2.20
(8s, 24H; 8COCH3), 2.52 (dd, 2J ˆ 12.6 Hz, J(3eq,4) ˆ 4.3 Hz, 1H; 3deq-H),
3.55 (dd, J(5,6) ˆ 10.7 Hz, J(6,7) ˆ 2.8 Hz, 1H; 6d-H), 3.60 (m, 1H; 5c-H),
59.42, H 7.50, N 1.25; found C 59.68, H 7.26, N 1.80%.
[17-(1,2-Di-O-hexadecyl-sn-glycer-3-oxy)-3,6,9,12,15-pentaoxaheptadec-1-
yl]-O-(methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-glycero-a-
d-galacto-2-nonulopyranosylonate)-(2 !3)-(2,4-di-O-acetyl-6-O-benzyl-
b-d-galactopyranosyl)-(1 !4)-[(3,4-di-O-acetyl-2-O-benzyl-a-l-fucopyra-
nosyl)-(1 !3)]-6-O-benzoyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylami-
no)-b-d-glucopyranoside (27c): A solution of 4c (326 mg, 405 mmol) and 26
(420 mg, 242 mmol) in dry dichloromethane (8 mL) was treated at room
temperature with a solution of trimethylsilyl trifluoromethanesulfonate
(0.25m) in dichloromethane (100 mL, 25 mmol). After stirring for 30 min,
the mixture was neutralized with triethylamine and concentrated in vacuo.
The residue was purified by flash chromatography (toluene/acetone 3:1 to
2:1) to give 27c (321 mg, 56%) as a colorless foam. Rf ˆ 0.28 (toluene/
2
3.73 (dd, J ˆ 10.4 Hz, J(5,6) ˆ 6.4 Hz, 1H; 6c-H), 3.76 (s, 3H; COOCH3),
3.80 (dd, J(2,3) ˆ 10.5 Hz, J(1,2) ˆ 3.3 Hz, 1H; 2b-H), 3.91 ± 4.05 [m, 5H;
H,H-COSY: 3.91 (d, 9d-H), 3.95 (dd, 6c-H), 3.98 (dd, 5d-H), 4.00 (d,
CHHCCl3), 4.05 (brd, 5a-H)], 4.13 ± 4.14 (m, 2H; 2a-, 3a-H), 4.31 ± 4.36 [m,
3H; H,H-COSY: 4.32 (s, 4c-H), 4.33 (dd, 4a-H), 4.36 (d, 6a-H)], 4.52 ± 4.66
2
2
[m, 5H; H,H-COSY: 4.53 (d, J ˆ 12.3 Hz, CHHPh), 4.54 (d, J ˆ 11.6 Hz,
CHHPh), 4.62 (d, 9d-H), 4.64 (d, CHHPh), 4.65 (d, CHHCCl3)], 4.75 (d, 2J
ˆ12.3 Hz, 1H; CHHPh), 4.81 (m, 1H; 4d-H), 4.84 (d, 2J ˆ 11.1 Hz, 1H; 6a-
H), 4.89 (m, 1H; 4c-H), 4.93 ± 4.98 [m, 3H; H,H-COSY: 4.94 (d, 1c-H), 4.94
(Nd-H), 4.97 (dd, 2c-H)], 5.05 (m, 1H; 5b-H), 5.24 ± 5.28 [m, 3H; H,H-
COSY: 5.24 (s, 1a-H), 5.25 (d, 3b-H), 5.28 (s, 4b-H)], 5.42 (dd, J(6,7) ˆ
2.6 Hz, J(7,8) ˆ 10.1 Hz, 1H; 7d-H), 5.45 (d, J(1,2) ˆ 2.9 Hz, 1H; 1b-H),
5.61 (d, J(7,8) ˆ 10.0 Hz, 1H; 8d-H), 6.36 (d, J(2,N) ˆ 5.7 Hz, 1H; Na-H),
7.21 ± 7.94 (m, 15H; 3C6H5); 13C NMR (151 MHz, CDCl3): d ˆ 16.09 (6b-C),
20.42 ± 23.21 (9COCH3), 37.24 (3d-C), 49.01 (5d-C), 53.11 (OCH3), 56.55
(2a-C), 62.04 (9d-C), 63.05 (6a-C), 64.06 (5b-C), 65.69 (7d-C), 67.30 (8d-C),
67.98 (4c-C), 68.17 (6c-C), 69.43 (5a-C), 69.52 (4d-C), 70.11 (3b-C), 70.20
(2c-C), 71.57 (6d-C), 71.68 (3c-C), 72.05 (4b-C), 72.11 (3a-C), 72.17
(CH2Ph), 73.02 (2b-C), 73.36 (5c-C), 73.63 (CH2Ph), 73.93 (4a-C), 74.91
(CH2CCl3), 92.27 (1a-C), 96.91 (2d-C), 97.41 (1b-C), 99.67 (1c-C), 127.36 ±
138.77 (phenyl-C), 154.75 (COCH2CCl3), 167.36 ± 171.78 (1d-C, 10COMe/
Ph); C70H85Cl3N2O33 (1588.8): calcd C 52.92, H 5.39, N 1.76; found C 53.24,
H 5.62, N 2.05%.
acetone 2:1); [a]D
ˆ
15.6 (c ˆ 1.0 in CHCl3); C117H179Cl3N2O41 (2376.1):
calcd C 59.14, H 7.59, N 1.18; found C 59.40, H 7.75, N 1.31%.
[26-(1,2-Di-O-hexadecyl-sn-glycer-3-oxy)-3,6,9,12,15,18,21,24-octaoxahex-
acos-1-yl]-O-(methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-
glycero-a-d-galacto-2-nonulopyranosylonate)-(2 !3)-(2,4-di-O-acetyl-6-
O-benzyl-b-d-galactopyranosyl)-(1 !4)-[(3,4-di-O-acetyl-2-O-benzyl-a-l-
fucopyranosyl)-(1 !3)]-6-O-benzoyl-2-deoxy-2-(2,2,2-trichloroethoxycar-
bonylamino)-b-d-glucopyranoside (27d):
A solution of 4d (487 mg,
520 mmol) and 26 (450 mg, 260 mmol) in dry dichloromethane (10 mL)
was treated at room temperature with solution of trimethylsilyl
trifluoromethanesulfonate (0.25m) in dichloromethane (100 mL, 25 mmol).
After stirring for 30 min, the mixture was neutralized with triethylamine
and concentrated in vacuo. The residue was purified by flash chromatog-
raphy (ethyl acetate/methanol 9:1) to give 27d (380 mg, 58%) as a colorless
a
O-(Methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-glycero-a-d-
galacto-2-nonulopyranosylonate)-(2 !3)-(2,4-di-O-acetyl-6-O-benzyl-b-d-
galactopyranosyl)-(1 !4)-[(3,4-di-O-acetyl-2-O-benzyl-a-l-fucopyrano-
syl)-(1 !3)]-6-O-benzoyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylami-
no)-a-d-glucopyranosyl trichloroacetimidate (26): Trichloroacetonitrile
(520 mL, 5.2 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (4 mL) was
added to a solution of 25 (817 mg, 514 mmol) in dry dichloromethane
(10 mL). After 1 h the mixture was concentrated in vacuo. Flash
chromatography (toluene/acetone 3:1‡1% triethylamine) furnished 26
foam. Rf ˆ 0.48 (ethyl acetate/methanol 9:1); [a]D
ˆ
13.8 (c ˆ 1.0 in
1
3
CHCl3); H NMR (600 MHz, CDCl3): d ˆ 0.88 (t, J ꢀ 7.0 Hz, 6H; 2CH3),
1.20 (d, J(5,6) ˆ 6.9 Hz, 3H; 6b-CH3), 1.25 (brs, 52H; 26CH2), 1.55
(quintet, 3J ꢀ 6.8 Hz, 4H; 2OCH2CH2), 1.69 ± 1.71 (m, 1H; 3dax-H), 1.70,
1.83, 1.90, 1.99, 2.02, 2.05, 2.08, 2.10, 2.22 (9s, 27H; 9COCH3), 2.56 (dd, 2J ˆ
12.6 Hz, J(3eq,4) ˆ 4.5 Hz, 1H; 3deq-H), 3.40 ± 3.90 [m, 57H; H,H-COSY:
3.39 (2a-H), 3.59 (5c-H), 3.59 (6d-H), 3.64 (5a-H), 3.70 (6c-H), 3.70
(CHHPh), 3.79 (s, 3H; COOCH3), 3.84 (2b-H), 3.84 (6'c-H), 3.90
(CHHPh), 18CH2 spacer, 5H glycerol, 2OCH2CH2], 4.02 (ddd, J(4,5) ˆ
J(5,6) ˆ J(5,N) ˆ 10.4 Hz, 1H; 5d-H), 4.11 (dd, J(3,4) ˆ J(4,5) ꢀ 9.0 Hz,
1H; 4a-H), 4.18 ± 4.25 [m, 3H; H,H-COSY: 4.18 (dd, 2J ˆ 12.9 Hz, J(8,9) ˆ
3.7 Hz, 9d-H), 4.20 (dd, 3a-H), 4.23 (dd, 2J ˆ 12.1 Hz, J(5,6) ˆ 4.3 Hz, 6a-
(828 mg, 93%) as a colorless foam. Rf ˆ 0.56 (toluene/acetone 1:1); [a]D
ˆ
6.5 (c ˆ 1.0 in CHCl3); 1H NMR (250 MHz, CDCl3): d ˆ 1.20 (d, J(5,6) ˆ
6.5 Hz, 3H; 6b-CH3), 1.6 ± 1.7 (m, 1H; 3dax-H), 1.75 ± 2.21 (m, 27H;
9COCH3), 2.56 (dd, 2J ˆ 12.6 Hz, J(3ex,4) ˆ 4.6 Hz, 1H; 3deq-H), 3.59 (dd,
J(5,6) ˆ 10.7 Hz, J(6,7) ˆ 2.9 Hz, 1H; 6d-H), 3.66 ± 5.60 (m, 32H; 2 1-H, 3
2-H, 3 3-H, 4 4-H; 4 5-H, 4 6-H, 7-H, 8-H, 2 9-H, 2NH, 2CH2Ph, CH2CCl3),
6.35 (d, J(1,2) ˆ 3.6 Hz, 1H; 1a-H), 7.23 ± 7.97 (m, 15H; 3C6H5), 8.69 (s, 1H;
2
H)], 4.29 (dd, J ˆ 12.9 Hz, J(8,9') ˆ 2.6 Hz, 1H; 9'd-H), 4.48 (dd, J(2,3) ꢀ
9.0 Hz, J(3,4) ꢀ 3.4 Hz, 1H; 3c-H), 4.53 (d, 2J ˆ 11.6 Hz, 1H; CHHPh),
4.58, 4.74 (2d, 2J ˆ 11.8 Hz; 1H; CH2CCl3 conformational exchange), 4.60,
4.78 (2d, 2J ˆ 12.0 Hz, 1H; CH2CCl3 conformational exchange), 4.61 (d,
2J ˆ 11.6 Hz, 1H; CHHPh), 4.84 (ddd, J(3ax,4) ˆ 11.9 Hz, J(3eq,4) ˆ 4.6 Hz,
J(4,5) ˆ 10.5 Hz, 1H; 4d-H), 4.95 ± 5.05 [m, 6H; H,H-COSY: 4.95 (d,
J(1,2) ꢀ 8.0 Hz; 1a-H), 4.96 (d, 1c-H), 4.96 (dd, 2c-H), 4.99 (s, 4c-H), 5.02 (d,
NdH), 5.03 (d, 6a-H)], 5.08 (q, J(5,6) ˆ 6.6 Hz, 1H; 5b-H), 5.28 ± 5.32 [m,
ˆ
NH); C72H85Cl6N3O33 (1733.2): calcd C 49.90, H 4.94, N 2.42; found C
49.96, H 5.14, N 2.64%.
(1,2-Di-O-hexadecyl-sn-3-glyceryl)-O-(methyl-5-acetamido-4,7,8,9-tetra-
O-acetyl-3,5-dideoxy-d-glycero-a-d-galacto-2-nonulopyranosylonate)-
(2 !3)-(2,4-di-O-acetyl-6-O-benzyl-b-d-galactopyranosyl)-(1 !4)-[(3,4-
Chem. Eur. J. 2000, 6, No. 1
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
0947-6539/00/0601-0119 $ 17.50+.50/0
119

R. R. Schmidt et al.
FULL PAPER
3H; H,H-COSY: 5.29 (d, 3b-H), 5.32 (s, 4b-H), 5.34 (s, 1b-H)], 5.41 (dd,
J(6,7) ˆ 2.8 Hz, J(7,8) ˆ 9.5 Hz, 1H; 7d-H), 5.57 (ddd, J(7,8) ˆ 9.5 Hz,
J(8,9) ˆ J(8,9') ˆ 3.2 Hz, 1H; 8d-H), 5.76 (brd, 1H; NaH), 7.23 ± 7.98 (m,
15H; 3C6H5); 13C NMR (151 MHz, CDCl3): d ˆ 14.09 (2CH3), 15.91 (6b-
C), 20.72 ± 23.17 (9COCH3), 20.07 ± 31.89 (CH2 alkyl), 37.40 (3d-C), 49.04
(5d-C), 53.11 (OCH3), 59.18 (2a-C), 61.96 (9d-C), 62.32 (6a-C), 64.16 (5b-
C), 66.58 (7d-C), 67.45 (8d-C), 67.92 (CH2Ph), 68.03 (4c-C), 68.70 (6c-C),
69.46 (4d-C), 70.07 (2c-C), 70.24 (3b-C), 70.37 ± 71.39 (C spacer, C glycerol,
5a-C, 5c-C, 6d-C), 71.54 (3c-C), 72.02 (4b-C), 72.94, 74.49 (CH2CCl3
conformational exchange), 73.32 (CH2Ph), 73.70 (2b-C), 74.06 (3a-C),
74.26 (4a-C), 95.71 (CH2CCl3), 96.96 (2d-C), 97.09 (1b-C), 99.56 (1c-C),
100.22 (1a-C), 127.32 ± 138.69 (phenyl-C), 154.01 (COCH2CH3), 165.63
(COPh), 167.65 (1d-C), 169.33, 169.41, 169.44, 169.83, 170.20, 170.37, 170.62,
170.72, 170.76 (9COMe); C123H191Cl3N2O44 ´ 2H2O (2544.3): calcd C 58.07,
H 7.72, N 1.10; found C 58.00, H 7.71, N 1.04%.
80:20:2:0.1 to 70:30:5:0.1) yielded 1a (110 mg, 65%) as a colorless powder
after lyophilization from water. Rf ˆ 0.23 (CHCl3/methanol/0.2% aqueous
CaCl2 70:30:5, HPTLC); [a]D
ˆ
30.1 (c ˆ 1.0 in CHCl3); 1H NMR
(600 MHz, SDS/D2O): d ˆ 0.84 (m, 6H; 2CH3), 1.20 (d, J(5,6) ˆ 6.3 Hz,
3H; 6b-CH3), 1.25 ± 1.36 (m, 52H; 26CH2), 1.41 (t, 9H; N(CH2CH3)3), 1.60
(brs, 4H; 2OCH2CH2), 1.85 (t, 1H; 3dax-H), 2.06, 2.07 (2s, 6H; 2COCH3),
2.78 (dd, 1H; 3deq-H), 3.24 (q, 3J ˆ 7.3 Hz, 6H; N(CH2Me)3), 3.40 ± 4.15 (m,
30H), 4.53 (d, J(1,2) ˆ 7.5 Hz, 1H; 1c-H), 4.59 (brs, 1H; 1a-H), 4.84 (q,
J(5,6) ˆ 6.5 Hz, 1H; 5b-H), 5.13 (brd, 1H; 1b-H); C72H137N3O25 ´ 4H2O
(1564.9): calcd C 55.26, H 9.34, N 2.69; found C 55.33, H 9.68, N 3.08%.
[8-(1,2-Di-O-hexadecyl-sn-glycer-3-oxy)-3,6-dioxaoct-1-yl]-O-(triethylam-
monium-5-acetamido-3,5-dideoxy-d-glycero-a-d-galacto-2-nonulopyrano-
sylonate)-(2 !3)-(b-d-galactopyranosyl)-(1 !4)-[a-l-fucopyranosyl)-
(1 !3)]-2-acetamido-2-deoxy-b-d-glucopyranoside (1b): Compound 28b
(100 mg, 52 mmol) was treated as described above to furnish 1b (45.4 mg,
55%) as a colorless powder. Rf ˆ 0.24 (CHCl3/methanol/0.2% aqueous
(1,2-Di-O-hexadecyl-sn-3-glyceryl)-O-(methyl-5-acetamido-4,7,8,9-tetra-
O-acetyl-3,5-dideoxy-d-glycero-a-d-galacto-2-nonulopyranosylonate)-
(2 !3)-(2,4-di-O-acetyl-b-d-galactopyranosyl)-(1 !4)-[(3,4-di-O-acetyl-
a-l-fucopyranosyl)-(1 !3)]-2-acetamido-6-O-benzoyl-2-deoxy-b-d-gluco-
pyranoside (28a): A solution of 27a (360 mg, 242 mmol) in THF/acetic
anhydride/acetic acid (3:2:1, 12 mL) was treated with activated zinc powder
(420 mg, activation with 2% CuSO4 in water for 5 min). The mixture was
stirred overnight at room temperature and then filtered and washed with
THF. The solvent was evaporated under reduced pressure, and the residue
was separated from zinc salts by flash chromatography (toluene/acetone
2:1). The product was dissolved in THF (8 mL) and acetic acid (2 drops).
Palladium on charcoal (150 mg, 10% Pd) was added, and the solution was
stirred vigorously under a hydrogen atmosphere for 2 days. The catalyst
was filtered off and washed with THF. Evaporation and purification by
flash chromatography (toluene/acetone 2:1) gave 28a (233 mg, 76%) as a
CaCl2 70:30:5, HPTLC); [a]D
ˆ
27.2 (c ˆ 1.0 in CHCl3); 1H NMR
(600 MHz, SDS/D2O): d ˆ 0.80 (m, 6H; 2CH3), 1.17 (d, J(5,6) ˆ 6.3 Hz,
3H; 6b-CH3), 1.21 ± 1.32 (m, 61H; 26CH2, N(CH2CH3)3), 1.57 (brs, 4H;
2OCH2CH2), 1.81 (t, 1H; 3dax-H), 2.02, 2.04 (2s, 6H; 2COCH3), 2.76 (dd,
1H; 3deq-H), 3.20 (q, 3J ˆ 7.3 Hz, 6H; N(CH2Me)3), 3.43 ± 4.13 (m, 42H),
4.53 (d, J(1,2) ˆ 7.7 Hz, 1H; 1c-H), 4.61 (d, J(1,2) ˆ 6.4 Hz, 1H; 1a-H), 4.83
(q, J(5,6) ˆ 6.3 Hz, 1H; 5b-H), 5.12 (d, J(1,2) ˆ 3.6 Hz, 1H; 1b-H);
C78H149N3O28 ´ 5H2O (1667.1): calcd C 56.20, H 9.61, N 2.52; found C
56.00, H 9.63, N 2.14%.
[17-(1,2-Di-O-hexadecyl-sn-glycer-3-oxy)-3,6,9,12,15-pentaoxaheptadec-1-
yl]-O-(triethylammonium-5-acetamido-3,5-dideoxy-d-glycero-a-d-galac-
to-2-nonulopyranosylonate)-(2 !3)-(b-d-galactopyranosyl)-(1 !4)-[a-l-
fucopyranosyl)-(1 !3)]-2-acetamido-2-deoxy-b-d-glucopyranoside (1c):
Compound 28c (182 mg, 88 mmol) was treated as described above to
colorless foam. Rf ˆ 0.33 (toluene/acetone 1:1, HPTLC); [a]D
ˆ
28.8 (c ˆ
furnish 1c (107 mg, 71%) as
methanol/0.2% aqueous CaCl2 70:30:5, HPTLC); [a]D
a
colorless powder. Rf ˆ 0.23 (CHCl3/
1.0 in CHCl3); C90H144N2O34 ´ H2O (1816.2): calcd C 59.52, H 8.10, N 1.54;
found C 59.60, H 8.32, N 1.92%.
ˆ
20.2 (c ˆ 1.0 in
CHCl3); 1H NMR (600 MHz, SDS/D2O): d ˆ 0.81 (m, 6H; 2CH3), 1.17 (d,
J(5,6) ˆ 6.3 Hz, 3H; 6b-CH3), 1.19 ± 1.32 (m, 61H; 26CH2, N(CH2CH3)3),
1.56 (brs, 4H; 2OCH2CH2), 1.80 (t, 1H; 3dax-H), 2.02, 2.04 (2s, 6H;
2COCH3), 2.75 (dd, 1H; 3deq-H), 3.20 (q, 3J ˆ 7.3 Hz, 6H; N(CH2Me)3),
3.44 ± 4.10 (m, 54H), 4.52 (d, J(1,2) ˆ 7.7 Hz, 1H; 1c-H), 4.61 (d, J(1,2) ˆ
8.3 Hz, 1H; 1a-H), 4.83 (q, J(5,6) ˆ 6.5 Hz, 1H; 5b-H), 5.12 (d, J(1,2) ˆ
3.4 Hz, 1H; 1b-H); C84H161N3O31 ´ 5H2O (1799.3): calcd C 56.07, H 9.58, N
2.34; found C 56.08, H 9.64, N 2.10%.
[8-(1,2-Di-O-hexadecyl-sn-glycer-3-oxy)-3,6-dioxaoct-1-yl]-O-(methyl-5-
acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-glycero-a-d-galacto-2-non-
ulopyranosylonate)-(2 !3)-(2,4-di-O-acetyl-b-d-galactopyranosyl)-
(1 !4)-[(3,4-di-O-acetyl-a-l-fucopyranosyl)-(1 !3)]-2-acetamido-6-O-
benzoyl-2-deoxy-b-d-glucopyranoside (28b): Compound 27b (346 mg,
154 mmol) was treated as described above to furnish after flash chroma-
tography (toluene/acetone 1:1) pure 28b (102 mg, 34%) as a colorless
foam. Rf ˆ 0.20 (toluene/acetone 1:1, HPTLC); C96H156N2O37 (1930.3).
[17-(1,2-Di-O-hexadecyl-sn-glycer-3-oxy)-3,6,9,12,15-pentaoxaheptadec-1-
yl]-O-(methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-glycero-a-
d-galacto-2-nonulopyranosylonate)-(2 !3)-(2,4-di-O-acetyl-b-d-galacto-
pyranosyl)-(1 !4)-[(3,4-di-O-acetyl-a-l-fucopyranosyl)-(1 !3)]-2-acet-
amido-6-O-benzoyl-2-deoxy-b-d-glucopyranoside (28c): Compound 27c
(259 mg, 109 mmol) was treated as described above to furnish after flash
chromatography (toluene/acetone 2:3) pure 28c (182 mg, 81%) as a
colorless foam. Rf ˆ 0.15 (toluene/acetone 2:3, HPTLC); C102H168N2O40
(2062.4).
[26-(1,2-Di-O-hexadecyl-sn-glycer-3-oxy)-3,6,9,12,15,18,21,24-octaoxahex-
acos-1-yl]-O-(triethylammonium-5-acetamido-3,5-dideoxy-d-glycero-a-d-
galacto-2-nonulopyranosylonate)-(2 !3)-(b-d-galactopyranosyl)-(1 !4)-
[a-l-fucopyranosyl)-(1 !3)]-2-acetamido-2-deoxy-b-d-glucopyranoside
(1d): Compound 28d (135 mg, 61.5 mmol) was treated as described above
to furnish pure 1d (92 mg, 77%) as a colorless powder. Rf ˆ 0.25 (CHCl3/
methanol/0.2% aqueous CaCl2 70:30:5, HPTLC); [a]D
ˆ
19.7 (c ˆ 1.0 in
CHCl3); 1H NMR (600 MHz, SDS/D2O): d ˆ 0.81 (m, 6H; 2CH3), 1.17 (d,
J(5,6) ˆ 6.4 Hz, 3H; 6b-CH3), 1.22 ± 1.28 (m, 52H; 26CH2), 1.31 (t, 9H;
N(CH2CH3)3), 1.56 (brs, 4H; 2OCH2CH2), 1.80 (t, 1H; 3dax-H), 2.02, 2.04
(2s, 6H; 2COCH3), 2.76 (dd, 1H; 3deq-H), 3.21 (q, 3J ˆ 7.3 Hz, 6H;
N(CH2Me)3), 3.44 ± 4.09 [m, 66H; H,H-COSY: 3.52 (2c-H), 3.58 (5a-H),
3.59 (5c-, 7d-H), 3.64 (9d-H), 3.67 (4d-H), 3.68 (6d-H), 3.70 (2b-H), 3.76
(4b-H), 3.77 (3a-H), 3.85 (5d-H), 3.88 (6a-, 3b-, 6'c-, 6c-H), 3.89 (9'd-H),
3.90 (4a-, 8d-H), 3.91 (2a-H), 3.93 (4c-H), 4.00 (6'a-H), 18CH2 spacer, 5H
glycerol, 2OCH2CH2], 4.09 (dd, J(2,3) ˆ 9.6 Hz, J(3,4) ˆ 2.1 Hz, 1H; 3c-H),
4.52 (d, J(1,2) ˆ 7.7 Hz, 1H; 1c-H), 4.61 (d, J(1,2) ˆ 7.8 Hz, 1H; 1a-H), 4.83
(q, J(5,6) ˆ 6.5 Hz, 1H; 5b-H), 5.11 (d, J(1,2) ˆ 3.5 Hz, 1H; 1b-H);
C90H173N3O34 ´ 5H2O (1931.4): calcd C 55.97, H 9.55, N 2.18; found C
55.94, H 9.45, N 2.39%.
[26-(1,2-Di-O-hexadecyl-sn-glycer-3-oxy)-3,6,9,12,15,18,21,24-octaoxahex-
acos-1-yl]-O-(methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-
glycero-a-d-galacto-2-nonulopyranosylonate)-(2 !3)-(2,4-di-O-acetyl-b-
d-galactopyranosyl)-(1 !4)-[(3,4-di-O-acetyl-a-l-fucopyranosyl)-
(1 !3)]-2-acetamido-6-O-benzoyl-2-deoxy-b-d-glucopyranoside
(28d):
Compound 27d (310 mg, 124 mmol) was treated as described above to
furnish after flash chromatography (ethyl acetate to ethyl acetate/methanol
9:1) pure 28d (140 mg, 51%) as a colorless foam. Rf ˆ 0.32 (ethyl acetate/
‡
methanol 5:1); MALDI: m/z: 2218 [M‡Na] ; C108H180N2O43 (2194.6).
(1,2-Di-O-hexadecyl-sn-3-glyceryl)-O-(triethylammonium-5-acetamido-
3,5-dideoxy-d-glycero-a-d-galacto-2-nonulopyranosylonate)-(2 !3)-(b-d-
galactopyranosyl)-(1 !4)-[a-l-fucopyranosyl)-(1 !3)]-2-acetamido-2-de-
oxy-b-d-glucopyranoside (1a): Compound 28a (213 mg, 118 mmol) was
dissolved in dry methanol, and a solution of sodium methoxide (1m) in
methanol (200 mL) was added. After stirring overnight at room temper-
[26-(1,2-Di-O-hexadecyl-sn-glycer-3-oxy)-3,6,9,12,15,18,21,24-octaoxahex-
acos-1-yl]-O-(methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-
glycero-a/b-d-galacto-2-nonulopyranosyl)onate (29a/b): A solution of 8[25]
(550 mg, 900 mmol) and acceptor 4d (550 mg, 580 mmol) in dry acetonitrile/
dichloromethane (1:1, 25 mL) was treated at 08C with trimethylsilyl
trifluoromethanesulfonate (50 mL, 270 mmol). After stirring for 45 min, the
reaction mixture was neutralized with triethylamine and concentrated in
vacuo. The residue was purified by flash chromatography (ethyl acetate/
methanol 1:0 to 30:1) to give 29a/b (420 mg, 51%) as a colorless foam in a
ratio of a/b ˆ 1:1. Rf ˆ 0.56 and 0.63 (CHCl3/methanol 15:1); 1H NMR
(600 MHz, C6D6): 29a: d ˆ 0.88 (t, 6H; 2CH3), 1.32 (brs, 52H; 26CH2),
‡
ature, the solution was neutralized with Amberlite IR120 (H ), filtered,
and evaporated. The residue was dissolved in dioxane/water 1:1 (10 mL),
and an aqueous solution of potassium hydroxide (0.2m, 1 mL) was added.
After stirring overnight, carbon dioxide was added and the solution was
lyophilized. Flash chromatography (CHCl3/methanol/water/triethylamine
120
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
0947-6539/00/0601-0120 $ 17.50+.50/0
Chem. Eur. J. 2000, 6, No. 1

Laterally Clustered Neoglycolipids
111±122
1.51 ± 1.57 (m, 4H; 2OCH2CH2), 1.57 (s, 3H; COCH3), 1.60 (m, 1H; 3ax-H),
1.68 ± 2.13 (4s, 12H; 4COCH3), 2.81 (dd, 2J ˆ 12.7 Hz, J(3eq,4) ˆ 4.5 Hz,
1H; 3eq-H), 3.23 ± 4.14 (m, 48H; 18CH2 spacer, 2OCH2CH2CH2, 5H
glycerol, COOCH3), 4.48 (dd, J(8,9) ˆ 8.9 Hz, 2J ˆ 12.2 Hz, 1H; 9-H), 4.68
(m, 1H; 5-H), 4.95 (dd, J(5.6) ˆ 10.7 Hz, J(6,7) <1 Hz, 1H; 6-H), 5.46 ±
5.49 (m, 1H; 9'-H), 5.53 (ddd, J(3ax,4) ˆ J(4,5) ꢀ 10.9 Hz, J(3ax,4) ˆ 4.9 Hz,
1H; 4-H), 5.80 (brd, J(8,9) ˆ 8.8 Hz, 1H; 8-H), 5.87 (s, 1H; 7-H), 6.48 (d,
J(5,N) ˆ 10.2 Hz, 1H; NH); 29b: d ˆ 0.88 (t, 6H; 2CH3), 1.32 (brs, 52H;
26CH2), 1.51 ± 1.57 (m, 4H; 2OCH2CH2), 1.57 (s, 3H; COCH3), 1.65 (m,
1H; 3ax-H), 1.68 ± 2.13 (4s, 12H; 4COCH3), 2.62 (dd, 2J ˆ 12.7 Hz,
J(3eq,4) ˆ 4.9 Hz, 1H; 3eq-H), 3.23 ± 4.14 [m, 50H; 18CH2 spacer,
2OCH2CH2CH2, 5H glycerol, COOCH3, H,H-COSY: 4.11 ± 4.14 (6-H,
NH)], 4.31 (dd, J(8,9) ˆ 6.3 Hz, 2J ˆ 12.4 Hz, 1H; 9-H), 4.41 (ddd, J(4,5) ˆ
J(5,6) ˆ J(5,N) ꢀ 10.5 Hz, 1H; 5-H), 4.65 ± 4.68 (m, 1H; 9'-H), 4.84 (ddd,
J(3ax,4) ˆ J(4,5) ꢀ 10.9 Hz, J(3eq,4) ˆ 4.9 Hz, 1H; 4-H), 5.47 ± 5.49 (m, 1H;
7-H), 5.84 (m, 1H; 8-H); C73H135NO24 (1410.9): calcd C 62.15, H 9.64, N
0.99; found C 61.90, H 9.52, N 0.82%.
9.5 Hz, 1H; NH); C68H126Cl3NO21 (1400.1): calcd C 58.33, H 9.07, N 1.00;
found C 58.44, H 9.32, N 1.28%.
[26-(1,2-Di-O-hexadecyl-sn-glycer-3-oxy)-3,6,9,12,15,18,21,24-octaoxahexa-
cos-1-yl]
2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-d-glucopyranoside
(33): A solution of 32 (252 mg, 180 mmol) in THF/acetic anhydride/acetic
acid (4:2:1; 14 mL) was treated with activated zinc powder (200 mg;
activation with 2% CuSO4 in water for 5 min). The mixture was stirred for
18 h at room temperature, then diluted with toluene, filtered, and washed
with toluene. Evaporation and flash chromatography (toluene/acetone 1:1
to 2:3) furnished 33 (186 mg, 82%) as a colorless solid. Rf ˆ 0.25 (toluene/
acetone 1:1); [a]D
ˆ
7.6 (c ˆ 1.0 in CHCl3); 1H NMR (250 MHz, CDCl3):
d ˆ 0.88 (t, 3J ˆ 6.5 Hz, 6H; 2CH3), 1.25 (brs, 52H; 26CH2), 1.51 ± 1.57 (m,
4H; 2OCH2CH2), 2.01, 2.02, 2.06, 2.08 (4s, 12H; 4COCH3), 3.40 ± 3.92 (m,
47H; 18CH2 spacer, 2OCH2CH2CH2, 5H glycerol, 5-H), 4.06 ± 4.17 (m,
2H; 2-, 6-H), 4.27 (dd, 2J ˆ 12.2 Hz, J(5,6') ˆ 4.5 Hz, 1H; 6'-H), 4.87 (d,
J(1,2) ˆ 8.4 Hz, 1H; 1-H), 5.06 (dd, J(3,4) ˆ J(4,5) ˆ 9.4 Hz, 1H; 4-H), 5.15
(dd, J(2,3) ˆ J(3,4) ˆ 10.0 Hz, 1H; 3-H), 6.94 (brs, 1H; NH); C67H127NO20
´
[26-(1,2-Di-O-hexadecyl-sn-glycer-3-oxy)-3,6,9,12,15,18,21,24-octaoxahex-
acos-1-yl]-O-(methyl-5-acetamido-3,5-dideoxy-d-glycero-a/b-d-galacto-2-
nonulopyranosyl)onate (30a/b): A solution of sodium methoxide (0.5m) in
methanol (110 mL) was added to a solution of 29a/b (387 mg, 274 mmol) in
dry methanol (10 mL). After 3 hours the solution was neutralized with
2H2O (1266.74): calcd C 61.77, H 10.13, N 1.08; found C 61.80, H 10.22, N
1.08%.
[26-(1,2-Di-O-hexadecyl-sn-glycer-3-oxy)-3,6,9,12,15,18,21,24-octaoxahex-
acos-1-yl] 2-acetamido-2-deoxy-b-d-glucopyranoside (34):
A catalytic
quantity of sodium methoxide was added to a solution of 33 (186 mg,
147 mmol) in dry methanol (9 mL). After 3 hours the solution was
‡
Amberlite IR120 (H ), filtered, and evaporated. Flash chromatography
(CHCl3/methanol ˆ 9:1) yielded 30a/b (250 mg, 72%) as a colorless foam
in a ratio of a/b ˆ 1:1. Rf ˆ 0.35 (CHCl3/methanol 9:1); 1H NMR (250 MHz,
CDCl3): d ˆ 0.88 (t, 6H; 2CH3), 1.26 (brs, 52H; 26CH2), 1.52 ± 1.57 (m, 4H;
2OCH2CH2), 1.60 (m, 1H; 3ax-H), 2.03, 2.05 (2s, 3H; NCOCH3), 2.43, 2.78
(2dd, 1H; 3eq-H), 3.38 ± 4.15 (m, 55.5H; 18CH2 spacer, 2OCH2CH2CH2,
5H glycerol, COOCH3, 4-, 5-, 6-, 7-, 8-, 9-, 9'-H, 0.5HN), 6.43 (d, J(5,N) ˆ
8.4 Hz, 0.5H; 0.5NH); C65H127NO20 ´ 1.5H2O (1269.74): calcd C 61.49, H
10.32, N 1.10; found C 61.56, H 10.33, N 1.05%.
‡
neutralized with Amberlite IR120 (H ), filtered, and evaporated. Flash
chromatography (CHCl3/methanol ˆ15:1) yielded 34 (160 mg, 96%) as a
colorless solid. Rf ˆ 0.32 (CHCl3/methanol 9:1); [a]D
ˆ
15.7 (c ˆ 0.30 in
1
3
CHCl3); H NMR (250 MHz, CDCl3): d ˆ 0.88 (t, J ˆ 6.6 Hz, 6H; 2CH3),
1.25 (brs, 52H; 26CH2), 1.51 ± 1.57 (m, 4H; 2OCH2CH2), 2.07 (s, 3H;
COCH3), 3.40 ± 3.96 (m, 51H; 18CH2 spacer, 2OCH2CH2CH2, 5H
glycerol, 2-, 3-, 4-, 5-, 6-, 6'-H), 4.64 (d, J(1,2) ˆ 8.4 Hz, 1H; 1-H), 7.16 (d,
1H; NH); C61H121NO17 (1140.6): calcd C 64.22, H 10.70, N 1.23; found C
64.11, H 10.52, N 1.60%.
[26-(1,2-Di-O-hexadecyl-sn-glycer-3-oxy)-3,6,9,12,15,18,21,24-octaoxahex-
acos-1-yl]-O-(potassium-5-acetamido-3,5-dideoxy-d-glycero-a/b-d-galac-
to-2-nonulopyranosyl)onate (31a) and (31b): Aqueous potassium hydrox-
ide (0.2m, 680 mL) was added to a solution of 30a/b (115 mg, 90.6 mmol) in
water/dioxane (1:1; 6 mL). After stirring for 2 hours, carbon dioxide was
added and the solution was lyophilized. The two anomers were separated
by flash chromatography (CHCl3/methanol/water 85:15:1 to 80:20:2) to
give 31a (58 mg, 49%) and 31b (58 mg, 48%) as colorless solids.
Preparation of supported planar bilayers: Supported planar bilayers were
prepared by using the Langmuir ± Blodgett technique. Microscope slides
(glass, diameter of 18 mm, thickness of 0.2 mm) were used as transparent
supports. Slides were first cleaned to achieve a highly homogeneous surface
by the following procedure. Slides were treated with a conc. H2SO4/H2O2
mixture (7:3) at 808C for 30 minutes under ultrasonic conditions and were
then rinsed with ultrapure water for 30 minutes. To increase the density of
silanole groups at the surface, a cleaning procedure with NH3/H2O2/H2O
(1:1:5) followed, before finally rinsing with ultrapure water and drying the
slides. The first step in forming a supported bilayer is the covalent binding
of monochlorodimethyloctadecylsilane (Sigma, Deisenhofen, Germany) at
508C for 30 minutes to produce the first monolayer on the slide. The bilayer
was completed by transferring the preformed lipid film at the Langmuir
trough. The lipid mixtures were transferred at a lateral pressure of
Compound 31a: Rf ˆ 0.48 (CHCl3/methanol/0.2% aqueous CaCl2 80:20:2,
HPTLC); [a]D
ˆ
5.3 (c ˆ 1.0 in CHCl3); 1H NMR (600 MHz, CDCl3/
CD3OD/D2O 65:25:4): d ˆ 0.89 (t, 6H; 2CH3), 1.27 (brs, 52H; 26CH2),
2
1.55 ± 1.58 (m, 4H; 2OCH2CH2), 1.62 (dd, J ˆ J(3ax,4) ˆ 11.4 Hz, 1H; 3ax-
H), 2.04 (s, 3H; COCH3), 2.77 (dd, 2J ˆ 12.7 Hz, J(3eq,4) ˆ 3.8 Hz, 1H; 3eq
-
H), 3.45 ± 3.90 (m, 53H; 18CH2 spacer, 2OCH2CH2CH2, 5H glycerol, NH,
4-, 5-, 6-, 7-, 8-, 9-, 9'-H); C64H124KNO20 ´ 1.5H2O (1293.8): calcd C 59.41, H
9.89, N 1.08; found C 59.36, H 9.88, N 0.69%.
1
1
38 mNm and a speed of 0.5 mmmin to hydrophobic substrates as a
X-type monolayer. The transfer ratios were between 0.95 and 1. Freshly
prepared supported bilayers were immediately used for experiments in the
flow chamber.
Compound 31b: Rf ˆ 0.34 (CHCl3/methanol/0.2% aqueous CaCl2 80:20:2,
HPTLC); [a]D
ˆ
9.1 (c ˆ 1.0 in CHCl3); 1H NMR (600 MHz, CDCl3/
CD3OD/D2O 65:25:4): d ˆ 0.89 (t, 6H; 2CH3), 1.27 (brs, 52H; 26CH2),
1.55 ± 1.58 (m, 4H; 2OCH2CH2), 1.77 (t, 1H; 3ax-H), 2.04 (s, 3H; COCH3),
2.34 (dd, 1H; 3eq-H), 3.46 ± 3.97 (m, 53H; 18CH2 spacer, 2OCH2CH2CH2,
5H glycerol, NH, 4-, 5-, 6-, 7-, 8-, 9-, 9'-H); C64H124KNO20 ´ 4H2O (1338.8):
calcd C 57.27, H 9.94, N 1.05; found C 57.18, H 9.68, N 0.79%.
Laminar flow experiments: The parallel-plate flow chamber used in these
studies has been described in detail in our previous investigations.[36] The
flow apparatus was mounted onto an inverted fluorescent microscope
Axiovert 135 of a laser scanning microscope (LSM 410 invert, Carl Zeiss,
Germany).
[26-(1,2-Di-O-hexadecyl-sn-glycer-3-oxy)-3,6,9,12,15,18,21,24-octaoxahex-
acos-1-yl] 3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylami-
no)-b-d-glucopyranoside (32): A solution of 19[29] (523 mg, 837 mmol) and
acceptor 4d (523 mg, 558 mmol) in dry dichloromethane (10 mL) was
treated at room temperature with a solution of trimethylsilyl trifluorome-
thanesulfonate (0.24m) in dichloromethane (82 mL, 20 mmol). After stirring
for 30 min, the reaction mixture was neutralized with triethylamine
(100 mL) and concentrated in vacuo. The residue was purified by flash
chromatography (toluene/acetone 4:1) to give 32 (654 mg, 84%) as a
Adhesion experiments were performed at 258C in a temperature-con-
trolled manner to maintain the lateral structure of the model membrane.
MEM-a medium was used as flow medium at shear rate of 200 s 1 powered
by hydrostatic pressure. For the flow experiments, 106 fluorescently labeled
CHO-E cells in 100 mL medium were injected into the streaming medium
without dilution. Either, cell adhesion or rolling was analyzed immediately,
or the flow was stopped for 5 minutes to allow cells to interact with the
supported membrane. After this time, flow with the desired shear force was
continued and the adhesion behavior of the cells was monitored by a
sequence of images taken every 2 seconds. To characterize the cell
movement, 50 to 150 cells within an area of 630 Â 630 mm were analyzed
over a period of 20 seconds. Only those cells observed to directly contact
the membrane in absence of prior contacts with adherent cells were
counted and analyzed. The experiments for the presented data were
repeated four times under similar conditions.
colorless foam. Rf ˆ 0.68 (toluene/acetone 1:1); [a]D
ˆ
2.9 (c ˆ 1.0 in
1
3
CHCl3); H NMR (250 MHz, CDCl3): d ˆ 0.88 (t, J ˆ 6.6 Hz, 6H; 2CH3),
1.25 (brs, 52H; 26CH2), 1.51 ± 1.58 (m, 4H; 2OCH2CH2), 2.00, 2.01, 2.09
(3s, 9H; 3COCH3), 3.40 ± 3.89 (m, 47H; 18CH2 spacer, 2OCH2CH2CH2,
5H glycerol, 2-, 5-H), 4.12 (dd, J(5,6) ˆ 2.3 Hz, 2J ˆ 12.3 Hz, 1H; 6-H), 4.27
(dd, J(5,6') ˆ 4.7 Hz, 2J ˆ 12.3 Hz, 1H; 6'-H), 4.73 (s, 2H; CH2CCl3), 4.83
(d, J(1,2) ˆ 8.6 Hz, 1H; 1-H), 5.02 ± 5.15 (m, 2H; 3-, 4-H), 6.46 (d, J(2,N) ˆ
Chem. Eur. J. 2000, 6, No. 1
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R. R. Schmidt et al.
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Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft (SFB
197 and Stu 17/1) and the Fonds der Chemischen Industrie. We are grateful
to Dr. A. Geyer for his help in the structural assignments by NMR
experiments.
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Received: April 28, 1999 [F1752]
122
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