Synthesis and supramolecular characterization of a novel class of glycopyranosyl-containing amphiphiles

Article abstract of DOI:10.1021/jo991685s

A novel class of glycopeptidolipids is described, which potentially can be used as a novel antigen-delivery system. The compounds have been prepared by a combination of solid-supported and solution-based methods. The use of the orthogonally protected FmocLysDde derivative provided an opportunity to incorporate two different types lipids. It was found that the model compound 1 forms aggregates in aqueous media which can be described as rod or tubelike structures. The aggregates can be stabilized by topotactic photopolymerization. Studies on the structural analogues 2-5 revealed the effect of the carbohydrate, peptide, and lipid moiety on the aggregation properties. It is concluded that none of the structure elements can lay claim to be exclusively important for the formation of highly organized aggregates such as tubes, fibers, or helical ribbons from 1, but the presence of all of these structural elements afforded the most uniformly shaped extended structures.

Full text of DOI:10.1021/jo991685s

J . Org. Chem. 2000, 65, 3357-3366
3357
Syn th esis a n d Su p r a m olecu la r Ch a r a cter iza tion of a Novel Cla ss
of Glycop yr a n osyl-Con ta in in g Am p h ip h iles
Frank Reichel,‡ Annie M. Roelofsen,§ Hubertus P. M. Geurts,| Sjerry J . van der Gaast,
Martinus C. Feiters,§ and Geert-J an Boons*,†,‡
School of Chemistry, The University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
Received October 27, 1999
A novel class of glycopeptidolipids is described, which potentially can be used as a novel antigen-
delivery system. The compounds have been prepared by a combination of solid-supported and
solution-based methods. The use of the orthogonally protected FmocLysDde derivative provided
an opportunity to incorporate two different types lipids. It was found that the model compound 1
forms aggregates in aqueous media which can be described as rod or tubelike structures. The
aggregates can be stabilized by topotactic photopolymerization. Studies on the structural analogues
2-5 revealed the effect of the carbohydrate, peptide, and lipid moiety on the aggregation properties.
It is concluded that none of the structure elements can lay claim to be exclusively important for
the formation of highly organized aggregates such as tubes, fibers, or helical ribbons from 1, but
the presence of all of these structural elements afforded the most uniformly shaped extended
structures.
In tr od u ction
established a correlation between molecular structure,
aggregation properties, and immunological activities of
a range lipopeptides and found that compounds, which
arrange in tubular or extended structures, are more
immunogenic than compounds that organize in vesicular
structures.3
Conjugates composed of bacterial poly(oligo)saccha-
rides and a carrier protein are successfully used as
vaccines against Haemophilus influenzae and are under
investigation as experimental vaccines against Strepto-
coccus pneumoniae, Neisseria meningitidis, and Strepto-
coccus group B.1
Here, we report an efficient synthetic methodology for
the preparation of a new class of amphiphiles that
contain a carbohydrate, a peptide, and a lipid moiety.
These compounds will be used as antigen delivery
systems and will have the additional advantage that their
aggregates can be stabilized by topotactic photopoly-
merization of the 10,12-pentacosadiynoic acid moiety. The
synthetic methodology was developed by the preparation
of model compound 1 (Chart 1). The self-assembly
properties of the glycopeptidolipids were studied by
transmission electron microscopy (TEM) and low-angle
X-ray powder diffraction (XRD). It was discovered that
compound 1 organized into fibers or tubular structures,
which can be stabilized by polymerization of a diacety-
lene-containing lipid moiety. The derivatives 2-5 (Chart
1) were prepared to reveal the structural motives of 1
that induce tubular or extended aggregate formation.
We are developing a new generation of saccharide-
containing vaccines based on self-assembling fully syn-
thetic glycopeptidolipids.2,3 These compounds contain a
carbohydrate moiety that will act as a B-epitope, a
peptide sequence which is a MHC class II restricted
recognition site for human T-cells and a lipid moiety,
which confers upon the compound the ability to self-
assemble to give macromolecular aggregates. The self-
assembly properties of the glycopeptidolipids are very
important because small molecules are unable to stimu-
late a sustainable antibody response.4 However, highly
immunological responses are observed when small hap-
tens are coupled at the surface of macromolecules.
Traditionally, antigenic macromolecules were obtained
by conjugation of a hapten to a carrier protein; however,
it is now recognized that self-assembled aggregates can
also function as efficient immunogens. Recently, we
Resu lts a n d Discu ssion
* To whom correspondence should be addressed.
† Present address: Complex Carbohydrate Research Center, 220
Riverbend Road, Athens, GA 30602. Tel.: 706-642-4401. Fax: 706-
542 4412. Email: gjboons@ccrc.uga.edu.
Syn th esis. The synthesis of the target compound 1
requires a highly convergent strategy and synthetic
procedures that are compatible with carbohydrate, pep-
tide, and lipid chemistry. It was envisaged that tetrapep-
tide ALFG (6) (A ) alanine, L ) leucine, F ) phenyl-
‡ The University of Birmingham.
§ Department of Organic Chemistry, NSR Institute for Molecular
Structure, Design and Synthesis, University of Nijmegen,
1 Toernooiveld, NL-6525 ED Nijmegen, The Netherlands.
| Central Microscopy Facility, Faculty of Science, University of
Nijmegen, 1 Toernooiveld, NL-6525 ED Nijmegen, The Netherlands.
Netherlands Institute for Sea Research (NIOZ), P.O. Box 59, NL-
1790 AB Den Burg, Texel, The Netherlands.
(1) J ennings, H. J .; Sood, R. K. In Neoglycoconjugates: preparation
and application; Lee, Y. C.; Lee, R. T., Eds.; Academic Press: New
York, 1994; p 325.
(4) (a) J ung, G.; Wiesmu¨ller, K.-H.; Becker, G.; Bu¨hring, H. J .;
Bessler, W. G. Angew. Chem., Int. Ed. Engl. 1985, 24, 872. (b) Deres,
K.; Schild, H.; Wiesmu¨ller, K. H.; J ung, G.; Rammensee, H. G. Nature
1989, 342, 561. (c) Reitermann, A.; Metzger, J .; Wiesmu¨ller, K.-H.;
J ung, G.; Bessler, W. G.; Biol. Chem. Hoppe-Seyler 1989, 370, 343. (d)
Metzger, J . W.; Wiesmu¨ller, K. H.; J ung, G. Int. J . Pept. Protein Res.
1991, 38, 545. (e) Wiesmu¨ller, K. H.; Bessler, W. G.; J ung, G. Int. J .
Pept. Protein Res. 1992, 40, 255. (f) DeOgny, L.; Pramanik, B. C.; Arndt,
L. L.; J ones, J . D.; Rush, J .; Slaughter, C. A.; Radolf, J . D.; Norgard,
M. V. Pept. Res. 1994, 7, 91
(2) Reichel, F.; Ashton, P. R.; Boons, G. J . Chem. Commun. 1997,
2087.
(3) Reichel, F.; Roelofsen, A. M.; Geurts, H. P. M.; Hamalainen, T.
I.; Feiters, M. C.; Boons, G. J . J . Am. Chem. Soc. 1999, 121, 7989
10.1021/jo991685s CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/04/2000

3358 J . Org. Chem., Vol. 65, No. 11, 2000
Reichel et al.
Sch em e 1. P r ep a r a tion of Com p ou n d 1a
Ch a r t 1
Ch a r t 2
a
Reagents and conditions: (i) 10 equiv of FmocGlyOH, 5 equiv
of DIPC, DMF/CH2Cl2 (5/2, v/v), then 9 and 1 equiv of DMAP; (ii)
20% piperidine/DMF; (iii) 5 equiv of FmocAA, 6 equiv of HOBt, 6
equiv of DIPC, CH2Cl2/DMF (5/2, v/v); (iv) 2 equiv of of 7, 2 equiv
of PyBOP, 2 equiv of DIPEA, CH2Cl2/DMF (5/2, v/v); (v) 4 equiv
of PalOH, 4 equiv of PyBOP, 4 equiv of DIPEA, CH2Cl2/DMF
(5/2, v/v); (vi) 2 % hydrazine monohydrate/DMF; (vii) 4 equiv of
10,12-pentacosadiynoic acid, 4 equiv of PyBOP, 4 equiv of DIPEA,
CH2Cl2/DMF (5/2, v/v); (viii) 1% TFA/CH2Cl2; (ix) 8, 3 equiv of
EDC, 3 equiv of HOBt, 3 equiv of DIPEA, CH2Cl2/DMF (5/2, v/v).
aminopropyl moiety of saccharide 8 to give the target
compound 1.
alanine, G ) glycine) linked to a copolystyrene solid
support, containing a hyperacid labile 4-(hydroxymethyl)-
3-(methoxyphenyloxy)butyric acid 4-methylbenzhydroxyl-
amine (HMPB-MBHA) linker, the quasi-orthogonally
protected lysine derivative FmocLysDdeOH 7 (Dde: 1-(4,4-
dimethyl-2,6-dioxycyclohex-1-ylidene)ethyl, Fmoc: fluo-
renylmethoxycarbonyl) and the aminopropyl derivatized
galactoside 8 (Chart 2), would be suitable building blocks
for a combined solid- and solution-phase synthesis of 1
(Scheme 1).
The following strategy was devised: in the last cycle
of peptide synthesis, the commercially available FmocLys-
DdeOH derivative 7 will be coupled with polymer-bound
tetrapeptide ALFG 6. The Fmoc and Dde group of the
lysine moiety are quasi-orthogonal which enables the
selective cleavage of the NR-Fmoc group (piperidine/DMF)
in the presence of the Nꢀ-Dde group.5 On the other hand,
the Dde group can be removed under mild conditions
using hydrazine in DMF. Thus, a key aspect of the
synthetic approach is that the two amino functionalities
of the lysine moiety of the pentapeptide can be selectively
derivatized with different lipids. The HMPB-MBHA
linker allows cleavage of the lipopeptide from the solid
support using mild acidic conditions (1% TFA in CH2-
Cl2). Finally, in solution, the revealed carboxylic acid of
the released lipopeptide will be condensed with the
The polymer-linked tetrapeptide ALFG (6) was pre-
pared on a co-polystyrene solid support containing a
HMPB-MBHA linker (9, Scheme 1). Attachment of the
first amino acid was achieved by condensation of the
symmetrical anhydride of Fmoc-protected glycine, with
co-polystyrene resin 9 in the presence of the acylating
reagent DMAP to give glycine-derivatized polymer 10.
The symmetrical anhydride was prepared by dehydration
of Fmoc-glycine in the presence of diisopropylcarbodi-
imide (DIPC). The Fmoc protecting group of 10 was
removed under standard conditions (20% piperidine in
DMF) to give 11. The amino acids alanine, leucine, and
phenylalanine were subsequently introduced to give 6 by
employing Fmoc-protected amino acids (FmocAA), N-
hydroxybenzotriazole (HOBt)/DIPC as chain elongating
reagents, and 20% piperidine in DMF to cleave Fmoc
protecting groups.6 In the last step of the peptide as-
sembly, the commercially available quasi-orthogonally
protected lysine derivative FmocLysDdeOH 7 was coupled
with the amino functionality of 6 by applying (benzo-
triazol-1-yloxy)tris(pyrrolidino)phosphonium hexafluoro-
phosphate (PyBOP)/diisopropylethylamine (DIPEA) as
condensation reagent to give peptide 12. The condensa-
tion reagent PyBOP/DIPEA ensured efficient coupling
using low equivalents of the expensive amino acid 7. The
(5) Bycroft, B. W.; Chan, W. C.; Ram Chhabra, S.; Hone, N. D. J .
Chem., Soc., Chem. Commun. 1993, 778.
(6) Atherton, E.; Sheppard, R. C. Solid-phase peptide synthesis, a
practical approach; IRL Press: Oxford, 1989.

Glycopyranosyl-Containing Amphiphiles
J . Org. Chem., Vol. 65, No. 11, 2000 3359
Sch em e 2a
Fmoc protecting group of 12 was selectively cleaved with
20% piperidine in DMF, and palmitoylation of the
NR-amino functionality of the resulting compound 13 was
achieved with palmitic acid in the presence of PyBOP/
HOBT to give the lipopeptide 14. The Dde group of 14
was removed by treatment with hydrazine in DMF to
give 15 and the commercially available 10,12-penta-
cosadiynoic acid (PDA-OH) was coupled with the
Nꢀ-amino group to afford polymer-bound lipopeptide 16.
Treatment of 16 with 1% TFA/CH2Cl2 cleaved the lipo-
petide from the solid support and gave, after purification
by Sephadex LH-20 size exclusion column chromatogra-
phy, compound 2 in an overall yield of 67% (based on
initial amino acid loading). Finally, the target compound
1 was obtained by 1-(3-(dimethylamino)propyl)-3-ethyl-
carbodiimide (EDC)/HOBT-mediated coupling of 2 with
the aminopropyl spacer of galactoside 8. Purification of
the crude material was accomplished by gel filtration on
Sephadex LH-20, and the fully deprotected conjugate was
isolated in a yield of 53%. 1D- and 2D-NMR spectroscopy
and mass spectrometry confirmed the molecular struc-
ture of 1. FAB mass analysis showed a peak at m/z 1370.8
corresponding to [M + Na]+, and signals at v ) 2375 and
2325 [cm-1] in the IR spectrum confirmed the presence
of the diacetylenic unit.
The preparation of compounds 3-5 is detailed in
Scheme 2. Compound 3 was prepared by a solution-based
method. Thus, the O-acetylated aminopropyl galactoside
17 was coupled with FmocLysDdeOH (7) to give 18. The
NR-Pal and Nꢀ-PDA were installed by a five-step proce-
dure. Thus, treatment of 18 with piperidine in DMF
cleaved the Fmoc group and gave 19 in almost a
quantitative yield. The revealed NR-amino group of 19
was palmitoylated with palmitic acid in the presence of
DIPC to afford 20. Treatment of 20 with hydrazine in
DMF removed the Dde group and gave 21 in a yield of
92%. The O-acetyl groups of the sugar moiety of 21 were
cleaved by reaction with Et3N in methanol to give 22.
Finally, the target compound 3 was obtained by coupling
the Nꢀ-amino group of 22 with 10,12-pentacosadiynoic
acid (PDA-OH). The latter reaction exploits the higher
reactivity of the amino group compared to the hydroxyls
of the sugar moiety.
Compound 4 was easily available by coupling glycine-
derivatized polymer 11 (Scheme 1) with FmocLysDdeOH
7 to give 23 which was converted into a NR-Pal and
Nꢀ-PDA functionalized derivative 27 according to a
similar procedure used for the preparation of 1. Thus,
the Fmoc protecting group of 23 was cleaved under
standard conditions, and the resulting NR-amino func-
tionality of 24 was palmitoylated to give 25. The Dde
group of 25 was removed by treatment with hydrazine,
and the Nꢀ-amino group of 26 was derivatized with a
10,12-pentacosadiynoyl moiety to afford 27. Target li-
popeptide 4 was obtained by treatment of 27 with 1%
TFA in CH2Cl2 followed by purification of the crude
product by LH-20 size-exclusion column chromatography.
Finally, compound 5 was obtained according to a
similar method used for the preparation of 1; however,
instead of lysine derivative 7, FmocLysFmocOH 28 was
used in the last cycle of peptide assembly to give
compound 29. The two Fmoc groups of 29 were cleaved
under standard conditions, and palmitoylation of the NR
and Nꢀ moieties of 30 with palmitoyl chloride and
pyridine gave lipopeptide 31. Polymer-bound 31 was
cleaved from the solid support, and the revealed carbox-
a
(i) 1.5 equiv of DIPC, 1.5 equiv of HONB, CH2Cl2/DMF (2/1,
v/v); (ii) 20% piperidine/DMF; (iii) 2 equiv of palmitic acid, 2 equiv
of HONB, 2 equiv of DIPC, CH2Cl2/DMF (1/1, v/v); (iv) 2%
hydrazine monohydrate/DMF; (v) Et3N/MeOH/H2O (2/5/5, v/v/v);
(vi) 1.5 equiv of 10,12-pentacosaiynoic acid, 2 equiv of EDC, 2 equiv
of HOBt, CH2Cl2/DMF (1/1, v/v); (vii) 2 equiv of PyBOP, 2 equiv
of DIPEA, CH2Cl2/DMF (5/2, v/v); (viii) 5 equiv of PalOH, 5 equiv
of PyBOP, 5 equiv of DIPEA, CH2Cl2/DMF (5/2, v/v); (ix) 5 equiv
of 10,12-pentacosadiynoic acid, 5 equiv of PyBOP; 5 equiv of
DIPEA, CH2Cl2/DMF (5/2, v/v); (x) 1% TFA/ CH2Cl2; (xi) 5 equiv
of FmocLysFmocOH, 5 equiv of PyBOP, 5 equiv of DIPEA, CH2Cl2/
DMF (5/2, v.v); (xii) 20 equiv of PalCl, pyridine/CH2Cl2 (1/1, v/v);
(xiii) 11 equiv of 8, 11 equiv of DIPC, 11 equiv ofHONB, CH2Cl2/
DMF (1/1, v/v)
ylic acid of 32 was coupled with the aminopropyl galac-
toside 8 to give target compound 5.
Aggr ega tion P r op er ties. The aggregation properties
of 1 were studied by transmission electron microscopy
(TEM). A water suspension of 1, obtained by a modified
ethanol injection method and prepared for TEM by drying
and Pt shadowing, showed the formation of uniformly
shaped extended structures (Figure 1a). The structures
appear to be cylindrical, and on the basis of the observed
diameters (15-20 nm), it is likely that they consist of
cylindrical micelles, a few monolayers, or a single bilayer
of 1. On the basis of Figure 1a, it may be considered that
tubules, i.e., aggregates with an aqueous inner compart-

3360 J . Org. Chem., Vol. 65, No. 11, 2000
Reichel et al.
tubules, but they have been reported as intermediates
in tubule formation.7 Alternatively, the morphology can
be interpreted as tubules with flat sections. In the freeze-
fracturing experiment (Figure 1c), however, 1 appeared
as extended structures that are less cylindrical than those
in Figure 1a. In view of the fact that our XRD studies
(see below) show that 1 preferentially packs as stacks of
monolayers, not bilayers, the morphology of 1 in the
negative staining (Figure 1b) and freeze fracture (Figure
1c) experiments is best described as ribbons of stacked
monolayers, whereas the morphology in the Pt shadowing
experiments is best accounted for by a few concentric
monolayers, possibly around a micellar fiber and there-
fore are better described as ribbons or rods. Saccharide
amphiphiles generally assemble into spheroidal su-
pramolecular assemblies such as micelles and vesicles8
and only a very few reports3,9 deal with other morphol-
ogies such as tubules.
Tubular or rodlike structures are starting points for
the development of new materials.10 They are considered
to be more ordered than vesicles and several theories of
tube formation have been proposed.11 Also, it has been
shown that in some cases extended aggregates are more
immunogenic than spherical ones.3 Some molecular
structural features such as chirality and diacetylenic
lipids are known to promote tubular or rodlike aggrega-
tion, but the limited number of compounds known to
organize into these structures makes it difficult to predict
this mode of assembly. There is an earlier report12 of a
glycopeptidolipid, based on lysine with a hydrocarbon and
fluorocarbon tail and a lactobiose headgroup, which was
found to display a rich aggregation chemistry with
stacked disklike assemblies, liposomes, helical twisted
tapes, and tubuli in a single micrograph although no
explanation for the variation in morphology was given.
A successful polymerization, as judged from a color
change from transparent to purple, of a water suspension
of compound 1 exposed to UV light (254 nm) corroborated
the highly ordered nature of the supramolecular as-
sembly. It is well-known13 that topotactic photopolymer-
ization of diacetylenes is only possible when they are part
of highly ordered structures, and the diacetylenic moi-
eties are appropriately positioned relative toward each
other. TEM of the precipitated and polymerized material
showed fibers at the edge of a polymer drop (Figure 1d)
which are much longer than the tubes/rods observed in
the unpolymerized water suspension. Therefore, poly-
merization occurs along the axis of the tubes/rods as
depicted in Scheme 3. Probably hydrogen bonding be-
tween the amide moieties along the axis of the tubular
structure helps to fulfill the structural requirements for
polymerization. It is noteworthy that for some reported
examples, a tubular aggregation disappeared upon
polymerization.9a
F igu r e 1. Electron micrographs of compounds 1-5. Bar
represents 500 nm. For concentrations see Table 3. (A) Pt-
shadowing of 1; (B) negative staining of 1; (C) freeze fracturing
of 1; (D) Pt-shadowing of polymerized 1; (E) Pt-shadowing of
freshly prepared aggregate of 3; (F) Pt-shadowing of aged (3
days) aggregate of 3; (G and H) Pt-shadowing of 4.
ment, are formed. This would be a reasonable expectation
on the basis of the known aggregation chemistry of
diacetylenic phospholipids.10 To define in more detail the
morphology of 1, we applied other preparation tech-
niques. With negative staining (Figure 1b), 1 appeared
as twisted ribbons. Such structures are distinct from
(9) (a) Fuhrhop, J .-H.; Helfrich, W. Chem. Rev. 1993, 93, 1565 and
references cited. (b) Sommerdijk, N. A. J . M.; Feiters, M. C.; Nolte, R.
J . M.; Zwanenburg, B. Recl. Trav. Chim. Pays-Bas 1994, 113, 194. (c)
Giulieri, F.; Guillod, F.; Greiner, J .; Krafft, M. P.; Riess, J . G. Chem.
Eur. J . 1996, 2, 1335. (d) Shimizu, T.; Masuda, M. J . Am. Chem. Soc.
1997, 119, 2812.
(10) Schnur, J . M.; Shashidar, R. Adv. Mater. 1994, 6, 971.
(11) (a) Helfrich, W. J . Chem. Phys. 1986, 85, 1085. (b) Helfrich,
W.; Prost, J . Phys. Rev. E. 1988, 38, 3065. (c) Selinger, J . V.;
MacKintosh, F. C.; Schnur, J . M. Phys. Rev. E 1996, 53, 3804.
(12) Zarif, L.; Gulik-Krzwycki, T.; Riess, J . G.; Pucci, B.; Guedj, C.;
Pavia, A. A. Colloids Surf. A 1993, 84, 107.
(7) Nakashima, N.; Asakuma, S.; Kunitake, T. J . Am. Chem. Soc.
1985, 107, 59.
(8) For a recent example of saccharide micelles and vesicles, see:
Hafkamp, R. J . H.; Feiters, M. C.; Nolte, R. J . M. Angew. Chem., Int.
Ed. 1994, 33, 986. For a recent review on amphiphilic carbohydrates,
see: (a) Boullanger, P. Topics Curr. Chem. 1997, 187, 275. (b) Feiters,
M. C.; Nolte, R. J . M. In Advances in Supramolecular Chemistry; Gokel,
G., Ed.; J AI Press Inc./Ablex Publishing Corp./Elsevier Science:
Stanford, 2000; Vol. VI, pp 41-156.
(13) (a) Wegner, G. Makromol. Chem. 1972, 154, 35. (b) Kuo, T.;
O’Brien, D. F. Macromolecules 1990, 23, 3225.

Glycopyranosyl-Containing Amphiphiles
J . Org. Chem., Vol. 65, No. 11, 2000 3361
Sch em e 3. Top ota ctic P olym er iza tion of Tu bu la r
Dia cetylen e Aggr ega tes
F igu r e 2. X-ray diffractograms of dried suspensions of
nonpolymerized 1 at different relative humidities. Solid line,
0%; dashed line, 50%; dotted line, 90% relative humidity.
samples were polymerized by illumination during drying
after dispersion in the dark, as well as illuminated while
being dispersed. In both cases, polymerization had oc-
curred as judged from the purple color. This is remark-
able in view of the topotactic requirements for polymer-
ization.13 Effects of dehydration of aggregates of di-
acetylenic amphiphiles have been reported before, for
example, a decrease of spacing from 65.5 to 67.5 Å has
been reported for a diacetylenic phopholipid.17 Effects of
polymerization have also be noted.18 All X-ray diffraction
results for 1 are in agreement with a lamellar packing
with spacing in the range 82.5-94.9 Å. In view of the
length of the molecule as estimated from molecular
models (approximately 80 Å) this points to a packing in
monolayers with varying degrees of hydration. The
slightly higher value of periodicity at 0% relative humid-
ity of the nonpolymerized material compared to poly-
merized material can be explained by a tilt in the
molecule upon polymerization (see Scheme 3). The hy-
dration patterns of the nonpolymerized sample and the
sample that was polymerized on the wafer are rather
similar, with extensions of the periodicity of 7.0 and 5.7
Å, respectively, going from 0 to 90% relative humidity,
corresponding to incorporation of five or four water
molecules per monolayer. The sample that was poly-
merized in dispersion expands significantly more upon
hydration (12.4 Å), suggesting a more open structure in
the packed monolayers. In all cases, the region of the
diffractogram where reflections due to the periodicities
of alkyl tail packing are expected (approximately 4.2 Å,
2θ range 20-25°) were also studied, but no strong
reflections were observed.
Ta ble 1. P er iod icities Der ived fr om X-r a y Diffr a ction of
La m ella r Str u ctu r es of Non p olym er ized a n d P olym er ized
1
rel humidity, nonpolymerized, polymerized in polymerized on
%
Å
suspension, Å
Si-wafer, Å
0
50
90
84.0
86.5
91.0
82.5
91.0
94.9
82.5
84.8
88.2
The packing of the amphiphile 1 was also investigated
by X-ray powder diffraction (XRD). For the study of the
self-assembly of molecules of the size described here, it
is necessary to use angles smaller than what is typically
possible on commercial diffractometers (2θ value ap-
proximately 3°, corresponding to d ) 29 Å at the
wavelength of Cu KR, 1.54 Å), in particular if detection
of the first-order reflection of head-to-tail or head-to-head
packing as well as its unambiguous assignment are
required. A possible disadvantage of reflection geometry
for the study of dispersions is the necessity of drying the
sample, with possible introduction of artifacts. In the
study presented here, this problem is counteracted in
part by the possibility of controlling the relative humidity
during the measurements, as well as drying the sample
in an environment of controlled relative humidity rather
than in a desiccator.14 In earlier studies using this
approach, the reversible binding of one layer of water
molecules between gemini bisphosphate monolayers was
demonstrated.15,16 Compound 1 was investigated under
a variety of conditions (Table 1 and Figure 2). To avoid
polymerization, the dispersion of the amphiphile was
kept in the dark during preparation and drying. Also,
To investigate the importance of particular molecular
features for tube or rod formation of 1, the self-assembly
properties of compounds 2-5 were studied. As expected,
the omission of the carbohydrate and peptide parts as in
compound 2 and 3, respectively, or of both as in com-
pound 4 and variation of the nature of the lipid tail as in
compound 5 were found to affect the morphology of the
supramolecular structures. The water suspension of
lipopeptide 2 exhibited mainly the formation of vesicular
structures with a typical diameter of 38 nm whereas only
a few extended structures were present (not shown).
(14) Kuehnel, R. A.; van der Gaast, S. J . Advances in X-ray Analysis;
Gilfrich, J ., Ed.; Plenum Press: New York, 1993; Vol. 36, p 439.
(15) Duivenvoorde, F. L.; Feiters, M. C.; van der Gaast, S. J .;
Engberts, J . B. F. N. Langmuir 1997, 13, 3737
(16) Klug, H. P.; Alexander, L. E. X-ray diffraction Analysis; 2nd
ed.; Wiley-Interscience: New York, 1974; p 687.
(17) Thomas, B.; Safynia, C. R.; Plano, R. J .; Clark, N. A.; Ratna,
B. R.; Shasidar, R. Mater. Res. Soc. Symp. Proc. 1992, 248, 83.
(18) Rhodes, D. G.; Xu, Z. Biochim. Biophys. Acta 1992, 1128, 93.

3362 J . Org. Chem., Vol. 65, No. 11, 2000
Reichel et al.
Ta ble 2. Su p r a str u ctu r es Obser ved by TEM
Å were observed up to n ) 1, consistent with a lamellar
packing with the glycopeptidolipid (extended length 60
Å) in intercalated bilayers with a tilt of 30°. Analysis of
the diffraction peak widths using the Scherrer formula16
gives a value of 560 Å for the diameter of the independent
domains at 0% RH, which increases to 760 Å at 90%
relative humidity. An alternative and more likely inter-
pretation of the broadening of the diffraction peaks at
low relative humidity is an increase of stress in packing
upon dehydration.
suprastructures
suprastructures
tubular structures
vesicles, few tubular structures
intertwined rods
rods, boomerang shaped structures,
tubular structures
after aging*
1
2
3
4
no changes
no changes
helical ribbons
no changes
5
nonuniformly shaped elongated structures helical ribbons
*Ageing for 3 and 5 days.
Thus, the sugar moiety of 1 seems to be important for
tube formation. On the other hand, glycolipid 3 missing
the tetrapeptide unit formed bundles of tubes or rods,
which are intertwined and reached a length of up to 20
µm and a width of around 270 nm (Figure 1e). Upon
aging for 3 days, very long ribbons were observed (Figure
1f), and the direction of their winding appeared to be left-
handed. It has been suggested that ribbons are precur-
sors and/or side products of tube formation.7,19 Surpris-
ingly, lipopeptide 4 lacking the carbohydrate and pos-
sessing only a short peptide moiety exhibited the forma-
tion of long clustered tubes/rods of lengths of up to a few
micrometers and a width of about 150 nm (Figure 1g).
Furthermore, smaller tubular and “boomerang” shaped
structures were observed (Figure 1h). Recently, it was
shown20 that particular histidine surfactants assemble
into boomerang shaped structures from bilayers upon
addition of copper ions, and it was suggested that they
are intermediates for the formation of helical structures.
The dipalmitoylated glycopeptidolipid 5 allowed us to
assess the importance of the diacetylenic unit. Compound
5 showed formation of extended structures which were
not uniformly shaped and had a tendency to cluster. In
an aged sample (3 days), ribbon type structures were seen
(not shown).
Water suspensions of the compounds 2-4 could be
polymerized by UV light (254 nm), but TEM showed
amorphous and not very dense structures (not shown).
Comparison of the different supramolecular behaviors of
compounds 1-5 gives some information about the im-
portance of the different moieties for self-assembly
behavior (Table 2).
X-ray diffraction patterns of compound 2 showed a
pattern of reflection that decreased in intensity when the
relative humidity was increased but showed no shift in
peak position and was best accounted for by assuming a
hexagonal packing with an interccolumnar distance D
of 98 Å. As the length of the extended molecule is 75 Å,
such packing would have to involve intercalation of the
alkyl chains, which is likely in view of the expected large
diameter of the peptide part. The hexagonal packing is
difficult to reconcile, however, with the observation of
vesicles in the microscope; apparently, slight variations
in the substrates (silicon wafer for the X-ray diffraction,
carbon-coated Formvar for the electron microscopy) has
a large effect on the result.
Con clu sion s
An efficient synthesis of the novel glycopyranosyl-
containing amphiphiles 1 is described which is based on
a combination of solid-phase and solution-based methods.
The use of the orthogonally protected FmocLysDde
derivative provided an opportunity to incorporate two
different lipids in the amphiphiles. The synthetic meth-
odology allowed the efficient synthesis of the derivatives
2-5.
It was found that compound 1 forms tubular or rodlike
structures in aqueous media, which can be stabilized by
topotactic photopolymerization. We conclude that none
of the structure elements we have omitted in the deriva-
tives 2-5 can lay claim to be exclusively important for
the formation of highly organized aggregates such as
tubes, fibers, or helical ribbons from 1, but the presence
of all of these structural elements afforded the most
uniformly shaped structures. The absence of the galactose
in 2 appears to have a dramatic influence, as mainly
vesicles are formed, which are considered to be a lower
form of organization than tubules. A possible explanation
for the effect of the galactose residue is that the tetrapep-
tide actually introduces a mismatch in the intermolecular
hydrogen bonding network, leading to vesicle formation
for 2, which is counteracted by the presence of the
galactose, leading to tubules for 1. The X-ray diffraction
studies of some of the glycopeptide surfactants described
here have shown that lamellar monolayer, lamellar
intercalated bilayer, and hexagonal packing can be found
depending on the functionalization.
As part of a future project, a derivative of 1 will be
prepared which contains an antigenic saccharide and
peptide moiety. This compound will be used for the
formation of coaggregates with the glycopeptidolipid 1.
Polymerization of a tubular or rodlike aggregate may
provide a highly immunogenic material. Results will be
compared with the immunological properties of an ag-
gregate, which is composed of one derivative only.
The methodology discussed in the paper will also
provide a powerful approach to reveal structural motives
which may promote tube/rod formation since a range of
compounds can easily be synthesized which differ in
saccharide, peptide, and lipid composition.
Exp er im en ta l Section
The XRD pattern of compound 3 also showed a slight
decrease in relative intensity with increasing relative
humidity but no change in peak positions. At 0% relative
humidity, reflections of a lamellar packing with d ) 76.3
Syn th esis. Gen er a l Meth od s. Gravity column chroma-
tography was performed on silica gel 60 (Merck, 70-230 mesh)
and flash column chromatography on silica gel 60 (Merck,
230-240 mesh). Gel filtration was performed on Sephadex LH
20 (Pharmacia) or on Sephadex G15 (Pharmacia). TLC analy-
sis was conducted on Kieselgel 60 F254 (Merck) plates, and
compounds were detected by UV light (254 nm), by charring
with concentrated sulfuric acid/methanol (1/20, v/v), molybdate
reagent (0.02 M solution of ammonium cerium(IV) sulfate
(19) (a) Frankel, D. A.; O’Brien, D. F. J . Am. Chem. Soc. 1991, 113,
7436. (b) Frankel, D. A.; O’Brien, D. F. J . Am. Chem. Soc. 1994, 116,
10057.
(20) Sommerdijk, N. A. J . M.;Lambermon, M. H. L.; Feiters, M. C.;
Nolte, R. J . M.; Zwanenburg, B. Chem. Commun. 1997, 455.

Glycopyranosyl-Containing Amphiphiles
J . Org. Chem., Vol. 65, No. 11, 2000 3363
dihydrate, and ammonium molybdate(VI) tetrahydrate in
aqueous 10% sulfuric acid) or ninhydrin reagent (3 mL acetic
acid, 0.3 g of ninhydrin in 100 mL of n-butanol). Dichlo-
romethane and acetonitrile were purchased from Fisher
Scientific, distilled from calcium hydride, and stored over 4 Å
molecular sieves under argon. N,N-Dimethylformamide was
purchased from Fisher Scientific, distilled from calcium hy-
dride under reduced pressure, and stored over 4 Å molecular
sieves under argon. Petroleum ether (60-80 °C) and ethyl
acetate were purchased from Fisher Scientific as well as
dimethyl sulfoxide (GPC grade) for HPLC. Amino acids,
PyBOP, and Fmoc-Gly-HMPB-MBHA resin were purchased
from NovaBiochem, piperidine, diisopropylcarbodiimide (DIPC),
and DMSO from Fluka, and all other chemicals from Aldrich.
Reactions were conducted under anhydrous conditions, under
inert gas atmosphere (N2), and at room temperature if not
mentioned otherwise. LSI and HR mass spectra were recorded
using a VG Zabspec mass spectrometer. All empirical formulas
are within the capability of the HRMS operating at a resolution
of 10.000.
(S )-Ala n yl-(S )-le u cin yl-(S )-p h e n yla la n yl-glyce n yl-
HMP B-MBHA (6) Atta ch m en t of th e F ir st Am in o Acid .
Fmoc-Gly-OH (832 mg, 2.8 mmol) was suspended in CH2Cl2
(13 mL), and DMF was added until a clear solution was
obtained. The solution was cooled (0 °C) and DIPC (0.22 mL,
1.43 mmol) added. Subsequently, the reaction mixture was
stirred at 0 °C for 20 min. The precipitated symmetrical
anhydride was redissolved by addition of DMF and stirring
continued for 10 min at 0 °C. The mixture was concentrated
under reduced pressure and the residue dissolved in a minimal
amount of DMF and subsequently added to preswollen (30
min) HMPB-MBHA resin (500 mg) in DMF. After adding
DMAP (35 mg, 0.285 mmol), the mixture was agitated for 1 h.
About 20 mg of resin was removed to measure the extent of
the first amino acid attachment. It was washed with DMF (5
mL) and CH2Cl2 (5 mL) and dried in vacuo.
the resin washed with CH2Cl2/DMF (5/2, v/v, 3 × 28 mL, 10
min). The resin 12 was treated with piperidine/DMF (2/8, v/v,
5 mL) for 30 min. A positive Kaiser test indicated removal of
the Fmoc-protecting group and formation of 13. The solvents
were removed by filtration, the resin was washed with DMF
(4 × 20 mL, 10 min) and, subsequently, suspended in CH2-
Cl2/DMF (5/2, v/v, 28 mL). Palmitic acid (97 mg, 0.38 mmol),
PyBOP (198 mg, 0.38 mmol), and DIPEA (49 mg, 0.38 mmol)
were added, and the mixture was agitated for 5 h. A negative
Kaiser test indicated the completion of the coupling reaction
and formation of 14. The solvent were removed by filtration,
and the resin was washed with CH2Cl2/DMF (5/2, v/v, 3 × 28
mL, 10 min) and treated with hydrazine monohydrate/DMF
(2/98, v/v, 5 mL) for 30 min. A positive Kaiser test indicated
cleavage of the Dde-protecting group and formation of 15. After
filtration, the resin was washed with DMF (4 × 10 mL, 10
min) and suspended in CH2Cl2/DMF (5/2, v/v, 28 mL). 10,12-
Pentacosadiynoic acid (142 mg, 0.38 mmol), PyBOP (198 mg,
0.38 mmol), and DIPEA (49 mg, 0.38 mmol) were added, and
the mixture was agitated in the dark for 6 h. A negative Kaiser
test indicated the complete formation of 16. After filtration
and washing with CH2Cl2/DMF (5/2, v/v, 3 × 28 mL, 10 min),
polymer-bound lipopeptide 16 was released from the solid
support following the general procedure for cleavage of the
HMPB-MBHA linker. The compound containing fractions were
combined and concentrated in vacuo, and the residue was
purified by gel filtration (Sephadex LH-20, MeOH/CH2Cl2, 1/1,
v/v) to afford 2 (58 mg, 67%) as a colorless sticky foam. [R]21.5
D
) -9.7° (c ) 2.2 mg/mL, CH2Cl2/MeOH, 2/1, v/v). IR (KBr): ν
2924, 2854 (CH2), 2375, 2325 (C/C-triple bond), 1636, 1548,
1461 (C(O)NH). UV (CH2Cl2/MeOH, 2/1, v/v): λmax 235.6 (ꢀ
1
4674). H NMR (CDCl3/CD3OD, 2/1, v/v, 500 MHz): δ 7.22-
7.08 (m, 5H), 4.60 (brs, 1H), 4.14 (brs, 2H), 3.99 (brs, 1H), 3.97
(s, 2H), 3.29-3.16 (m, 2H), 3.07-3.00 (m, 1H), 2.93-2.83 (m,
1H), 2.23 (t, 2H, J ) 7.7 Hz), 2.18 (t, 4H, J ) 7.0 Hz), 2.13 (t,
2H, J ) 7.7 Hz), 1.72-1.61 (m, 2H), 1.61-1.50 (m, 6H), 1.50-
1.35 (m, 9H), 1.35-1.15 (m, 55H), 0.84-0.79 (m, 9H), 0.74 (d,
3H, J ) 6.2 Hz). 13C NMR (CDCl3/CD3OD, 2/1, v/v, 125 MHz)
δ 175.7, 175.1, 174.6, 174.4, 174.1, 173.2, 172.2 (CO), 137.3,
129.3, 128.4, 126.7, 120.0 (Phe-aromat), 65.3, 65.2 (C/C-triple),
55.3 (Leu-CHR), 54.8 (Phe-CHR), 53.2, 52.1, 50.3 (Gly-CH2, Lys-
CHR, Ala-CHR), 46.2, 36.5, 36.4, 35.9 (alkyl-CH2, Lys-NCH2),
31.9, 29.7, 29.5, 29.3, 29.2, 29.1, 29.0, 28.9, 28.8, 28.7, 28.4,
25.9, 25.7, 25.6, 24.8 (Alkyl-CH2, Leu-CH, Lys-CH2, Leu-CH2),
22.7 (Leu-CH3), 21.3 (Leu-CH3), 19.2 (alkyl-CH3), 14.0 (alkyl-
CH3), 8.5 (Ala-CH3). MALDI TOF MS: m/z ) 1152 ([M + Na]+,
retinoic acid), 1151 ([M + Na]+, indolacrylic acid). LSIMS: m/z
) 1151 (M + Na)+. HRMS (LSIMS): calcd for C67H112N6NaO8
(M + Na)+ 1151.8439, found 1151.8444.
Deter m in a tion of Am in o Acid Atta ch m en t. Dry resin
(17.5 mg) was treated with piperidine/DMF (1/4, v/v, 30 mL)
for 10 min after which an aliquot of 3 mL was removed and
the UV absorbance measured at 290 nm against piperidine/
DMF (1/4, v/v, 3 mL). Comparison with a correlation table
showed an attachment of >95%.
Gen er a l P r oced u r e for P ep tid e Ch a in Elon ga tion .
Solid-phase synthesis was performed manually, using a cy-
lindrical reaction vessel equipped with a sintered filter at the
bottom to enable the removal of solvents and the agitation of
the resin by a nitrogen flow.
Sta n d a r d P r oced u r e. (1) Washing the resin with CH2Cl2
(39 mL) for 10 min. (2) Cleavage of the Fmoc-group by
treatment with piperidine/DMF (1/4, v/v, 10 mL) for 30 min.
(3) Kaiser test.2121 (4) Elongation of the peptide chain by
addition of CH2Cl2/DMF (5/2, v/v, 28 mL), Fmoc amino acid (5
equiv), HOBt (5 equiv), DIPC (5 equiv). Agitation of the
mixture for 3-16 h. (5) Kaiser test.21 (6) Washing with CH2-
Cl2/DMF (5/2, v/v, 28 mL) for 3 × 10 min.
Nr-P a lm it oyl-NE-10,12-p en t a cosa d iyn oyl-(S)-lysin yl-
(S)-a la n yl-(S)-leu cin yl-(S)-p h en yla la n yl-glycin e[3-(â-D-
ga la ctop yr a n osyloxy)p r op yl]a m id e (1). Lipopeptide 2 (56
mg, 0.049 mmol) was dissolved in CH2Cl2/DMF (3/1, v/v, 4 mL),
and HOBt (40 mg, 0.298 mmol) in CH2Cl2/DMF (1/1, v/v, 1
mL) and DIPC (38 mg, 0.298 mmol) in CH2Cl2 (0.5 mL) added.
Galactoside 8 (59 mg, 0.248 mmol) in DMF (2.5 mL) was added
dropwise to the stirred solution, in a manner that all com-
pounds remained in solution. After the reaction mixture was
stirred for 5 h in the dark, it was concentrated in vacuo, and
the residue purified by gel filtration (Sephadex LH-20, MeOH/
CH2Cl2, 1/1, v/v) to afford 1 (35 mg, 53%) as a colorless gum.
[R]22D: -2.1° (c ) 3.2 mg/mL, CH2Cl2). IR (KBr): ν 2923, 2851
(CH2), 2471, 2426 (C/C-triple bond), 1746, 1632, 1548, 1467
(C(O)NH). UV (CH2Cl2/MeOH, 1/1, v/v): λmax 269.4 nm (ꢀ 986).
1H NMR (CDCl3/CD3OD, 2/1, v/v, 400 MHz): δ 7.05-6.74 (m,
5H), 4.31 (dd, 1H, J ) 10.1 Hz, J ) 4.8 Hz), 3.99 (d, 1H, J )
7.0 Hz), 3.97-3.88 (m, 2H), 3.77 (t, 1H, J ) 7.1 Hz), 3.74-
3.49 (m, 6H), 3.45-3.23 (m, 4H), 3.45-3.23 (m, 4H), 2.97-
2.82 (m, 1H), 2.72 (dd, 1H, J ) 14.0 Hz, J ) 10.2 Hz), 2.13-
1.90 (m, 8H), 1.62-0.98 (m, 72H), 0.88 (d, 3H, J ) 6.5 Hz),
0.70-0.61 (m, 6H), 0.59 (d, 3H, J ) 6.5 Hz). 13C NMR (CDCl3/
CD3OD, 2/1, v/v, 100 MHz): δ 175.5, 174.2, 174.2, 173.6, 172.2,
169.7 (CO), 136.8, 128.8, 128.0, 126.4, 126.3, 116.7 (Phe-
aromat), 103.0 (C-1), 74.6 (C-3), 73.3 (C-5), 71.1 (C-2), 68.8 (C-
4), 66.7 (C-6), 65.0, 64.8 (C/C-triple), 61.2 (Sp-OCH2), 55.2, 55.0
Gen er a l P r oced u r e for Clea va ge fr om th e HMP B-
MBHA Resin . The resin was treated with TFA/CH2Cl2 (1/99,
v/v). After 1 min, the solvent was removed into a flask
containing pyridine/MeOH (1/9, v/v, double the volume of the
TFA solution). This procedure was repeated several times, and
the product containing fractions (TLC analysis) were combined
and concentrated in vacuo.
Nr-P a lm it oyl-NE-10,12-p en t a cosa d iyn oyl-(S)-lysin yl-
(S)-a la n yl-(S)-leu cin yl-(S)-p h en yla la n yl-glycin e (2). Resin
6 (152 mg, 0.076 mmol of attached tetrapeptide) was sus-
pended in CH2Cl2/DMF (5/2, v/v, 28 mL), and NR-Fmoc-Nꢀ-Dde-
(S)-Lys (7) (121 mg, 0.228 mmol), PyBOP (119 mg, 0.228
mmol), and DIPEA (30 mg, 0.228 mmol) were added. The
reaction mixture was agitated for 4 h after which a negative
Kaiser test indicated completion of the coupling reaction and
formation of 12. The solvents were removed by filtration and
(21) Kaiser, E.; Colescott, R. L.; Bessinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 595.

3364 J . Org. Chem., Vol. 65, No. 11, 2000
Reichel et al.
(Phe-CHR, Leu-CHR), 52.9 (Ala-CHR), 50.4 (Lys-CHR), 42.6 (Gly-
CH2), 39.3 (alkyl-CH2), 37.7 (Lys-NCH2), 36.6 (Phe-CH2), 36.2
(Sp-NCH2), 36.0, 35.4, 31.5, 31.2, 31.0 29.8, 29.2, 29.1, 28.9,
28.6, 28.5, 28.4, 27.9, 25.5, 25.1, 24.4, 22.6, 22.2 (alkyl-CH2,
Leu-CH, Leu-CH2, Lys-CH2, Sp-CH2), 20.7 (Leu-CH3), 18.6
(alkyl-CH2), 16.1 (Ala-CH3), 13.4 (alkyl-CH3). MALDI TOF
MS: 1373 ([M + Na]+, retinoic acid). LSIMS: m/z ) 1370.8
(M + Na)+. HRMS (LSIMS): calcd for C76H129N7O13Na (M +
Na)+ 1370.9546, found 1370.9559.
material. The mixture was concentrated in vacuo. The residue
was dissolved in CH2Cl2 (15 mL) and washed with water (3 ×
5 mL), and the combined water layers extracted with CH2Cl2
(3 mL). The combined organic layers were dried (MgSO4),
filtered, and concentrated in vacuo. The residue was purified
by flash column chromatography (5 g of silica, 1-3% MeOH/
CH2Cl2) to afford 20 (137.6 mg, 92%) as a colorless gum. Rf:
0.41 (MeOH/CH2Cl2, 5/95, v/v). [R]22 ) -8.0° (c ) 10.3 mg/
D
mL, CH2Cl2). 1H NMR (CDCl3, 300 MHz): δ 13.4 (brs, 1H),
6.52 (t, 1H, J ) 5.7 Hz), 6.41 (d, 1H, J ) 8.5 Hz), 5.37 (d, 1H,
J ) 2.9 Hz), 5.14 (dd, 1H, J ) 10.5 Hz, J ) 7.9 Hz), 5.00 (dd,
1H), 4.54-4.44 (m, 1H), 4.41 (d, 1H), 4.15 (dd, 1H, J ) 11.2
Hz, J ) 6.4 Hz), 4.08 (dd, 1H, J ) 6.8 Hz), 4.00-3.85 (m, 2H),
3.56-3.46 (m, 1H), 3.41-3.27 (m, 4H), 2.50 (s, 3H), 2.32 (s,
4H), 2.20 (t, 2H, J ) 7.5 Hz), 2.12, 2.06, 2.01, 1.96 (s, 12H),
1.92-1.52 (m, 8H), 1.50-1.39 (m, 2H), 1.21 (s, 24H), 0.99 (s,
6H), 0.84 (t, 3H, J ) 6.6 Hz). 13C NMR (CDCl3, 75 MHz): δ
173.4, 171.1, 170.1 (CO), 108.9 (C/C-double), 101.4 (C-1), 70.8,
70.6 (C-3, C-5), 69.0 (C-2), 68.9 (Sp-OCH2), 67.0 (C-4), 61.1 (C-
6), 52.5 (Lys-CHR), 43.1, 37.5 (Sp-NCH2, Lys-NCH2), 36.6 (Dde-
Cq), 28.3 (C/C-double-CH3), 25.7 (Pal-CH2), 20.7 (Ac-CH3), 17.9
(Dde-CH3), 14.1 (Pal-CH3). MALDI TOF MS: m/z ) 959 ([M
+ Na]+, retinoic acid). LSIMS: m/z ) 958.5 (M + Na)+. HRMS
(LSIMS): calcd for C49H82N3O14 (M + H)+ 936.5797, found
936.5751.
Nr-(F lu or en ylm eth oxyca r bon yl)-NE-(4,4-d im eth yl-2,6-
d ioxocycloh exylid en e)eth yl-(S)-lysin e[3-(2,3,4,6-tetr a -O-
a cetyl-â-D-ga la ctop yr a n osyloxy)p r op yl]a m id e (18). Fmoc-
LysDdeOH (7) (166 mg, 0.312 mmol) was dissolved in CH2-
Cl2/DMF (2/1, v/v, 3 mL), and HONB (84 mg, 0.468 mmol) in
CH2Cl2/DMF (1/1, v/v, 0.5 mL), DIPC (59 mg, 0.468 mmol) in
CH2Cl2 (0.5 mL), and compound 17 (189 mg, 0.468 mmol) in
CH2Cl2 (1 mL) were added. After stirring for 18 h, the reaction
mixture was diluted with CH2Cl2 (15 mL) and washed with
water (3 × 5 mL). The combined water layers were extracted
with CH2Cl2 (5 mL). The combined organic layers were dried
(MgSO4), filtered, and concentrated in vacuo. The residue was
purified by flash column chromatography (8 g of silica, 0-3%
MeOH/CH2Cl2) to afford 18 (280 mg, 97%) as a colorless gum.
Rf: 0.15 (MeOH/CH2Cl2, 3/97, v/v). [R]17.5 ) -6.25° (c ) 18.3
D
1
mg/mL, CH2Cl2). H NMR (CDCl3, 300 MHz): δ 13.48-13.36
(m, 1H), 7.74 (d, 2H, J ) 7.7 Hz), 7.65-7.50 (m, 2H), 7.38 (t,
2H, J ) 7.4 Hz), 7.29 (t, 2H), 6.50 (brs, 1H), 5.75 (d, 1H, J )
8.1 Hz), 5.37 (d, 1H, J ) 2.9 Hz), 5.18 (dd, 1H, J ) 10.3 Hz, J
) 8.1 Hz), 5.01 (dd, 1H), 4.50-3.94 (m, 8H), 3.90-3.80 (m,
1H), 3.60-3.48 (m, 1H), 3.44-3.31 (m, 4H), 2.51 (s, 3H), 2.31
(s, 4H), 2.07, 2.03, 2.02, 1.95 (s, 12H), 1.84-1.60 (m, 6H), 1.57-
1.40 (m, 2H), 0.99 (s, 6H). 13C NMR (CDCl3, 75 MHz): δ 173.5,
171.5, 170.3, 170.1, 170.0 (CO), 156.2 (Fmoc-CO), 143.8, 143.8,
141.3, 127.7, 127.1, 126.8, 125.1, 120.0 (Fmoc-aromat), 107.8
(C/C-double), 101.4 (C-1), 70.8, 70.6 (C-3, C-5), 69.2 (C-2), 68.9
(Sp-OCH2), 67.0 (Fmoc-OCH2), 66.9 (C-4), 61.2 (C-6), 54.5 (Lys-
CHR), 52.9 (brs, Dde-CH2), 47.2 (Fmoc-CH), 42.8, 37.6 (Sp-
NCH2, Lys-NCH2), 32.5 (Dde-Cq), 32.0, 29.2, 28.6 (Sp-CH2, Lys-
CH2), 28.2 (Dde-CH3), 22.8 (Lys-CH2), 20.8, 20.6, 20.5, (Ac-
CH3), 17.8 (C/C-double-CH3). LSIMS: m/z ) 942 (M + Na)+.
HRMS (LSIMS): calcd for C48H61N3NaO15 (M + Na)+ 942.4000,
found 942.4019.
Nr-P a lm it oyl-(S)-lysin e[3-(â-D-ga la ct op yr a n osyloxy)-
p r op yl]a m id e (22). Compound 20 (28 mg, 0.03 mmol) was
dissolved in hydrazine monohydrate/DMF (2/98, v/v, 2 mL) and
the reaction mixture stirred for 30 min. The reaction mixture
was concentrated in vacuo to obtain 21 which was subse-
quently treated with a mixture of NEt3/MeOH/water (2/5/5,
v/v/v, 5 mL). After stirring for 3 h, the reaction mixture was
concentrated in vacuo and the residue coevaporated with
toluene (3 × 3 mL). The residue was purified by gel filtration
(Sephadex LH-20, MeOH/CH2Cl2, 1/1, v/v) to afford 22 (11.9
mg, 67%) as a colorless gum. [R]22D: -20.4° (CH2Cl2/MeOH,
2/1, v/v, c ) 10.3 mg/mL). 1H NMR (CDCl3/MeOD, 3/1, v/v,
500 MHz): δ 4.29 (dd, 1H, J ) 7.9 Hz, J ) 6.1 Hz), 4.21 (d,
1H, J ) 7.2 Hz), 4.15-3.83 (m, 5H), 3.79-3.69 (m, 3H), 3.64-
3.57 (m, 1H), 3.53-3.29 (m, 2H), 2.93-2.80 (m, 2H), 2.17 (t,
2H, J ) 7.8 Hz), 1.81-1.17 (m, 34H), 0.82 (t, 3H, J ) 7.0 Hz).
13C NMR (CDCl3/MeOD, 3/1, v/v 125 MHz): δ 174.9, 172.4
(CO), 103.6 (C-1), 75.0, 73.7, 71.7 (C-2, C-3, C-5), 69.3 (C-4),
68.9 (Sp-OCH2), 61.7 (C-6), 53.7 (Lys-CHR), 39.7 (Lys-NCH2),
37.9 (Sp-NCH2), 36.5, 32.1, 31.9, 29.9, 29.7, 29.5, 28.8, 26.7,
25.9, 23.3, 22.8, 22.4 (Pal-CH2, Sp-CH2, Lys-CH2), 14.1 (Pal-
CH3). MALDI TOF MS: m/z ) 602 ([M + H]+, retinoic acid),
602 ([M + H]+, indolacrylic acid). LSIMS: m/z ) 626 (M +
Na)+. HRMS (LSIMS): calcd for C31H61N3O8Na (M + Na)+
626.4356, found 626.4350.
NE-(4,4-Dim et h yl-2,6-d ioxocycloh exylid en e)et h yl-(S)-
lysin e[3-(2,3,4,6-tetr a -O-a cetyl-â-D-ga la ctop yr a n osyloxy)-
p r op yl]a m id e (19). Compound 19 (100 mg, 0.11 mmol) was
dissolved in piperidine/DMF (2/8, v/v, 3 mL) and the reaction
mixture stirred for 30 min. The reaction mixture was concen-
trated in vacuo and the residue purified by flash column
chromatography (8 g of silica, MeOH/CH2Cl2, 7.5/92.5, v/v) to
afford 19 (67 mg, 95%) as a colorless sticky foam. Rf: 0.35
(MeOH/CH2Cl2, 1/9, v/v), [R]20.5D: -14.4° (c ) 9.44 mg/mL, CH2-
Cl2). 1H NMR (CDCl3, 300 MHz): δ 13.42 (brs, 1H), 7.44-7.35
(m, 1H), 5.38 (d, 1H, J ) 3 Hz), 5.18 (dd, 1H, J ) 10 Hz, J )
8.0 Hz), 5.01 (dd, 1H), 4.45 (d, 1H), 4.19 (dd, 1H, J ) 11 Hz,
J ) 7 Hz), 4.10 (dd, 1H, J ) 6.7 Hz), 4.00-3.85 (m, 2H), 3.61-
3.51 (m, 1H), 3.45-3.20 (m, 5H), 2.54 (s, 3H), 2.35 (s, 4H), 2.16,
2.15, 2.06, 2.04 (s, 12H), 1.90-1.64 (m, 8H), 1.60-1.44 (m, 2H),
1.00 (s, 6H). 13C NMR (CDCl3, 75 MHz): δ 174.8, 173.5, 173.4,
170.3, 170.1, 170.0, 169.6 (CO), 107.8 (C/C-double), 101.3 (C-
1), 70.8, 70.7 (C-3, C-5), 68.9 (C-2), 68.3 (Sp-OCH2), 67.2 (C-
4), 61.2 (C-6), 54.9 (Lys-CHR), 52.9 (brs, Dde-CH2), 43.1, 36.6
(Lys-NCH2, Sp-NCH2), 34.5 (Dde-Cq), 30.0, 29.6, 29.4, 28.8 (Sp-
CH2, Lys-CH2), 28.2 (Dde-CH3), 23.2 (Lys-CH2), 20.8, 20.6, 20.5
(Ac-CH3), 17.8 (C/C-double-CH3). MALDI TOF MS: m/z ) 698
([M + H]+, retinoic acid); m/z ) 698 ([M + H]+, indolacrylic
acid). LSIMS: m/z ) 720.3 (M + Na)+.
Nr-P a lm it oyl-NE-10,12-p en t a cosa d iyn oyl-(S)-lysin e[3-
(â-D-ga la cto-p yr a n osyloxy)p r op yl]a m id e (3). Compound
22 (11.9 mg, 002 mmol) was dissolved in CH2Cl2 (2 mL), and
10,12-pentacosadiynoic acid (11.2 mg, 0.03 mmol) in CH2Cl2
(0.2 mL), EDC (8 mg, 0.04 mmol) in CH2Cl2/DMF (1/1, v/v,
0.2 mL), and HOBt (5 mg, 0.04 mmol) in CH2Cl2/DMF (1/1,
v/v, 0.2 mL) were added. The reaction mixture was stirred in
the dark for 18 h. MALDI TOF MS analysis (indolacrylic acid)
indicated completion of the coupling reaction. Subsequently,
the reaction mixture was concentrated in vacuo and the
residue purified by gel filtration (Sephadex LH-20, MeOH/CH2-
Cl2, 1/1, v/v) to afford 3 (9.4 mg, 50%) as a sticky foam. [R]22
:
D
-7.8° (CH2Cl2, c ) 5.7 mg/mL). IR (KBr): ν 3422 (OH), 2921,
2849 (CH2), 2361, 2343 (C/C-triple bond), 1646, 1550, 1466
1
(C(O)NH). H NMR (CDCl3, 400 MHz): δ 7.39 (brs, 1H), 6.70
(brs, 1H), 5.94 (brs, 1H), 4.40-4.35 (m, 2H), 4.10-3.99 (m, 2H),
3.98-3.78 (m, 3H), 3.76-3.58 (m, 3H), 3.57-3.52 (m, 1H),
3.51-3.31 (m, 2H), 3.29-3.15 (m, 2H), 2.28-2.12 (m, 8H),
1.85-1.75 (m, 2H), 1.74-1.47 (m, 8H), 1.45-1.19 (m, 52H),
0.91-0.85 (m, 6H). 13C NMR (CDCl3, 125 MHz): δ not seen
(CO), 103.2 (C-1), 77.6 (C/C-triple), 74.4, 73.6, 71.5 (C-5, C-3,
C-2), 69.3 (C-4), 69.0 (Sp-OCH2), 65.2 (C/C-triple), 62.2 (C-6),
52.9 (Lys-CHR), 38.8 (Lys-NCH2), 37.6, (Sp-NCH2), 36.8, 36.5,
31.9, 31.6, 29.7, 29.4, 29.3, 29.2, 29.1, 29.1, 28.9, 28.8, 28.6,
28.4, 28.3, 25.7, 22.5, 19.2 (alkyl-CH2, Lys-CH2, Sp-CH2, Alkyl-
Nr-P a lm it oyl-NE-(4,4-d im et h yl-2,6-d ioxocycloh exyli-
d en e)eth yl-(S)-lysin e[3-(2,3,4,6-tetr a -O-a cetyl-â-D-ga la c-
top yr a n osyloxy)p r op yl]a m id e (20). Compound 19 (112 mg,
0.16 mmol) was dissolved in CH2Cl2 (3 mL), and palmitic acid
(82 mg, 0.32 mmol) in CH2Cl2 (0.5 mL), HONB (57 mg, 0.32
mmol) in CH2Cl2/DMF (1/1, v/v, 0.2 mL) and DIPC (40.3 mg,
0.32 mmol) in CH2Cl2 (0.1 mL) were added. The reaction
mixture was stirred for 18 h. MALDI TOF analysis (retinoic
acid) indicated formation of product and absence of starting

Glycopyranosyl-Containing Amphiphiles
J . Org. Chem., Vol. 65, No. 11, 2000 3365
Ta ble 3. Con cen tr a tion s of Wa ter Su sp en sion s a s Used
for TEM a n d of Or ga n ic Gels
CH3), 14.1 (alkyl-CH3). MALDI TOF MS: m/z ) 983 ([M +
Na]+, indolacrylic acid), 983 ([M + Na]+, gentisic acid).
LSIMS: m/z ) 982.6 (M + Na)+. HRMS (LSIMS): calcd for
negative
freeze
C
56H101N3O9Na (M + Na)+ 982.7436, found 982.7443.
Pt-shadowing, staining, fracturing,
Nr-P a lm itoyl-NE-(10,12-p en ta cosa d iyn oyl)-(S)-lysin yl-
% (w/v)
% (w/v)
0.53
% (w/v)
solvent (v/v)
(S)-glycin e (4). Fmoc-Gly-HMPB-MBHA resin (10) (120 mg,
0.06 mmol of attached amino acid) was preswollen in DMF (5
mL) for 30 min. After removal of the solvent, the Fmoc-group
was cleaved by treatment with piperidine/DMF (2/8, v/v, 10
mL) for 30 min. The solvents was removed by filtration and
the resin washed with DMF (4 × 20 mL, 10 min) and CH2-
Cl2/DMF (5/2, v/v, 28 mL). The resulting resin 11 was
suspended in CH2Cl2/DMF (5/2, v/v, 28 mL), and FmocLysDde-
OH (7) (65 mg, 0.12 mmol), PyBOP (63 mg, 0.12 mmol), and
DIPEA (41 µL, 0.24 mmol) were added. The mixture was
agitated for 4 h, when a negative Kaiser test indicated
completion of the coupling reaction to give 23. The solvents
were removed by filtration, and the resin was washed with
CH2Cl2/DMF (5/2, v/v, 3 × 28 mL, 10 min). The Fmoc-group
of 23 was cleaved by treatment with piperidine/DMF (2/8, v/v,
10 mL) for 30 min. A positive Kaiser test indicated cleavage
of the protecting group and formation of 24. The solvents were
removed by filtration, and the resin washed with DMF (4 ×
20 mL, 10 min). The resin 24 was resuspended in CH2Cl2/DMF
(5/2, v/v, 28 mL), palmitic acid (78 mg, 0.3 mmol), PyBOP (157
mg, 0.3 mmol), and DIPEA (101 µL, 0.6 mmol) were added,
and the mixture was agitated for 5 h. A negative Kaiser test
indicated complete formation of 25. After removal of the
solvent by filtration, the resin 25 was washed with CH2Cl2/
DMF (5/2, v/v, 3 × 28 mL, 10 min) and treated with hydrazine
monohydrate/DMF (2/98, v/v, 10 mL) for 30 min. A positive
Kaiser test indicated successful cleavage of the Dde-protecting
group and formation of 26. After filtration and washing with
DMF (4 × 20 mL, 10 min), the resin 26 was suspended in CH2-
Cl2/DMF (5/2, v/v, 28 mL), and 10,12-pentacosadiynoic acid
(114 mg, 0.3 mmol), PyBOP (157 mg, 0.3 mmol), and DIPEA
(101 µL, 0.6 mmol) were added. The mixture was agitated in
the dark for 6 h when a negative Kaiser test indicated the
completion of the reaction and formation of 27. After filtration
and washing with CH2Cl2/DMF (5/2, v/v, 3 × 28 mL, 10 min),
the immobilized lipodipeptide 27 was released from the solid
support following the general procedure for cleavage of the
HMPB-MBHA linker. Purification of the residue by gel filtra-
tion (Sephadex LH-20, MeOH/CH2Cl2, 1/1, v/v) afforded 4 (33.1
mg, 68%) as a colorless gum [R]22D: -4.1° (CH2Cl2/MeOH, 2/1,
v/v, c ) 5.85 mg/mL). IR (KBr): ν 3309 (OH), 2921, 2848 (CH2),
2358, 2337 (C/C-triple bond), 1645, 1558, 1467 (C(O)NH). UV
1
2
3
4
5
0.59
0.39
0.26
0.15
0.15
0.66
0.84
0.7
THF/EtOH (1/1)
THF/EtOH (8/2)
THF/EtOH (1/1)
THF/EtOH (1/1)
THF/EtOH (8/2)
0.7
solvent was removed by filtration and the resin washed with
CH2Cl2 (30 mL, 4 × 10 min). Lipopeptide 31 was released from
the HMPB-MBHA resin according to the general procedure.
The combined product containing fractions were concentrated
in vacuo, and the residue was purified by gel filtration
(Sephadex LH-20, MeOH/CH2Cl2, 1/1, v/v) to afford pure 32
(147 mg, 58%) as a white powder. [R]22D: -19.6° (CH2Cl2/
MeOH, 1/1, v/v, c ) 3.73 mg/mL). mp 185-190 °C (decomp).
1H NMR (CD3OD/CDCl3, 1/1, v/v, 500 MHz): δ 7.20-7.05 (m,
5H), 4.59 (dd, 1H, J ) 10.1 Hz, J ) 4.4 Hz), 4.20-4.09 (m,
2H), 4.01-3.90 (m, 1H), 3.96, 3.80 (AB, 2H), 3.23-3.14 (m,
2H), 3.05-2.98 (m, 1H), 2.84 (dd, 1H, J ) 13.9 Hz), 2.23 (t,
2H, J ) 7.7 Hz), 2.10 (t, 2H, J ) 7.6 Hz), 1.71-1.60 (m, 2H),
1.59-1.48 (m, 6H), 1.47-1.35 (m, 4H), 1.28-1.13 (m, 52H),
0.82-0.77 (m, 9H), 0.71 (d, 3H, J ) 6.2 Hz). 13C NMR (CDCl3/
CD3OD, 1/1, v/v, 125 MHz): δ 175.3, 174.9, 173.4, 172.8, 172.0
(CO), 136.8, 128.8, 127.8, 126.1 (Phe-aromat), 54.7 (Lys-CHR),
54.2 (Phe-CHR), 52.5, 49.8 (Leu-CHR, Ala-CHR), 40.7 (Gly-CH2),
39.7, 37.8 (Pal-CH2, Lys-NCH2), 37.1 (Phe-CH2), 29.2, 29.0,
29.0, 28.9, 28.8, 28.5, 28.2, 25.5, 25.3, 25.1, 24.3 (Pal-CH2, Lys-
CH2, Leu-CH, Leu-CH2), 22.2 (Leu-CH3), 22.1 (Pal-CH2), 20.6
(Leu-CH3), 16.1 (Ala-CH3), 13.3 (Pal-CH3). MALDI TOF MS:
m/z ) 1034 ([M + Na]+, retinoic acid), 1034 ([M + Na]+,
indolacrylic acid). LSIMS: m/z ) 1055.7 ([M + 2Na - H]+).
HRMS (LSIMS): calcd for C58H102N6NaO8 (M + Na)+ 1033.7657,
found 1033.7659.
N,N′-Dip a lm it oyl-(S)-lysin yl-(S)-a la n yl-(S)-leu cin yl-
(S)-p h en yla la n yl-glycin e[3-(â-D-ga la ct op yr a n osyloxy)-
p r op yl]a m id e (5). Lipopeptide 32 (76 mg, 0.075 mmol) was
suspended in CH2Cl2/DMF (2/1, v/v, 9 mL), and DIPC (103 mg,
0.820 mmol) in CH2Cl2 (1 mL) and HONB (147 mg, 0.820
mmol) in CH2Cl2/DMF (1/1, v/v, 1 mL) were added. Galactoside
8 (195 mg, 0.820 mmol) in DMF (2 mL) was added dropwise
together with DMF in a manner that all compounds remained
in solution. The mixture was stirred for 7 days and subse-
quently concentrated in vacuo. The residue was purified by
gel filtration (Sephadex LH-20, MeOH/CH2Cl2, 1/1) to afford
1
(CH2Cl2/MeOH, 2/1, v/v): λmax 233.2 (ꢀ 902). H NMR (CDCl3,
500 MHz): δ 7.37 (brs, 1H), 6.84 (brs, 1H), 6.03 (brs, 1H),
4.60-4.50 (m, 1H), 4.00 (s, 2H), 3.32-3.10 (m, 2H), 2.26-2.13
(m, 8H), 1.88-1.76 (m, 1H), 1.75-1.64 (m, 1H), 1.64-1.55 (m,
4H), 1.55-1.64 (m, 6H), 1.41-1.32 (m, 6H), 1.32-1.20 (m,
46H), 0.88 (t, 6H, J ) 7 Hz). 13C NMR (CDCl3, 125 MHz): δ
174.4, 174.0, 172.7 (CO), 65.4, 65.3 (C/C-triple bond), 53.4 (Lys-
CHR), 50.7 (Gly-CH2), 38.9 (Lys-NCH2), 36.7, 36.4, 29.7, 29.7,
29.6, 29.5, 29.4, 29.3, 29.2, 29.1, 29.0, 28.6, 26.7, 24.5 (alkyl-
CH2, Lys-CH2), 19.7 (alkyl-CH3), 14.1 (alkyl-CH3). MALDI TOF
MS: m/z ) 820 ([M + Na]+, retinoic acid). LSIMS: m/z ) 820.6
(M + Na)+. HRMS (LSIMS): calcd for C49H87N3NaO5 (M +
Na)+ 820.6543, found 820.6541.
5 (12.8 mg, 13%) as a sticky gum. [R]21 ) -6.6° (CH2Cl2/
D
MeOH, 2/1, v/v, c ) 0.87 mg/mL). 1H NMR (CDCl3/CD3OD,
1/1, v/v, 500 MHz): δ 7.24-7.10 (m, 5H), 4.49 (dd, 1H, J )
10.1 Hz, J ) 4.8 Hz), 4.21-4.01 (m, 4H), 3.97-3.69 (m, 6H),
3.60-3.41 (m, 4H), 3.29-3.02 (m, 5H), 2.90 (dd, 1H, J ) 14.6
Hz), 2.28 (t, 2H, J ) 7.7 Hz), 2.17-2.11 (m, 2H), 1.82-1.00
(m, 66H), 0.87-0.70 (m, 12H). 13C NMR (CDCl3/CD3OD, 1/1,
v/v, 125 MHz): 176.0, 175.4, 174.8, 174.1, 172.7, 170.1 (CO),
137.2, 129.2, 128.4, 126.7, 126.6 (Phe-aromat), 103.5 (C-1),
75.0, 73.7 (C-3, C-5), 71.5 (C-2), 69.2 (C-4), 67.1 (C-6), 61.6 (Sp-
OCH2), 55.8 (Leu-CHR), 55.5 (Phe-CHR), 53.4, 53.0 (Ala-CHR,
Lys-CHR), 50.7 (Gly-CH2), 39.7, 38.1, 36.6 (Pal-CH2, Sp-NCH2,
Lys-NCH2), 37.0 (Phe-CH2), 36.4, 35.8, 31.9, 30.1, 30.0, 29.7,
29.5, 29.4, 29.3, 29.0, 28.5, 26.7, 26.0, 25.5, 24.8 (Pal-CH2, Sp-
CH2, Lys-CH2, Leu-CH2, Leu-CH), 22.8, 22.6 (Pal-CH2, Leu-
CH3), 21.1 (Leu-CH3), 16.5 (Ala-CH3), 13.8 (Pal-CH3). MALDI
TOF MS: m/z ) 1254 ([M + Na]+, retinoic acid). LSIMS: m/z
) 1253 (M + Na)+. HRMS (LSIMS): calcd for C67H119N7NaO13
1252.8764 (M + Na)+, found 1252.8720.
N,N′-Dip a lm it oyl-(S)-lysin yl-(S)-a la n yl-(S)-leu cin yl-
(S)-p h en yla la n yl-glycin e (32). The resin 6 (500 mg, 0.25
mmol) was suspended in CH2Cl2/DMF (5/2, v/v, 28 mL), N,N′-
Bis-Fmoc-LysOH 28 (738 mg, 1.25 mmol), HOBt (203 mg, 1.5
mmol), and DIPC (189 mg, 1.5 mmol) were added, and the
reaction mixture was agitated for 8 h to give resin bound 29.
The solvents were removed by filtration, the resin was washed
with CH2Cl2/DMF (5/2, v/v, 28 mL, 3 × 10 min). The Fmoc
protecting groups of 29 were removed by treatment with
piperidine/DMF (2/8, v/v, v/v, 15 mL) for 30 min to give 30.
After washing with DMF (30 mL, 3 × 10 min), the resin was
suspended in pyridine/CH2Cl2 (1/1, v/v, 30 mL), palmitoyl
chloride (1.374 g, 5 mmol) in CH2Cl2 (1 mL) was added, and
the mixture was agitated for 5 h. A negative Kaiser test
indicated completion of the reaction and formation of 31. The
E lect r on Micr oscop y. Gen er a l P r oced u r e for P t -
Sh a d ow in g. The dry compound was dissolved in an appropri-
ate mixture of EtOH and THF. An aliquot of this solution was
injected into hot (72 °C) vortexed water resulting in the
concentration listed in Table 3. One drop of the resulting
homogeneous water suspension was applied to a carbon-coated
copper grid. After allowing the aggregates to settle for 1 min,

3366 J . Org. Chem., Vol. 65, No. 11, 2000
Reichel et al.
the excess water was drained with filter paper. After drying,
the aggregates were shadowed with Pt atoms. Transmission
electron microscopy (TEM) was performed on a Philips EM201
instrument using an acceleration voltage of 60 kV. Pt shadow-
ing was performed using an Edwards 306 system.
X-r a y P ow d er Diffr a ction . Samples were prepared by
placing a drop of an aggregate dispersion on a silicon wafer.
For the studies presented here, the best results were obtained
by drying in an environment of controlled relative humidity,
and not in a desiccator. The diffraction experiments were
carried out on a commercial Philips X-ray powder diffracto-
meter of the Bragg Brentano type that was optimized for
measurements at low angle. The X-ray tube was ceramic with
a long fine focus and gave Cu KR radiation (generator 40 kV,
40 mA). The goniometer had a variable divergence and
antiscatter slits, with the receiving slits set at 0.1 mm. The
detector was of the Peltier-cooled Si/Li type. During the
measurements, the sample was mounted in a chamber of
which the relative humidity could be controlled by a humidify-
ing instrument flushed with N2 gas.
Gen er a l P r oced u r e for Nega tive Sta in in g. The water
suspension was prepared and applied to a copper grid as
described above. Subsequently, a uranyl acetate solution (1/
49, v/v) was added for 1 min and then removed by filter paper.
Gen er a l P r oced u r e for F r eeze F r a ctu r in g a n d Etch -
in g. Aqueous suspensions were prepared as described for Pt-
shadowing. A freshly cleaned gold (400 mesh) grid was
immersed in the suspension and placed between two freshly
cleaned copper plates. The grid and copper plates were rapidly
frozen in liquid propane and fractured at 10-6 Torr and -105
°C. After etching (2 min), Pt-shadowing (2 nm), and carbon
deposition (20 nm), the resulting replicas were cleaned ca. 28
h with 70% H2SO4 and subsequently transferred to water
(several times) and finally dried.
General procedure for polymerization of compounds contain-
ing a diacetylenic unit: Water suspensions were prepared as
described above and transferred to a Quartz cuvette and placed
under an UV lamp. Irradiation at 254 nm was continued for
18 h. A color change from colorless to purple/blue indicated a
successful polymerization. A drop of the polymer suspension
was applied to a carbon-coated copper grid and treated in a
way as described for Pt-shadowing. Freeze fracturing experi-
ments were performed in a Balzers Freeze Etching System
BAF 400 D, with samples rapidly frozen in a BAL-TEC J FD
030 J et-Freeze Device.
Ack n ow led gm en t. The authors thank Dr. Neil
Spencer and Mr. Peter Ashton who have performed the
NMR experiments and the MS measurements, respec-
tively. We also thank Professor Roeland J . M. Nolte for
helpful suggestions. The Medical Research Council
(MRC) and the Novartis Foundation have provided
financial support.
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
spectra for all compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
J O991685S