Synthesis and supramolecular assemblies of bipolar archaeal glycolipid analogues containing a cis-1,3-disubstituted cyclopentane ring

Article abstract of DOI:10.1021/ja049805n

Unsymmetrical archaeal tetraether glycolipid analogues 1-2 incorporating a 1,3-disubstituted cyclopentane ring into the bridging chain have been synthesized. The cyclopentane has been introduced with a totally controlled cis configuration, either into the

Full text of DOI:10.1021/ja049805n

Published on Web 07/23/2004
Synthesis and Supramolecular Assemblies of Bipolar
Archaeal Glycolipid Analogues Containing a
cis-1,3-Disubstituted Cyclopentane Ring
Mickae¨lle Brard,† Walter Richter,‡ Thierry Benvegnu,*,† and Daniel Plusquellec†
Contribution from the Ecole Nationale Supe´rieure de Chimie de Rennes (ENSCR),
Synthe`ses et ActiVations de Biomole´cules, CNRS UMR 6052, Institut de Chimie de Rennes,
AVenue du Ge´ne´ral Leclerc, 35700 Rennes, France, and Elektronenmikroskopisches Zentrum,
UniVersita¨tsklinikum Jena, Ziegelmu¨hlenweg 1, D-07740 Jena, Germany
Received January 12, 2004; E-mail: Thierry.Benvegnu@ensc-rennes.fr
Abstract: Unsymmetrical archaeal tetraether glycolipid analogues 1-2 incorporating a 1,3-disubstituted
cyclopentane ring into the bridging chain have been synthesized. The cyclopentane has been introduced
with a totally controlled cis configuration, either into the middle of the aliphatic chain or at three methylene
groups from the glycerol unit linked to the bulkier disaccharide residue. Freeze-fracture and cryotransmission
electron microscopy experiments clearly demonstrated unprecedented glycolipid supramolecular organiza-
tions involving two-by-two monolayer associations coupled with interconnection and fusion phenomena.
Furthermore, a significant difference in the hydration properties and in the lyotropic liquid crystalline behavior
of bipolar lipids 1-2 was found depending on the position of the cyclopentane residue.
Introduction
Lipids of thermophilic and thermoacidophilic Archaebacteria
are characterized by tetraether-type macrocyclic components
possessing one or two polar headgroups derived from phosphate
and/or sugar moieties at terminal ends of a lipidic backbone.1
These tetraethers can be divided into two classes.2 The first one
is called glycerol-dialkyl-glycerol-tetraethers (GDGT, Figure 1;
compounds a-d) and is formed by two biphytanyl ether chains
linked at both ends to a glycerol unit. In the second class of
molecules, glycerol-dialkyl-nonitol-tetraethers (GDNT, Figure
1; compounds a′-d′), a calditol group replaces one of the
glycerol units. Recently, Sinay¨ et al.3 demonstrated the poly-
hydroxylated cyclopentanic structure of this polyol. A particu-
larly attractive point concerns the presence of 0-4 pentacyclic
rings into each biphytanyl chain;4 the number and position of
the cyclopentanes were found to depend on the nature of the
lipids and vary with the growing temperature of microorganisms.
In fact, it has been shown that the extent of cyclization increases
† ENSCR.
‡ Universita¨tsklinikum Jena.
(1) (a) Kates, M. Archaebacterial Lipids: Structure, Biosynthesis and Function.
In The Archaebacteria: Biochemistry and Biotechnology; Danson, M. J.,
Hough, D. W., Lunt, G. G., Eds.; Portland Press: London and Chapel Hill,
1992; pp 51-72. (b) De Rosa, M.; Gambacorta, A. Prog. Lipid Res. 1988,
27, 153-175. (c) Sprott, G. D. J. Bioenerg. Biomembr. 1992, 24, 555-
566. (d) Eguchi, T.; Arakawa, K.; Kakinuma, K.; Rapp, G.; Ghosh, S.;
Nakatami, Y.; Ourisson, G. Chem.-Eur. J. 2000, 6, 3351-3357.
(2) De Rosa, M.; Trincone, A.; Nicolaus, B.; Gambarcorta, A. In Life under
Extreme Conditions; Di Prisco, G., Ed.; Springer-Verlag: Heidelberg,
Germany, 1991; pp 61-87.
Figure 1. Typical basic structures of GDGT-type (a-d) or GDNT-type
(a′-d′) tetraethers found in methanogenic and thermoacidophilic archaeal
membrane lipids.
(3) Untersteller, E.; Fritz, B.; Ble´riot, Y.; Sinay, P. C. R. Acad. Sci., Ser. IIc
1999, 429-433.
when the species are grown at increasing temperatures. The
regular disposition of these cycles at two or six methylene
groups starting from the glycerol or calditol units could result
(4) (a) De Rosa, M.; Esposito, E.; Gambarcorta, A.; Nicolaus, B.; Bu’Lock, J.
D. Phytochemistry 1980, 19, 827-831. (b) Russel, N. J.; Fukunaga, N.
Microbiol. ReV. 1990, 75, 171-182. (c) Itoh, Y. H.; Sugai, A.; Uda, I.;
Itoh, T. AdV. Space Res. 2001, 28, 719-724.
9
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J. AM. CHEM. SOC. 2004, 126, 10003-10012
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A R T I C L E S
Brard et al.
backbone characterized by the presence of (1) a bridging chain
attached to two glycerols at sn-3 and sn-3′ positions and
containing a cis-1,3-cyclopentane, either into the middle of the
chain or located at three methylene groups from the sn-3 position
of the glycerol unit linked to the lactosyl residue, and (2) two
dihydrocitronellyl chains linked to glycerol at sn-2 and sn-2′
positions. The supramolecular assemblies formed by these
original amphiphiles with a totally controlled cis configuration
for the 1,3-disubstituted cyclopentane moiety were then inves-
tigated in dilute aqueous media to give further insights into the
mechanisms involved in the bilamellar vesicle formation and
to evaluate the influence of the cyclopentyl ring position on
lyotropic properties.
Figure 2. Synthetic unsymmetrical neutral archaeal glycolipid analogues
1-2.
Results and Discussion
from the mechanism of biosynthesis that operates on alkyl chains
starting from the middle of the isoprenoid system toward ether
bonds.2 Very little is known concerning the functional role of
cyclopentanes within the membrane apart from the fact that they
probably reduce the rotational freedom of the hydrocarbon
chains. It is not clear, however, whether their presence corre-
sponds to an adaptative response to environmental high tem-
peratures or whether it results from a particular biosynthetic
pathway. Seemingly, there does not appear to be any specific
structural feature of the polar lipids of thermophiles since
cyclopentyl rings were also found in lipids isolated from
psychrophilic archaebacteria living at low temperatures.5 These
points may be of fundamental importance in understanding the
structure-function relationship of natural archaeal membranes
and may help the development of bipolar tetraether liposomes
for biotechnological applications such as sterilization,6 drug or
antigen delivery,7 and membrane protein/peptide reconstitu-
tions.8
(I) Synthesis of Bipolar Lipids 1-2. Bisglycosides 1 and 2
were synthesized according to a strategy that initially involved
constructing the lipophilic monoprotected diols 17 or 24
(Scheme 1) followed by the sequential introduction of the
saccharidic moieties. This novel synthetic pathway warrants the
cis-configuration of the 1,3-disubstituted cyclopentane as well
as modulates the position of the carbocycle within the bridging
chain. Spacer 7 was prepared via a double Wittig reaction
between 6-benzyloxy-hexan-1-al 5 and the bisphosphonium salt
4 obtained from cis-1,3-diformylcyclopentane9b,c followed by
hydrogenation of the double bonds and removal of the benzyl
groups. Then, alkylation of the (S)-glycerol derivative 169b with
ditriflate 15, subsequent hydrogenolysis of the benzyloxy groups,
and monobenzylation of the corresponding diol provided the
monoprotected tetraether 17.
Our attention was next directed toward the synthesis of the
lipophilic backbone 24 possessing a nonsymmetrical spacer. The
additional difficulties in preparing such a structure lay in the
dissymmetrization of the molecule due to the position of the
cyclopentane ring in the spacer that required the introduction
of orthogonal protective groups. Our strategy for the synthesis
of the monoprotected spacer 14 was based upon two sequential
Wittig reactions involving phosphonium salts 9 and 12. Com-
pound 9 could be readily prepared by monoprotection of the
cis-1,3-bis-(hydroxymethyl) cyclopentane 3 and conversion of
the free alcohol unit to the corresponding phosphonium iodide.
Phosphonium salt 12 was prepared by connection of the 10-
benzyloxy-decan-1-al 10 with the cyclopentyl derivative 9 via
standard Wittig reaction conditions followed by removal of the
tetrahydropyranyl group and transformation of the hydroxyl unit
into phosphonium salt. Formation of spacer 14 was finally
accomplished by a second Wittig reaction between phosphonium
salt 12 and 2-O-THP-ethan-1-al 1310 and subsequent reduction
of the double bonds and removal of the tetrahydropyranyl group
under acidic conditions.
To provide further evidence for the crucial role of cyclopen-
tanes on the membrane properties, we launched a program of
synthesis and physicochemical evaluations of archaeal tetraether
glycolipid analogues. Preliminary freeze-fracture electron mi-
croscopy experiments were carried out from synthetic acyclic
tetraether-type glycolipids that incorporated a 1,3-disubstituted
cyclopentane ring at midpoint of their bridging chain, as a
diastereoisomeric mixture of cis-trans isomers.9c Interestingly,
cyclopentane ring was found to play an important role in the
organization of the bipolar lipids within the vesicle membranes.
Pursuing our efforts on understanding the relationships between
1,3-disubstituted cyclopentane rings and transmembrane orga-
nizing properties of the bipolar lipids, we now report on a novel
approach for the synthesis of the unsymmetrical tetraether-type
glycolipids 1-2 (Figure 2) that possess lactosyl and galacto-
furanosyl units as polar headgroups and an acyclic lipophilic
(5) (a) De Long, E. F.; Wu, K. Y.; Prezelin, B. B.; Jovine, R. V. M. Nature
1994, 371, 695-697. (b) Delong, E. F.; King, L. L.; Massana, R.; Cittone,
H.; Murray, A.; Schleper, C.; Wakeham, S. G. Appl. EnViron. Microbiol.
1998, 1133-1138.
Having the free alcohol 14 in hand, the last crucial step
involved the construction of the hemimacrocyclic backbone 24
via two consecutive alkylations with triflates 20 and 23 formed
from glyceryl derivatives 19 and 16, respectively, possessing
different allyloxy or benzyloxy groups at the sn-1 position
(Scheme 1). The major unexpected difficulty in this sequence
was the selective removal of the benzyl group from intermediate
(6) Choquet, G. C.; Patel, G. B.; Sprott, G. D. Can. J. Microbiol. 1996, 42,
183-186.
(7) (a) Sprott, G. D.; Tolson, D. L.; Patel G. B. FEMS Microbiol. Lett. 1997,
154, 17-22. (b) Patel, G. B.; Agnew, B. J.; Deschatelets, L.; Flemming,
L. P.; Sprott, G. D. Int. J. Pharm. 2000, 194, 39-49.
(8) Schuster, B.; Weigert, S.; Pum, D.; Sa´ra, M.; Sleytr, U. B. Langmuir 2003,
19, 2392-2397.
(9) (a) Auze´ly-Velty, R.; Benvegnu, T.; Plusquellec, D.; Mackenzie, G.; Haley,
J. A.; Goodby, J. W. Angew. Chem., Int. Ed. 1998, 37, 2511-2515. (b)
Lecollinet, G.; Auze´ly-Velty, R.; Danel, M.; Benvegnu, T.; Mackenzie,
G.; Goodby, J. W.; Plusquellec, D. J. Org. Chem. 1999, 64, 3139-3150.
(c) Lecollinet, G.; Gulik, A.; Mackenzie, G.; Goodby, J. W.; Benvegnu,
T.: Plusquellec, D. Chem.-Eur. J. 2002, 8, 585-593.
(10) Nicolaou, K. C.; Liu, J. J.; Yang, Z.; Ueno, H.; Sorensen, E. J.; Claiborne,
C. F.; Guy, R. K.; Hwang, C. K.; Nakada, M.; Nantermet, P. G. J. Am.
Chem. Soc. 1995, 117, 634-644.
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Bipolar Archael Glycolipid Analogues
A R T I C L E S
Scheme 1. Synthesis of the Monoprotected Alcohols 17 and 24a
21 in the presence of an allyl ether unit. Deprotection by using
selective standard conditions (Me2S-BCl3, CH2Cl2)11 was
ineffective. Surprisingly, oxidative deprotection with 2,3-
dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) under condi-
tions12 (CH2Cl2-H2O) specifically used for the cleavage of
mono- or dimethoxylbenzyl ethers efficiently provided monoal-
cohol 22. Compound 24 was next obtained in a two-step
procedure by introduction of the second glyceryl moiety and
selective removal of the allyl group.
The cis configuration of the 1,3-disubstituted cyclopentane
in compounds 17 and 24 was clearly demonstrated by the
1
presence of two distinct signals in H NMR for the protons of
the methylene group CHCH2CH for the cyclopentyl moiety,
whereas a single signal should have appeared for the trans
isomer.9b Monoprotected diols 17 and 24 were both obtained
as a mixture of two diastereoisomers because of the presence
of the (()-cis-disubstituted cyclopentyl ring. It is noteworthy
that (()-cis-cyclopentanes were characterized by unique signals
in NMR. At this stage, the sequential introduction of the â-D-
galactofuranosyl and lactosyl units was performed in the same
manner as previously described from lactosyl thioglycoside 25
and pent-4-enyl 2,3,5,6-tetra-O-R,â-D-galactofuranoside 269b
(Scheme 2) and permitted us to isolate the corresponding
glycolipids 1 and 2.
(II) Transmission Electron Microscopy Studies. The su-
pramolecular aggregates formed by these synthetic bipolar lipids
in distilled water were examined by transmission electron
microscopy (TEM). The membrane-spanning organization of
the lipids was shown by freeze-fracturing from the observation
of circles instead of convex and concave fracture faces visualized
in the case of bilayered systems.13 The absence of a fracturable
midplane in the membranes indicates that the bipolar lipids do
not adopt a U-bent shape but span the membrane to form a
monolayer. The cross-fracture behavior, which is the absence
of a preferential fracture plane, has further been shown to be
indicative for monomolecular vesicle membranes and membrane
spanning lipid molecules14 and also in recent investigations.15
Additional confirmation of the aggregate morphologies as-
sumed by these glycolipids was obtained by cryo-TEM experi-
ments that allowed the direct visualization of the lamellar
arrangements without any alteration. For glycolipid 1, we have
determined a membrane thickness of 3.0-3.5 nm in bent
vesicular membranes (Figure 3a). This is in good agreement
with the molecular length of the bolaamphiphiles 1-2 in a
stretched conformation estimated to be about 2.8-3.0 nm.
Compound 1, which possesses a cyclopentane in the middle
of the main chain, gave rise to oligolamellar or bilamellar
vesicles, but no multilamellar onionlike vesicles were observed
as in the case of a mixture of cis-trans isomers.9c A two-by-
two monolayer association frequently appeared from dispersed
systems prepared either in the presence (Figure 3a) or the
absence (see Supporting Information) of glycerol added as a
cryoprotectant for FFEM. We also checked that after storage
a
(i) NaBH4, CH3OH, 0 °C, 80%. (ii) (a) I2, imidazole, PPh3, CH3CN/
Et2O (1:3 v/v). (b) PPh3, CH3CN, reflux, 50% over two steps. (iii) n-BuLi,
THF, 0 °C, then BnO-(CH2)5-CHO 5, 55%. (iv) H2, Pd/C (10%), EtOH,
98%. (v) DHP, Dowex 50WX2, toluene, 81%. (vi) (a) I2, imidazole, PPh3,
CH2Cl2; (b) PPh3, K2CO3, CH3CN, 70% over two steps. (vii) (a) n-BuLi,
THF, 0 °C, then BnO-(CH2)9-CHO 10. (b) PTSA, CH3OH, 75% over
two steps. (viii) (a) See (vi) (a). (b) PPh3, CH3CN, 80% over two steps.
(ix) (a) n-BuLi, THF, 0 °C, then THPO-CH2-CHO 13. (b) H2, Pd/C (10%),
Et3N, AcOEt, 75% over two steps. (x) See (vii) (b), 77%. (xi) Tf2O, 2,6-
lutidine, CH2Cl2, 95%. (xii) (a) 16, KH, THF, then 15. (b) H2, Pd/C (10%),
AcOEt, 40% over two steps. (c) NaH, 15-5 crown-ether, BnBr, THF, 60%.
(xiii) (a) DHP, PTSA, CH2Cl2. (b) See (iv), 65% over two steps. (xiv) (a)
NaH, allylbromide, (Bu)4N+Br-, THF. (b) PTSA, CH3OH, 75% over two
steps. (xv) (see xi), 0 °C, 95%. (xvi) 14, KH, THF, 0 °C, then 20, 70%.
(xvii) DDQ, CH2Cl2/H2O (18:1 v/v), 70%. (xviii) (a) 22, KH, THF, 0 °C,
then 23, 50%. (b) (Ph3P)3RhCl, toluene/EtOH/H2O (1.25:2.65:0.4 v/v/v)
78%. Bn ) benzyl, DDQ ) 2,3-dichloro-5,6-dicyano-1,4-benzoquin-
one, DHP ) 2,3-dihydro-4H-pyran, THP ) tetrahydropyranyl, PTSA )
p-toluenesulfonic acid.
(11) Congreve, M. S.; Davison, E. C.; Fuhry, M. A.; Holmes, A. B.; Payne, A.
N.; Robinson, M. A.; Ward, S. E. Synlett 1993, 663-664.
(12) Oikawa, Y.; Yoshioka, T.; Yonemitsu, O. Tetrahedron Lett. 1982, 23, 885-
888.
(13) Lo, S. L.; Chang, E. L. Biochem. Biophys. Res. Commun. 1990, 167, 238-
245.
(14) Elferink, M. G. L.; de Wit, J. G.; Demel, R.; Driessen, A.; J. M., Konings,
W. N. J. Biol. Chem. 1992, 267, 1375-1381.
(15) Kanichay, R.; Boni, L. T.; Cooke, P. H.; Khan, T. K.; Chong, P. L.-G.
Archaea 1 2003, 175-183.
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Scheme 2. Introduction of the Glycosidic Polar Head Groups in Compound 1a
a
(i) NIS, Et3SiOTf, CH2Cl2, 48%. (ii) H2, Pd/C, EtOH, >99%. (iii) NIS, Et3SiOTf, CH2Cl2, 35%. (iv) MeONa, MeOH, >99%. NIS ) N-iodosuccinimide.
Under the same conditions, 25 + 24 (then 26) f f 2.
b
Figure 4. FFEM of compound 1 after gentle hydration at 94 wt % (a) and
Figure 3. Freeze-fracture electron microscopy (FFEM; a, b) and Cryo-
TEM (c, d) of glycolipid 1 after hydration by intensive stirring in excess
water (99 wt %). (a) Vesicles with two-by-two lamellar arrangement
showing an interconnection (arrowheads) between the two envelopes. (b)
Vesicle with two-by-two lamellar arrangement showing two tunnel-like
interconnections (arrowheads). (c) Cryo-TEM of vesicular two-by-two
lamellar arrangements. The inside vesicle looks like an invagination of the
outer envelope. (d) At higher magnification, a second interconnection of
the two membranes is clearly visible (arrow).
97 wt % (b-d) water content. (a) Projection with a regular pattern of a
cubic 3D structure. Repeat distance of about 18 nm of the orthogonally
arranged elements (tunnels). (b) Projection of a spongelike 3D structure
after storage over 10 days at room temperature. A transition to tightly
coupled lamellae is visible. (c) Structural transition from a spongelike
nucleus to peripheral two-by-two arranged lamellae. Interconnection at the
end of two lamellar loops (arrowhead). (d) Tangentially running fracture
plane near the surface of lamellar faces with a number of tunnel-like
structures (arrows). They may be descended from interconnections of the
sponge structure (arrowheads).
of the sample at room temperature and ultrasonication the same
phenomenon could be obtained. Moreover, a particularly striking
feature happened in several bilamellar systems. Surprisingly,
the outer envelope of many two-by-two arranged lamellae shows
openings such as invaginations favoring their connection with
the inner vesicle structure as illustrated by FFEM (Figure 3a,b)
and confirmed by cryo-TEM (Figure 3c,d). In many cases,
several (two or even more) interconnections were revealed by
TEM pictures (Figure 3b,d). The structures mainly appeared as
an “X” intersection in the two dimensions of the FFEM image
because the freeze fracture plane ran at the border of the opening
and not across its center. Nevertheless, the opening of these
bilamellar vesicle systems was shown at the upper part of Figure
3a and more clearly in the Supporting Information where the
magnification is higher. These unexpected contacts may involve
original fusion mechanisms that operate between two matched
monolayers through the possible existence of specific interac-
tions between sugar residues. A strong affinity between these
headgroups could exhibit a cooperative orientation of the
monomers within the lamellae, allowing a stronger coupling of
two close monolayers; this original phenomenon may appear
very early during the hydration process. To elucidate the
mechanism of invaginated vesicle formation, additional electron
microscopic experiments were then performed at lower hydra-
tion conditions.
Fresh dispersion of compound 1 with a 94 wt % water content
showed after gentle hydration a regular structure pattern16,17
(Figure 4a) resulting from a cubic 3D structure.9c After further
dilution (97 wt % water content) and prolonged storage at room
temperature (10 days), it exhibited a spongelike structure13,16
(Figure 4b) that was transforming into bilamellar arrangements
(16) Strey, R.; Jahn, W.; Porte, G.; Bassereau, P. Langmuir 1990, 6, 1635-
1639.
(17) Hoffmann, H.; Thunig, C.; Munkert, U.; Meyer, H. W.; Richter, W.
Langmuir 1992, 8, 2629-2638.
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Bipolar Archael Glycolipid Analogues
A R T I C L E S
cyclopentyl residue mixture used, since the same behavior would
have been observed from glycolipids 1 and 2. Indeed, the
presence of an identical (()-cis isomeric system in both
structures clearly demonstrates the profound positional effect
of the cyclopentane on the self-assembling properties of these
neutral archaeal glycolipid analogues. The presence of cyclo-
pentanes in precise positions within the bridging chains may
influence the orientation of the glycosidic polar headgroups
attached to the tetraether backbone, as recently shown by
molecular modeling.18 When containing eight cyclopentane
rings, the polar disaccharidic moiety of natural tetraether-type
lipids was shown to run parallel to the membrane surface,
whereas the headgroup is oriented perpendicularly in the absence
of cyclopentanes.18b Therefore, one may assume that the
orientation of the lactose residue of our synthetic glycolipids is
affected by the cyclopentane position. The increased hydration
for compound 1 in comparison with that of glycolipid 2 may
be associated with a more favorable arrangement of the bulkier
polar headgroup relative to the surface membrane for hydration.
Conclusions
Figure 5. FFEM of glycolipid 2 after hydration by intensive stirring in
excess water (a, d) and gentle hydration at 95 wt % water content (b, c).
(a) Multilamellar onionlike vesicles are predominant. At larger magnification
(inset), the multilamellar arrangement with a repeat distance of about 5 nm
is visible. (b) Two projections of a cubic 3D structure with a repeat distance
of about 23 nm of the hexagonally arranged elements (tunnels, left part).
(c) Coexistence of a cubic phase structure and a multilamellar vesicle.
Contrary to compound 1, spongelike structured particles have not been
found. (d) Rarely detectable spongelike structure after ultrasonication in
excess water.
The results obtained from this study clearly reveal the
importance of the cyclopentane incorporation into bridging
chains on the self-assembling behavior of neutral tetraether-
type glycolipids. Utilizing novel synthetic neutral bipolar lipids
possessing a 1,3-disubstituted cyclopentane with a controlled
cis configuration, we have demonstrated, for the first time to
our knowledge, the profound impact of the cycle position on
the physicochemical properties of the monolayer membranes.
Another major finding of this work concerns the formation of
the two-by-two lamellar arrangements via the existence of
primary tunnel-like connections between two matched mono-
layers. Consequently, the cyclopentane ring position within the
bridging chains may modulate interactions between neutral
tetraether-type membranes and the aqueous environment and
between two monolayers next to each other. Insights gained
into the structure-property relationships of these bolaam-
phiphiles can help explain the unusual organization and
functionality of archaeal lipids in natural membranes as well
as allow the design of new materials based upon cyclopentane
ring-containing lipids capable of inducing original supramo-
lecular systems. Applications of these monolayer systems for
the encapsulation and the delivery of biomaterials are under
investigation. Indeed, the positional effect of cyclopentanes may
represent a new method for the control of membrane perme-
ability and drug releasing.
at its periphery (Figure 4b,d). These two-by-two arranged
lamellae generally showed tunnel-like interconnections (Figure
4d). We assume that the interconnections are primary structures
of the 3D sponge arrangement that are preserved during the
transition to the vesicular lamellae. Because of a structural
genesis from cubic to sponge and curved lamellar (vesicles),
the frequent two-by-two arrangements of lamellae could be
favored by these primary connections initially observed in the
sponge-type aggregates (Figure 4d).
A great difference in the hydration properties of compounds
1 and 2 was observed depending on the position of the
cyclopentyl moiety on the bridging chain. Only a part of
glycolipid 2 could be dispersed in water, whereas compound 1
exhibited a complete dispersion under the same conditions.
Compound 2 did not nearly form oligolamellar or two-by-two
lamellar vesicles. The typical features were multilamellar
onionlike vesicles with tightly packed lamellae (Figure 5a). After
gentle hydration at 90 wt % water content, compound 2 gave a
regular cubic 3D structure (Figure 5b,c) as bisglycoside 1;
however, no spongelike aggregates were found, even after a
prolonged storage (14 days) and further dilution. A sponge
structure with narrow meshes could be induced by ultrasoni-
cation in excess water (Figure 5d), but it was found extremely
seldom. These results suggested that bipolar lipid 2 could adopt
the same transmembrane conformation as in compound 1, but
the swelling of the lipid by incorporation of a large amount of
water appeared to be much more restricted. Consequently, the
position of the (()-cis-1,3-disubstituted cyclopentane moiety
within the main chain seems to play a major role on the lyotropic
behavior of the tetraether-type lipids. The different dispersion
morphologies cannot simply be a consequence of the (()-cis-
Experimental Section
General. All commercially available chemicals were used without
further purification, and solvents were carefully dried and distilled prior
to use. Unless otherwise noted, nonaqueous reactions were carried out
under a nitrogen atmosphere. Analytical TLC was performed on Merck
60 F254 silica gel nonactivated plates. A solution of 5% H2SO4 in EtOH
was used to develop the plates. 1H and 13C NMR spectra were recorded
at 400 and 100 MHz, respectively. Fast-atom bombardement (FAB)
(18) (a) De Rosa, M.; Morana, A. In Neural Networks and Biomolecular
Engineering to Bioelectronics; Nicolini, C., Ed.; Plenum Press: New York,
1995; pp 217-222. (b) Gabriel, J. L.; Chong, P. L. G. Chem. Phys. Lipids
2000, 105, 193-200. (c) Nicolas, J.-P. A Molecular Dynamic Study of
Interfaces: from Pure Liquids to Biological Membranes. Ph.D. Thesis,
Faculty of Science, University of Amsterdam, The Netherlands, 2003.
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A R T I C L E S
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mass spectra were acquired on a MS/MS ZabSpec TOF Micromass
spectrometer with m-nitrobenzylic alcohol as the matrix. Merck 60 H
(5-40 µm) silica gel was used for column chromatography. For freeze-
fracturing, lipid dispersions (94-97 wt % and 99 wt % water content)
were prepared by gentle stirring (low water content) or intensive stirring
overnight at room temperature (high water content). Small amounts of
the dispersions were sandwiched, after addition of glycerol (2.3:10,
v/v, glycerol/water) as cryoprotectant, between copper profiles and were
then rapidly frozen in a liquid 1:1 ethane/propane mixture cooled by
liquid nitrogen. Fracturing and replication were carried out at -120
°C in a freeze-fracture apparatus BAF 400T (BAL-TEC, Liechtenstein)
equipped with electron beam evaporators. All fracture samples were
etched before Pt/C-shadowing to visualize the propagation patch much
more clearly. Freeze-etching was repeated after storage of the samples
at room temperature over 4 days (or more) and ultrasonication for 10
min in a cleaning batch immediately before freezing. Electron
micrographs were prepared with a CEM 902A electron microscope
(Zeiss, Germany). For cryo-TEM experiments, the CM 120 electron
microscope (FEI Co., The Netherlands) equipped with cryo-Transfer
(Gatan, USA) was used. Pent-4-enyl galactofuranoside 26, lactosyl
thioglycoside 25 and 1-O-benzyl-2-O-(R)-3,7-dimethyl-octyl-sn-glycerol
16 were prepared as previously described.9b The synthesis of 2-O-THP-
ethan-1-al 13 has already been described.10
The solution was subjected to an extractive workup (quenching with
water and extraction with petroleum ether/EtOAc (2:8 v/v)), and the
crude product was purified by silica gel column column chromatog-
raphy. Elution with a mixture of petroleum ether and ethyl acetate (99/1
v/v) yielded compound 6 as a colorless oil (150 mg, 55%); Rf ) 0.8
1
(EP/EtOAc 8:2 v/v); H NMR (400 MHz, CDCl3) δ 0.95-1.04 (m,
1H), 1.21-1.37 (m, 14H), 1.53-1.56 (m, 4H), 1.69-1.81 (m, 2H),
1.89-1.90 (m, 1H), 2.65-2.80 (m, 2H), 3.36-3.39 (t, 4H, J ) 6.6
Hz), 5.12 (s, 4H), 5.18-5.28 (m, 4H), 7.15-7.21 (m, 10H); 13C NMR
(400 MHz, CDCl3) δ 26.3-32.4, 32.8, 38.3, 42.2, 70.6, 72.9, 128-
134.5, 127.5, 127.7, 128.4, 138.6; elemental analysis calcd (%) for
C33H46O2: C, 83.49; H, 9.77. Found: C, 83.91; H, 10.20.
(cis-8,11-Methylidene)-octadecane-1,18-diol (7). A solution of 6
(383 mg, 0.81 mmol, 1 equiv) in ethanol (50 mL) was stirred in the
presence of 10% palladium on activated charcoal (153 mg) under an
atmosphere of hydrogen gas at room temperature for 24 h. The catalyst
was removed by filtration after the solvent was heated to 40 °C, and
the filtrate was concentrated to dryness under vacuum. The residue
was purified by flash silica gel column chromatography (petroleum
ether/EtOAc 7:3) to afford compound 7 as a colorless oil (232 mg,
1
97%); Rf ) 0.2 (EP/EtOAc 8:2 v/v); H NMR (400 MHz, CDCl3) δ
0.51-0.56 (m, 1H), 1.31-1.75 (m, 30H), 1.79-1.84 (m, 1H), 3.64-
3.68 (m, 4H); 13C NMR (400 MHz, CDCl3) δ 26.0-30.0, 31.9, 33.0,
36.9, 40.4, 63.2; elemental analysis calcd (%) for C19H38O2: C, 76.45;
H, 12.83. Found: C, 76.04; H, 12.96.
cis-1,3-Bis-(hydroxymethyl)cyclopentane (3). Sodium borohydride
(4.52 g, 120 mmol, 2 equiv) was added slowly at 0 °C to a stirred
solution of cis-1,3-diformylcyclopentane (7.5 g, 59.8 mmol) in methanol
(65 mL). After being stirred overnight at room temperature under N2
atmosphere, the reaction mixture was quenched with water, extracted
3 times with the ethyl acetate/butanol (9:1 v/v) mixture. The combined
extracts were dried (MgSO4) and concentrated under vacuum. The
residue was purified by flash column chromatography upon elution with
EtOAc to afford 3 as a colorless oil (4.4 g, 56%); Rf ) 0.3 (EtOAc);
1H NMR (400 MHz, CDCl3) δ 0.90-0.98 (dt, 1H, J ) 9.1, 12.7 Hz),
1.33-1.41 (m, 2H), 1.72-1.79 (m, 2H), 1.95 (s, 1H), 1.94-2.00 (m,
1H), 2.13-2.20 (m, 2H, J ) 6.6 Hz), 3.50-3.59 (m, 4H, J ) 3.6 Hz);
13C NMR (400 MHz, CDCl3) δ 28.3, 32.8, 42.2, 67.3.
cis-(1-Tetrahydropyranyloxymethyl-3-hydroxymethyl)-cyclopen-
tane (8). To 26.9 mmol (3.5 g) of compound 3 in a mixture of toluene
(150 mL) and dihydropyran (7.5 mL), resin Dowex 50X ×2 (6 g)
prewashed with methanol was added, and the reaction mixture was
heated at 40 °C for 2 h. The residue was filtered and concentrated
under vacuum. The crude product was chromatographed over silica
gel with petroleum ether/EtOAc (8:2 then 6:4) to give the monopro-
tected diol 8 as a colorless oil (4.66 g, 81%); Rf ) 0.3 (EP/EtOAc 8/2
1
v/v); H NMR (400 MHz, CDCl3) δ 0.88-0.99 (m, 1H), 1.32-1.84
(m, 10H), 1.94-2.04 (m, 1H, J ) 7.8 Hz), 2.11-2.19 (q, 1H, J ) 7.8
Hz), 2.19-2.28 (q, 1H, J ) 7.8 Hz), 3.27-3.34 (dt, 1H, J ) 6.6, 9.4
Hz), 3.48-3.54 (m, 3H), 3.61-3.81 (dt, 1H, J ) 7.6, 9.4 Hz), 3.84-
3.88 (dt, 1H, J ) 3.6, 7.6 Hz), 4.58 (s, 1H); 13C NMR (400 MHz,
CDCl3) δ 19.6, 25.5, 28.3, 28.8, 28.9, 30.7, 33.3, 33.4, 39.7, 42.3, 62.3,
67.4, 72.0, 72.1, 98.9, 99.0; elemental analysis calcd (%) for
C12H22O3: 0.1 H2O: C, 66.69; H, 10.35. Found: C, 66.45; H, 10.47.
[(cis-2,5-Methylidene)-hexyl]-1,6-di-triphenylphosphonium Bis-
iodide (4). To a solution of triphenylphosphine (19.4 g, 74 mmol, 2.2
equiv) and imidazole (5 g, 74 mmol, 2.2 equiv) in a mixture of
acetonitrile/ether (1/3 (40 mL:120 mL) v/v), diiode (18.8 g, 74 mmol,
2.2 equiv) was added at 0 °C. After stirring of the mixture at room
temperature in the dark for 10 min, we added a solution of diol 3 (4.38
g, 33.6 mmol) in ether (20 mL). The reaction mixture was stirred for
3 days at room temperature in the dark. The solution was washed with
10% aqueous sodium thiosulfate and extracted with diethyl ether. The
combined organic layers were washed with water, dried over MgSO4,
filtered, and concentrated to dryness. The crystallized triphenylphos-
phine oxide was filtered on silica gel with petroleum ether/EtOAc (8:2
v/v). The crude product (11.8 g, 33.6 mmol) was dissolved in
acetonitrile (90 mL), and triphenylphosphine (26 g, 100 mmol, 3 eq)
was added. The reaction mixture was refluxed for 2 days. Dilution with
toluene (50 mL) gave the desired salt 4 as a white solid (14.3 g, 50%),
which was recrystallized from a mixture of methanol and diethyl ether;
[(cis-2,5-Methylidene)-6-tetrahydropyranyloxy]-hexyl-1-triphe-
nylphosphonium Iodide (9). To a solution of triphenylphosphine (27.5
g, 105 mmol, 3 equiv) and imidazole (14.3 g, 210 mmol, 6 equiv) in
CH2Cl2 (80 mL), diiode (27.2 g, 105 mmol, 3 equiv) was added at 0
°C. The reaction mixture was stirred in the dark at room temperature
for 10 min. Alcohol 8 (7.5 g, 35 mmol) dissolved in CH2Cl2 was then
added at 0 °C, and the reaction mixture was stirred for 3 h. The solution
was washed with 10% aqueous Na2S2O3 and extracted with CH2Cl2.
The organic layer was washed with water, dried over MgSO4, filtered,
and concentrated. The crystallized triphenylphosphine oxide was
removed by filtration on silica gel with petroleum ether/EtOAc (8:2
v/v). To a stirred solution of the resulting crude product (10.9 g, 33.6
mmol) in acetonitrile (250 mL), triphenylphosphine (10.6 g, 40 mmol,
1.2 equiv) and potassium carbonate (5.6 g, 40 mmol, 1.2 equiv) were
added. The reaction mixture was refluxed for 2 days. A column
chromatography over silica gel with CH2Cl2 and CH2Cl2/MeOH (95/
5) gave the phosphonium salt 9 as a white solid (13.5 g, 66%); Rf )
1
Rf ) 0.2 (CH2Cl2/MeOH 96/4 v/v); mp > 260 °C (MeOH/Et2O); H
NMR (400 MHz, CDCl3) δ 1.47-1.53 (m, 2H), 1.57-1.65 (m, 2H),
1.88-1.96 (m, 1H), 2.07-2.14 (m, 1H), 2.36-2.43 (m, 2H), 3.59-
3.69 (m, 2H), 3.78-3.88 (m, 2H), 7.72-7.85 (m, 30H); 13C NMR (400
MHz, CDCl3) δ 28.2, 28.7, 32.66, 32.74 (d, J ) 8 Hz), 33.4, 41.8,
117.5-135.0; FABMS (m-nitrobenzyl alcohol matrix) calcd for [M2+,I-]+,
747.1807; found, 747.1809.
1
0.4 (CH2Cl2/MeOH 94/6 v/v); H NMR (400 MHz, CDCl3) δ 1.02-
1.14 (m, 1H, J ) 10.9 Hz), 1.38-1.69 (m, 10H), 1.71-1.80 (m, 1H),
1.97-2.05 (m, 1H), 2.10-2.18 (m, 1H), 3.12-3.18 (dt, 1H, J ) 6.4,
9.2 Hz), 3.37-3.43 (m, 1H), 3.45-3.53 (m, 1H), 3.55-3.80 (m, 3H),
4.42 (s, 1H), 7.64-7.80 (m, 15H); 13C NMR (400 MHz, CDCl3) δ
19.6, 25.2, 27.7, 27.8, 28.8, 30.4, 33.2, 33.3, 34.4, 38.2, 38.3, 38.6,
38.7, 62.0, 62.4, 71.0, 71.1, 98.6, 98.9, 117.8, 118.6, 130.3, 130.4, 133.4,
1,18-Dibenzyloxy-(cis-8,11-methylidene)-octadec-6,12-diene (6).
To a suspension of phosphonium diiodide 4 (500 mg, 0.57 mmol, 1
equiv) in dry THF (13 mL), butyllithium (0.8 mL, 1.2 mmol, 2.1 equiv)
was added at 0 °C and under N2 atmosphere. The reaction mixture
was stirred for 20 min at 0 °C, and 6-benzyloxy-hexan-1-al 5 (250
mg, 1.3 mmol, 2.3 equiv) dissolved in dry THF (13 mL) was added.
9
10008 J. AM. CHEM. SOC. VOL. 126, NO. 32, 2004

Bipolar Archael Glycolipid Analogues
A R T I C L E S
133.5, 135.0; FABMS (m-nitrobenzyl alcohol matrix) calcd for [M -
was added at 0 °C under a N2 atmosphere. The reaction mixture was
stirred for 15 min at 0 °C, and 2-O-THP-ethan-1-al 13 (217 mg, 1.51
mmol, 1.1 equiv) dissolved in dry THF (10 mL) was added. The
resulting mixture was decolorated, and the formation of a solid was
observed. The solution was subjected to an extractive workup (quench-
ing with water and extraction with diethyl ether), and the product was
purified by silica gel column chromatography. Elution with a mixture
of petroleum ether and ethyl acetate (the volume ratio was changed
from 98:2 to 8:2 v/v) yielded the unsaturated compound resulting from
the Wittig reaction as a colorless oil (480 mg, 75%); Rf ) 0.5 (EP/
EtOAc 95:5 v/v). A solution of this product (1.87 g, 4 mmol) in ethyl
acetate (19 mL) was stirred in the presence of triethylamine (667 µL,
4.8 mmol, 1.2 equiv) and 10% palladium on activated charcoal (190
mg) under an atmosphere of hydrogen gas at room temperature for 12
h. The catalyst was removed by filtration, and the filtrate was
concentrated to dryness under vacuum to give the compound resulting
from the reduction of the double bonds, as a colorless oil (1.9 g); Rf )
I-]+ 459.2453; found: 459.2451.
1-Benzyloxy-10-en-(cis-12,15-methylidene)-hexadecan-16-ol (11).
To a suspension of phosphonium salt 9 (1 g, 1.71 mmol) in dry THF
(12 mL), butyllithium in hexane (2 M) (940 µL, 1.88 mmol, 1.1 equiv)
was added at 0 °C and under a N2 atmosphere. The reaction mixture
was stirred for 15 min at 0 °C, and 10-benzyloxy-decan-1-al 10 (492
mg, 1.88 mmol, 1.1 equiv) dissolved in dry THF (13 mL) was added.
The mixture was decolorated, and a solid appeared. The solution was
subjected to an extractive workup (quenching with water and extraction
with petroleum ether/EtOAc (6:4 v/v)), and the crude product was
purified by silica gel column chromatography. Elution with a mixture
of petroleum ether and ethyl acetate (98:2 v/v) yielded the desired
compound as a colorless oil (600 mg, 80%); Rf ) 0.6 (EP/EtOAc 8/2
v/v); 1H NMR (400 MHz, CDCl3) δ 0.90-1.00 (m, 1H, J ) 6.4, 11.4
Hz), 1.27-1.28 (m, 14H), 1.49-1.83 (m, 10H), 1.91-1.99 (m, 1H),
2.00-2.03 (m, 2H), 2.21-2.28 (m, 1H), 2.74-2.81 (m, 1H), 3.25-
3.31 (dt, 1H, J ) 6.8, 9.1 Hz), 3.44-3.51 (m, 3H), 3.60-3.64 (m, 1H,
J ) 4.8, 6.8 Hz), 3.87 (m, 1H), 4.50 (s, 2H), 4.58 (s, 1H), 5.22-5.38
(m, 2H), 7.25-7.34 (m, 5H); 13C NMR (400 MHz, CDCl3) δ 19.6,
25.5, 26.2-32.8, 27.6, 30.8, 38.0, 38.3, 39.6, 62.2, 62.3, 70.5, 72.2-
72.3, 72.9, 98.9, 128.9-134.8, 127.5, 127.8, 128.4, 138.7; elemental
analysis calcd (%) for C29H46O3: C, 78.68; H, 10.47. Found: C, 79.67;
H, 10.35. To a solution of this protected alcohol (540 mg, 1.22 mmol)
in methanol (20 mL), p-toluenesulfonic acid (11.6 mg, 0.06 mmol, 0.05
equiv) was added. After being stirred for 2 h at room temperature, the
solution was concentrated under vacuum, and the residue was then
purified by column chromatography on silica gel (petroleum ether/
EtOAc (98:2)) to afford compound 11 as a colorless oil (410 mg, 94%);
1
0.5 (EP/EtOAc 95:5 v/v),); H NMR (400 MHz, CDCl3) δ 0.66 (m,
1H), 1.16-1.85 (m, 36H), 1.89-1.93 (m, 1H), 3.37 (1H), 3.46 (t, 2H,
J ) 6.6 Hz), 3.48-3.52 (m, 1H), 3.72 (m, 1H), 3.86-3.91 (m, 1H),
4.50 (s, 2H), 4.57 (s, 1H), 7.20-7.35 (m, 5H); 13C NMR (400 MHz,
CDCl3) δ 19.6-40.6, 62.2, 67.8, 70.5, 72.8, 98.7, 127.4, 127.5, 128.3,
138.6; elemental analysis calcd (%) for C31H52O3: C, 78.76; H, 11.09.
Found: C, 79.10; H, 11.09. To a solution of this protected alcohol
(2.08 g, 4.4 mmol) in methanol (50 mL), p-toluenesulfonic acid (84
mg, 0.44 mmol, 0.1 equiv) was added. The reaction mixture was stirred
overnight and concentrated under vacuum. The crude product was
purified by column chromatography on silica gel (petroleum ether/
EtOAc (from 95:5 to 8:2)) to give compound 14 (1.32 g, 77%) as a
white solid; Rf ) 0.3 (EP/EtOAc 95:5 v/v); 1H NMR (400 MHz, CDCl3)
δ 0.64 (m, 1H, J ) 11.4 Hz), 1.16-1.82 (m, 30H), 1.89-1.93 (m, 1H,
J ) 5.6 Hz), 3.46 (t, 2H, J ) 6.6 Hz), 3.61 (m, 2H, J ) 6.6 Hz), 4.50
(s, 2H), 7.26-7.34 (m, 5H); 13C NMR (400 MHz, CDCl3) δ 26.2-
40.7, 63.3, 70.6, 72.9, 127.5, 127.7, 128.4, 138.7; elemental analysis
calcd (%) for C26H44O2: C, 80.35; H, 11.42. Found: C, 80.01; H, 11.30.
1
Rf ) 0.2 (EP/EtOAc 8/2 v/v); H NMR (400 MHz, CDCl3) δ 0.87-
0.99 (m, 1H, J ) 10.1 Hz), 1.23-1.44 (m, 14H), 1.57-1.66 (m, 2H),
1.72-1.79 (m, 2H), 1.88-1.95 (m, 1H), 2.00-2.03 (m, 2H), 2.10-
2.20 (m, 1H), 2.42-2.46 (m, 1H), 2.70-2.74 (m, 1H), 3.44-3.51 (m,
4H), 4.50 (s, 2H), 5.25-5.37 (m, 2H), 7.26-7.32 (m, 5H); 13C NMR
(400 MHz, CDCl3) δ 27.5, 26.2-32.8, 37.5, 38.3, 42.1, 67.5, 70.5,
72.9, 129.1, 134.6, 127.5, 127.6, 128.3, 138.6.
3,3′-O-[1,18-Octadecan-(cis-8,11-methylidene)-methylene]-2,2′-di-
O-[3-(R)-7-dimethyloctyl]-1′-O-benzyl-sn-diglycerol] (17). A solution
of 2,6-lutidine (150 µL, 1.29 mmol, 3.8 equiv) in 20 mL of dry CH2-
Cl2 was cooled to 0 °C under N2. Triflic anhydride (217 µL, 1.29 mmol,
3.8 equiv) was slowly introduced, and after a few minutes diol 7 (100
mg, 0.34 mmol) was added. The mixture was stirred for 15 min before
water was added. The layers were separated, and the aqueous layer
was extracted twice with CH2Cl2. The combined organic layers were
washed with 5% aqueous HCl and 5% aqueous NaHCO3 and brine,
dried over MgSO4, filtered, and concentrated to dryness. The residue
was purified by silica gel column chromatography, eluting with a
mixture of petroleum ether and EtOAc (9:1) to afford ditriflate 15 as
a colorless oil (Rf ) 0.8 (petroleum ether/EtOAc 9:1). The crude product
was dissolved in dry tetrahydrofuran (3 mL), and a suspension of
alcohol 16 (471 mg, 1.02 mmol, 3 equiv) and potassium hydride (35%
in oil) (156 mg, 1.36 mmol, 4 equiv) in 3 mL of dry THF was added.
The suspension was stirred for a few minutes at room temperature
before we added water. The resulting mixture was extracted with
ether: the combined extracts were dried over MgSO4, and the solvent
was removed under reduced pressure. The residue was purified by
column chromatography on silica gel with a mixture of petroleum ether
and ethyl acetate (95:5) to yield the acyclic diol (125 mg, 42%) as a
yellow oil (Rf ) 0.8 (EP/EtOAc 9:1 v/v)). A solution of this diol (1.77
g, 1.2 mmol) in ethyl acetate (50 mL) was stirred overnight in the
presence of 10% palladium on activated charcoal (500 mg) under an
atmosphere of H2 at room temperature. The catalyst was removed by
filtration, and the filtrate was concentrated to dryness under vacuum
to give the deprotected diol as a colorless oil (780 mg, 89%); Rf ) 0.3
(EP/EtOAc 8:2 v/v); [R]20D +11.0° (c 0.5, CHCl3); 1H NMR (400 MHz,
CDCl3) δ 0.58-0.66 (m, 1H), 0.84-0.89 (m, 18H), 1.05-1.83 (m,
48H), 1.86-1.95 (m, 1H), 3.4-3.7 (m, 18H); 13C NMR (400 MHz,
1-Benzyloxy-10-en-(cis-12,15-methylidene)-hexadecyl-16-triphe-
nylphosphonium iodide (12). To a solution of alcohol 11 (1.240 g,
3.46 mmol) in CH2Cl2 (25 mL), imidazole (500 mg, 7.37 mmol, 2.1
equiv) and diiode (1.27 g, 4.91 mmol, 1.4 equiv) were added at 0 °C,
and the resulting reaction mixture took a red coloration. Triphenylphos-
phine (1.09 g, 4.2 mmol, 1.2 equiv) in CH2Cl2 (25 mL) was added
slowly at 0 °C; the solution became yellow, and the formation of a
solid was observed. After being stirred 5 h in the dark, the solution
was quenched with saturated aqueous sodium hydrogenocarbonate and
extracted with CH2Cl2. The combined organic layers were washed with
water, dried over MgSO4, filtered, and concentrated to dryness. A
column chromatography on silica gel (CH2Cl2) gave the desired product
(1.39 g, 86%). This product (5.9 g, 12.7 mmol) and additional
triphenylphosphine (4.0 g, 15.24 mmol, 1.2 equiv) were dissolved in
acetonitrile (140 mL). The reaction mixture was refluxed for 2 days.
A column chromatography over silica gel with CH2Cl2 and CH2Cl2/
MeOH (98:2) gave the phosphonium salt 12 as a white solid (7.24 g,
78%); Rf ) 0.5 (CH2Cl2/MeOH 95:5 v/v); mp ) 139-140 °C (MeOH/
1
Et2O); H NMR (400 MHz, CDCl3) δ 1.19-1.42 (m, 15H), 1.52-
1.69 (m, 4H), 1.74-1.83 (m, 1H, J ) 6.0 Hz), 1.89-1.97 (m, 2H),
2.18-2.31 (m, 1H), 2.58-2.65 (m, 1H), 3.45-3.48 (t, 2H, J ) 9.6
Hz), 3.74-3.88 (d, 2H), 4.50 (s, 2H), 5.21-5.29 (m, 2H), 7.26-7.34
(m, 5H), 7.70-7.88 (m, 15H); 13C NMR (400 MHz, CDCl3) δ 26.1-
29.7, 27.4, 29.2, 32.0, 33.2, 34.5, 37.5, 42.5, 70.4, 72.8, 129.4,133.7,
127.5, 127.6, 128.3, 138.6, 118.0, 118.8, 130.5, 130.6, 133.7, 133.8,
135.1, 135.2; FABMS (m-nitrobenzyl alcohol matrix) for C42H52OP:
calcd for [M+], 603.3756; found, 603.3755.
(cis-4,7-Methylidene)-18-benzyloxy-octadecan-1-ol (14). To a
suspension of phosphonium salt 12 (1 g, 1.37 mmol) in dry THF (15
mL), butyllithium in hexane (1.6 M) (943 µL, 1.51 mmol, 1.1 equiv)
9
J. AM. CHEM. SOC. VOL. 126, NO. 32, 2004 10009

A R T I C L E S
Brard et al.
CDCl3) δ 19.67, 19.74, 19.8, 22.7-22.8, 24.4-40.8, 39.4, 40.2, 63.1,
68.7, 71.0, 71.9, 78.3; elemental analysis calcd (%) for C45H90O6: C,
74.32; H, 12.47. Found: C, 73.92; H, 12.36. A solution of 270 mg of
this diol (0.37 mmol, 1 equiv) and 45 µL of crown ether 15-5 in 13
mL of tetrahydrofuran was stirred at room temperature for 10 min,
and sodium hydride (9 mg, 0.37 mmol, 1 equiv) was added. The reaction
mixture was stirred for 10 min followed by dropwise addition of benzyl
bromide (53 µL, 0.45 mmol, 1.2 equiv). The solution was refluxed
overnight and quenched with addition of water. The aqueous phase
was extracted with diethyl ether and ethyl acetate. The organic layers
were dried (MgSO4), filtered, and concentrated under vacuum. The
residue was purified by column chromatography on silica gel eluting
with CH2Cl2 and then CH2Cl2/MeOH (96:4) to give alcohol 17 (0.5 g,
59%) as a colorless oil; Rf ) 0.5 (EP/EtOAc 9:1 v/v); [R]20D +4.5° (c
2, CHCl3); 1H NMR (400 MHz, CDCl3) δ 0.51-0.59 (m, 1H), 0.75-
0.80 (m, 18H), 1.00-1.70 (m, 50H), 1.80-1.84 (m, 1H), 2.17 (s, 1H),
3.34-3.66 (m, 18H), 4.48 (s, 2H), 7.18-7.26 (m, 5H); 13C NMR (400
MHz, CDCl3) δ 19.6, 19.8, 22.7, 22.8, 24.4-40.4, 40.2, 63.1, 68.7,
68.9, 70.3, 70.8, 71.0, 71.7, 71.9, 73.4, 78.0, 78.4, 127.5, 127.6, 128.4,
138.5; elemental analysis calcd (%) for C52H96O6: C, 76.42; H, 11.84.
Found: C, 76.74; H, 12.06.
was cooled to 0 °C under N2. Triflic anhydride (1.04 mL, 6.16 mmol,
2 equiv) was slowly introduced, and after a few minutes, diol 19 (840
mg, 3.08 mmol) dissolved in 16 mL of CH2Cl2 was added dropwise.
The mixture was stirred for 15 min before water was added. The layers
were separated, and the aqueous layer was extracted twice with CH2-
Cl2. The combined organic layers were washed with 5% aqueous HCl,
5% aqueous NaHCO3, and brine, dried over MgSO4, filtered, and
concentrated to dryness. The residue was purified by silica gel column
chromatography, eluting with a mixture of petroleum ether and EtOAc
(9:1) to afford compound 20 (1.12 g, 90%) as a colorless oil (Rf ) 0.6
(petroleum ether/EtOAc 9:1). To a solution of this crude product in
dry tetrahydrofuran (10 mL), a suspension of alcohol 14 (811 mg, 2.09
mmol, 1 equiv) and potassium hydride (35% in oil) (311 mg, 2.7 mmol,
1.3 equiv) in 30 mL of dry THF was added at 0 °C. The suspension
was stirred for a few minutes at room temperature before water was
added, and the resulting mixture was extracted with ether. The combined
extracts were dried over MgSO4, and the solvent was removed at
reduced pressure. The residue was purified by column chromatography
on silica gel with a mixture of petroleum ether and ethyl acetate (95/5)
to yield compound 21 (900 mg, 68%) as a colorless oil; Rf ) 0.6 (EP/
EtOAc 9/1 v/v); [R]20D +1.3° (c 1, CHCl3); 1H NMR (400 MHz, CDCl3)
δ 0.63 (m, 1H), 0.86 (m, 9H), 1.06-1.75 (m, 40H), 1.89-1.93 (m,
1H), 3.41-3.66 (m, 11H), 4.03 (m, 2H), 4.50 (s, 2H), 5.17 (d, 1H, J
) 10.4 Hz), 5.26 (d, 1H, J ) 17.3 Hz), 5.89 (m, 1H, J ) 6.4 Hz),
7.26-7.35 (m, 5H); 13C NMR (400 MHz, CDCl3) δ 19.7, 22.7, 22.8,
24.7-40.7, 69.0, 70.3, 70.6, 70.8, 71.9, 72.4, 72.9, 77.9, 116.9, 127.5,
127.7, 128.4, 134.9, 138.7.
2-O-(3-(R)-7-Dimethyloctyl)-3-O-tetrahydropyranyl-sn-glycerol
(18). To a solution of alcohol 17 (2 g, 6.2 mmol) and p-toluenesulfonic
acid (60 mg, 0.3 mmol, 0.5 equiv) in 32 mL of CH2Cl2, dihydropyran
(876 µL, 9.6 mmol, 1.5 equiv) was added dropwise at 0 °C. The solution
was stirred overnight at room temperature, and 88 µL of triethylamine
(0.63 mmol, 0.1 equiv) was added. After being stirred for 5 min, norit
was introduced, and the mixture was stirred for a few minutes. The
reaction mixture was concentrated and filtered on silica gel (CH2Cl2).
After removal of the solvent, 1.8 g (4.4 mmol) of the protected
compound was isolated. To this product dissolved in ethanol (45 mL),
10% palladium on activated charcoal (180 mg) was added, and the
solution was stirred under H2 atmosphere overnight. The catalyst was
removed by filtration, and the filtrate was concentrated to dryness to
3-O-[1-Octadecan-(cis-4,7-methylidene)-(18-hydroxy)-methylene]-
2-O-[3-(R)-7-dimethyloctyl]-1-O-allyl-sn-glycerol (22). To a solution
of compound 21 (1.8 g, 1.24 mmol) in CH2Cl2 (60 mL) and water (3.2
mL), 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (1.41 g, 6.2
mmol, 5 equiv) was added. The reaction mixture was stirred vigorously
for 20 h and then quenched with 5% aqueous of sodium carbonate (40
mL). The aqueous phase was extracted twice with CH2Cl2. The
combined organic layers were dried over MgSO4, filtered, and
concentrated to dryness under vacuum. The residue was purified by
column chromatography on silica gel (petroleum ether/EtOAc (the
volume ratio was changed from 99:1 to 9:1)) to give deprotected alcohol
give alcohol 18 (1.26 g, 66%) as a colorless oil; Rf ) 0.3 (EP/EtOAc
1
8:2 v/v); [R]20 +9.4° (c 1, CH2Cl2); H NMR (400 MHz, CDCl3) δ
D
0.76-1.04 (2s, 9H), 1.10-1.84 (m, 16H), 3.27-3.34 (dt, 1H, J ) 6.6,
9.4 Hz), 3.50-3.80 (m, 7H), 3.84-3.88 (dt, 1H, J ) 3.6, 7.6 Hz),
4.60 (s, 1H); 13C NMR (400 MHz, CDCl3) δ 19.6, 19.7, 22.7, 22.8,
24.8, 25.5, 28.0, 29.9, 30.7, 37.2, 37.5, 39.4, 62.3, 63.0, 69.1, 72.0,
78.4, 99.0.
22 as a colorless oil (470 mg, 68%); Rf ) 0.5 (EP/EtOAc 8/2 v/v);
1
[R]20 +1.5° (c 1, CHCl3); H NMR (400 MHz, CDCl3) δ 0.56 (m,
D
1H, J ) 11.7 Hz), 0.80 (m, 9H), 1.00-1.72 (m, 40H), 1.83 (m, 1H, J
) 5.6 Hz), 3.34-3.60 (m, 11H), 3.94 (d, 2H, J ) 5.6 Hz), 5.10 (dd,
1H, J ) 1.5, 10.4 Hz), 5.20 (dd, 1H, J ) 1.8, 17.3 Hz), 5.84 (m, 1H);
13C NMR (400 MHz, CDCl3) δ 19.7, 22.7, 22.8, 24.7-40.7, 63.2, 69.0,
70.3, 70,8, 72.0, 72.4, 78.0, 116.9, 134.9; elemental analysis calcd (%)
for C35H68O4: C, 76.03; H, 12.40. Found: C, 76.49; H, 12.31.
1-O-Allyl-2-O-(3-(R),7-dimethyloctyl)-sn-glycerol (19). To a solu-
tion of alcohol 18 (1.70 g, 5.4 mmol) in dry tetrahydrofuran (60 mL),
sodium hydride (60% in oil) (430 mg, 10.8 mmol, 2 equiv) was added
slowly. The solution was stirred for 15 min, and tetrabutylammonium
bromide (174 mg, 0.54 mmol, 0.1 equiv) and allyl bromide (701 µL,
8.1 mmol, 1.5 equiv) were then introduced. After being stirred for 5 h,
the mixture was diluted with ether, washed with water and brine, dried
over MgSO4, filtered, and concentrated to dryness under vacuum. To
this residue dissolved in methanol (50 mL), p-toluenesulfonic acid (100
mg, 0.53 mmol, 0.1 equiv) was added, and the resulting solution was
stirred overnight at room temperature. The reaction mixture was
concentrated under vacuum, and the residue was purified by silica gel
column chromatography elution with a mixture of petroleum ether/
EtOAc (9:1 then 8:2 v/v) to give alcohol 19 as a colorless oil (1.08 g,
75%); Rf ) 0.2 (EP/EtOAc 9/1 v/v); [R]20D -7.9° (c 1.2, CH2Cl2); 1H
NMR (400 MHz, CDCl3) δ 0.86-0.89 (2s, 9H), 1.12-1.63 (m, 10H),
2.28 (s, 1H), 3.50-3.73 (m, 7H), 4.00-4.01 (d, 2H, J ) 5.6 Hz), 5.19
(dd, 1H, J ) 1 Hz, 10.4 Hz), 5.27 (dd, 1H, J ) 1.6, 17.3 Hz), 5.88 (m,
1H, J ) 6.9 Hz); 13C NMR (400 MHz, CDCl3) δ 19.7, 22.7, 22.8,
24.7, 28.0, 29.8, 37.1, 37.4, 39.3, 62.9, 68.7, 70.1, 72.5, 78.5, 117.2,
134.5; elemental analysis calcd (%) for C16H32O3: C, 70.54; H, 11.84.
Found: C, 70.32; H, 11.76.
1-O-Benzyl-2-O-(3-(R)-7-dimethyloctyl)-3-O-triflate-sn-glycerol
(23). A solution of 2,6-lutidine (442 µL, 3.80 mmol, 2 equiv) in 40
mL of dry CH2Cl2 was cooled to 0 °C under N2. Triflic anhydride (639
µL, 3.80 mmol, 2 equiv) was slowly introduced, and after a few minutes
diol 16 (612.5 mg, 1.90 mmol) dissolved in 10 mL of CH2Cl2 was
added dropwise. The mixture was stirred for 15 min before water was
added. The layers were separated, and the aqueous layer was extracted
twice with CH2Cl2. The combined organic layers were washed with
5% aqueous HCl, 5% aqueous NaHCO3, and brine, dried over MgSO4,
filtered, and concentrated to dryness. The residue was purified by silica
gel column chromatography, eluting with a mixture of petroleum ether
and EtOAc (9:1) to afford compound 23 (691 mg, 80%) as a colorless
oil; Rf ) 0.6 (petroleum ether/EtOAc 9:1); 1H NMR (400 MHz, CDCl3)
δ 0.78 (m, 9H), 1.00-1.56 (m, 10H), 3.41-3.52 (m, 4H), 3.65 (m,
1H), 4.46 (s, 2H), 4.48 (d, 1H, J ) 6.1 Hz), 4.56 (dd, 1H, J ) 3.1,
10.4 Hz), 7.26-7.35 (m, 5H); 13C NMR (400 MHz, CDCl3) δ 19.5,
22.6, 22.7, 24.7, 28.0, 29.7, 36.8, 37.3, 39.3, 67.8, 69.3, 73.7, 75.9,
76.0, 118 (q, J ) 320 Hz), 127.8, 128.0, 128.6, 137.5.
3-O-[1-Octadecan-(cis-4,7-methylidene)-18-benzyloxy-methylene]-
2-O-[3-(R),7-dimethyloctyl]-1-O-allyl-sn-glycerol (21). A solution of
2,6-lutidine (720 µL, 6.16 mmol, 2 equiv) in 40 mL of dry CH2Cl2
3,3′-O-[1,18-Octadecan-(cis-4,7-methylidene)-methylene]-2,2′-di-
O-[3-(R)-7-dimethyloctyl]-1′-O-benzyl-sn-diglycerol (24). To a solu-
9
10010 J. AM. CHEM. SOC. VOL. 126, NO. 32, 2004

Bipolar Archael Glycolipid Analogues
A R T I C L E S
tion of triflate (691 mg, 1.52 mmol, 1.6 equiv) in dry tetrahydrofuran
(2 mL), a suspension of alcohol 22 (500 mg, 0.96 mmol, 1 equiv) and
potassium hydride (35% in oil) (150 mg, 1.15 mmol, 1.2 equiv) in 6
mL of dry THF was added at 0 °C. The suspension was stirred for a
few minutes at room temperature before water was added, and the
resulting mixture was extracted with ether. The combined extracts were
dried over MgSO4, and the solvent was removed under reduced pressure.
The crude product was purified by column chromatography on silica
gel with a mixture of petroleum ether and ethyl acetate (95/5) to yield
68.7, 69.0, 69.1, 70.2, 70.3, 70.5, 70.7, 71.0, 71.6, 71.7, 71.8, 72.5,
72.9, 76.2, 77.7, 78.3, 100.7, 101.0, 169.3, 169.7, 170.0, 170.3, 170.4,
170.6, 170.7; elemental analysis calcd (%) for C71H124O23: C, 63.37;
H, 9.29. Found: C, 63.09; H, 9.21.
3,3′-O-[1,18-Octadecan-(cis-8,11-methylidene)-methylene]-2,2′-di-
O-[3-(R)-7-dimethyloctyl]-1-O-[4-O-(â-D-galactopyranosyl)-â-D-glu-
copyranosyl]-1′-O-(â-D-galactofuranosyl)-sn-diglycerol (1). Com-
pound 27 (51 mg, 0.04 mmol, 1 equiv) and pent-4-enyl 2,3,5,6-tetra-
O-acetyl-R,â-D-galactofuranoside 26 (24 mg, 0.06 mmol, 1.5 equiv)
were combined, rotoevaporated twice with toluene, and then dried for
2 h under vacuum. A solution of this mixture in dry CH2Cl2 (2 mL)
was added to 4 Å molecular sieves. The mixture was treated at 0 °C
under N2 and in the dark with N-iodosuccinimide (34 mg, 0.16 mmol,
4 equiv) followed by dropwise addition of triethylsilyl trifluo-
romethanesulfonate (6.8 µL, 0.03 mmol, 0.8 equiv). The reaction was
quenched with a few drops of triethylamine after 10 min at room
temperature. The resulting solution was diluted with CH2Cl2, washed
successively with 10% aqueous sodium thiosulfate, water, and brine,
dried over MgSO4, and rotoevaporated. The crude product was purified
with silica gel column chromatography with petroleum ether/EtOAc
(7:3) to give the acetylated bisglycosylated lipid as a colorless oil (22
mg, 35%); Rf ) 0.3 (EP/EtOAc 6:4 v/v); [R]20D -11.5° (c 1, CHCl3);
1H NMR (400 MHz, CDCl3) δ 0.57 (m, 1H, J ) 10.2 Hz), 0.78-0.80
(m, 18H), 0.97-1.73 (m, 50H), 1.78-1.85 (m, 1H), 1.90-2.09 (m,
33H), 3.31-3.55 (m, 17H), 3.69 (q, 1H, J ) 4.0 Hz), 3.72 (t, 1H, J )
9.6 Hz), 3.79-3.84 (m, 2H), 3.99-4.09 (m, 3H), 4.15 (m, 1H), 4.18
(m, 1H), 4.26 (dd, 1H, J ) 4.3, 11.9 Hz), 4.40 (m, 2H), 4.48 (d, 1H,
J ) 7.9 Hz), 4.82-4.93 (m, 3H), 5.00 (m, 2H), 5.05 (m, 1H), 5.12 (t,
1H, J ) 9.2 Hz), 5.27 (d, 1H, J ) 3.1 Hz), 5.32 (m, 1H); 13C NMR
(400 MHz, CDCl3) δ 19.7, 19.8, 20.6, 20.9, 22.7, 22.8, 24.7-39.3,
40.2, 40.8, 60.8, 62.1, 62.8, 66.6, 67.5, 69.0, 69.2, 69.1, 69.3, 70.5,
70.7, 71.0, 71.7, 71.8, 72.6, 72.9, 76.4, 76.6, 77.7, 77.9, 79.9, 81.3,
100.9, 101.2, 105.8, 169.2, 170.6; elemental analysis calcd (%) for
C85H142O32: C, 60.91; H, 8.54. Found: C, 61.24; H, 8.72. A solution
of sodium methoxide in CH3OH (0.1 M, 131 µL) was added to a
solution of the acetylated bisglycosylated lipid (22 mg, 0.013 mmol)
in 2 mL of CH3OH. The mixture was stirred for 2 h at room
temperature, neutralized with a solution of acetic acid in methanol and
concentrated under vacuum. The crude product was purified by silica
gel column chromatography (CHCl3/MeOH/H2O 7:3:0.5) to yield 1 as
a pasty solid (16 mg, >95%); Rf ) 0.7 (CHCl3/MeOH/H2O 7:3:0.5
the corresponding acyclic compound (411.5 mg, 50%) as a colorless
1
oil; Rf ) 0.6 (EP/EtOAc 8:2 v/v); [R]20 -4.0° (c 0.8. CHCl3); H
D
NMR (400 MHz, CDCl3) δ 0.64 (m, 1H, J ) 10.7 Hz), 0.85 (m, 18H),
1.10-1.73 (m, 50H), 1.91 (m, 1H), 3.41-3.64 (m, 18H), 4.01 (d, 2H,
J ) 5.6 Hz), 4.55 (s, 2H), 5.17 (d, 1H, J ) 10.4 Hz), 5.27 (d, 1H, J )
17.0 Hz), 5.90 (m, 1H, J ) 5.6 Hz), 7.26-7.34 (m, 5H); 13C NMR
(400 MHz, CDCl3) δ 19.7, 22.7, 22.8, 24.7-40.2, 68.9, 69.0, 70.3,
70.8, 71.7, 71.9, 72.4, 73.4, 78.0, 116.9, 127.6, 127.7, 128.4, 138.5,
134.9. To a solution of this compound (50 mg, 0.077 mmol) in a mixture
of toluene/EtOH/H2O (1.25 mL:2.65 mL:400 µL), Wilkinson catalyst
(Ph3P)3RhCl (15 mg, 0.016 mmol, 0.2 equiv) was added. The reaction
mixture was heated at 100 °C for 24 h and stirred 12 h at room
temperature. After removal of solvent, the residue was dissolved in
diethyl ether. The organic layer was washed with water and brine, dried
(MgSO4), filtered, and concentrated under vacuum. The residue was
purified by silica gel column chromatography eluting with a mixture
of petroleum ether/EtOAc (95:5 v/v) to yield compound 24 (47 mg,
78%) as a colorless oil; Rf ) 0.3 (EP/EtOAc 8/2 v/v); [R]20D +12.2° (c
1
1.07, CHCl3); H NMR (400 MHz, CDCl3) δ 0.65 (m, 1H, J ) 10.9
Hz), 0.87 (m, 18H), 1.00-1.73 (m, 50H), 1.90 (m, 1H), 2.22 (s, 1H),
3.41-3.71 (m, 18H), 4.56 (s, 2H), 7.26-7.34 (m, 5H);13C NMR (400
MHz, CDCl3) δ 19.7, 22.7, 22.8, 24.7-40.7, 63.2, 68.7, 68.9, 70.3,
70.8, 71.0, 71.7, 72.2, 73.4, 78.0, 78.3, 127.6, 127.7, 128.4, 138.4;
elemental analysis calcd (%) for C52H96O6: C, 76.42; H, 11.84.
Found: C, 76.95; H, 11.98.
3,3′-O-[1,18-Octadecan-(cis-8,11-methylidene)-methylene]-2,2′-di-
O-[3-(R)-7-dimethyloctyl]-1-O-[2,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-
O-acetyl-â-D-galactopyranosyl)-â-D-glucopyranosyl]-sn-diglycerol (27).
Compound 17 (109 mg, 0.13 mmol) and lactosyl thioglycoside 25 (136
mg, 0.2 mmol, 1.5 equiv) were combined, rotoevaporated twice with
toluene, and then dried for 2 h under vacuum. A solution of this mixture
in dry CH2Cl2 (4.5 mL) was added to 4 Å molecular sieves. The mixture
was treated at 0 °C under N2 and in the dark with N-iodosuccinimide
(60 mg, 0.27 mmol, 2 equiv) followed by dropwise addition of
triethylsilyl trifluoromethanesulfonate (12 µL, 0.05 mmol, 0.4 equiv).
The reaction was quenched with a few drops of triethylamine after 5
min at room temperature. The resulting solution was diluted with CH2-
Cl2, washed successively with 10% aqueous sodium thiosulfate, water,
and brine, dried over MgSO4, and rotoevaporated. The crude product
was purified with silica gel column chromatography with petroleum
ether/EtOAc 7:3 to give the acetylated lactosylated lipid (89 mg, 48%);
Rf ) 0.5 (EP/EtOAc 6:4 v/v); [R]20D -5.2° (c 1, CHCl3) as a colorless
oil. To a solution of this product (55 mg, 0.04 mmol) in ethanol (2
mL), 10% palladium on activated charcoal (10 mg) was added. The
solution was stirred under H2 atmosphere overnight. The catalyst was
removed by filtration, and the filtrate was concentrated to dryness. A
purification by column chromatography on silica gel (petroleum ether/
EtOAc 7:3)) gave the deprotected monoglycosylated lipid 27 (51 mg,
v/v); mp ) 125-130 °C; [R]20 -16.4° (c 1, MeOH/CHCl3 1:1 v/v);
D
1H NMR (400 MHz, CDCl3) δ 0.59 (m, 1H), 0.80 (m, 18H), 0.95-
1.68 (m, 51H), 3.14-3.91 (m, 36H), 4.24 (2d, 2H, J ) 7.4 Hz), 4.80
(m, 1H); 13C NMR (400 MHz, CDCl3) δ 20.19, 20.23, 23.1, 23.2, 25.9-
40.5, 41.2, 41.4, 41.9, 61.9, 62.4, 64.5, 68.4, 69.6, 69.7, 70.2, 70.3,
70.0, 71.8, 72.4, 72.5, 72.6, 72.8, 74.7, 74.8, 76.3, 76.5, 77.1, 78.9,
79.2, 79.3, 80.5, 83.2, 84.6, 104.6, 105.1, 109.7; FABMS (m-nitrobenzyl
alcohol matrix) calcd for [M + Na]+, 1235.8220; found, 1235.8217.
3,3′-O-[1,18-Octadecan-(cis-4,7-methylidene)-methylene]-2,2′-di-
O-[3-(R)-7-dimethyloctyl]-1-O-[4-O-(â-D-galactopyranosyl)-â-D-glu-
copyranosyl]-1′-O-(â-D-galactofuranosyl)-sn-diglycerol (2). Follow-
ing the same strategy as for compound 1 and starting from alcohol 25,
compound 2 was obtained (first glycosylation: 50% yield; deprotec-
tion: 98% yield; second glycosylation: 58% yield; last deprotection:
86% yield), compound 2: Rf ) 0.7 (CHCl3/MeOH/H2O 7:3:0.5 v/v);
mp ) 196-199 °C, [R]20D -9.8° (c 0.86, MeOH); 1H NMR (400 MHz,
CDCl3) δ 0.59 (m, 1H), 0.80 (m, 18H), 0.95-1.68 (m, 51H), 3.14-
3.91 (m, 36H), 4.24 (2d, 2H, J ) 7.4 Hz), 4.80 (m, 1H); 13C NMR
(400 MHz, CDCl3) δ 20.19, 20.23, 23.1, 23.2, 25.9-40.5, 41.2, 41.4,
41.9, 61.9, 62.4, 64.5, 68.4, 69.6, 69.7, 70.2, 70.3, 70.0, 71.8, 72.4,
72.5, 72.6, 72.8, 74.7, 74.8, 76.3, 76.5, 77.1, 78.9, 79.2, 79.3, 80.5,
83.2, 84.6, 104.6, 105.1, 109.7; FABMS (m-nitrobenzyl alcohol matrix)
calcd for [M + Na]+, 1235.8220; found, 1235.8220.
>99%) as a colorless oil; Rf ) 0.4 (EP/EtOAc 6:4 v/v); [R]20 -3.2°
D
1
(c 1, CHCl3); H NMR (400 MHz, CDCl3) δ 0.52-0.61 (m, 1H, J )
9.9 Hz), 0.79-0.81 (m, 18H), 1.08-1.67 (m, 50H), 1.82-1.85 (m,
1H), 1.91-2.10 (6s, 21H), 3.31-3.63 (m, 18H), 3.75 (t, 1H, J ) 9.4
Hz), 3.82-3.86 (m, 2H), 4.00-4.10 (m, 3H), 4.40-4.45 (m, 2H), 4.49
(d, 1H, J ) 7.7 Hz), 4.82-4.86 (t, 1H, J ) 8.2 Hz), 4.89-4.92 (dd,
1H, J ) 3.3, 10.4 Hz), 5.01-5.06 (dd, 1H, J ) 8.1, 10.3 Hz), 5.13 (t,
1H, J ) 9.4 Hz), 5.28 (d, 1H, J ) 2.7 Hz); 13C NMR (400 MHz, CDCl3)
δ 19.6, 20.4, 20.8, 22.5, 22.6, 24.6-39.2, 40.7, 60.8, 62.0, 62.6, 66.6,
Acknowledgment. We thank Mrs. R. Kaiser for excellent
technical assistance in freeze-fracturing, Mr. F. Steiniger for
9
J. AM. CHEM. SOC. VOL. 126, NO. 32, 2004 10011

A R T I C L E S
Brard et al.
preparing the micrographs by cryo-TEM, and the french
Ministe`re de l′Education Nationale de la Recherche et de la
Technologie for a grant to M.B.
two-by-two vesicle membranes connected by a tunnel-like
opening. This material is available free of charge via the Internet
at http://pubs.acs.org.
Supporting Information Available: FFEM of compound 1
in the absence of glycerol and FFEM of compound 1 showing
JA049805N
9
10012 J. AM. CHEM. SOC. VOL. 126, NO. 32, 2004