A new type of soft vesicle-forming molecule: An amino acid derived guanidiniocarbonyl pyrrole carboxylate zwitterion

Article abstract of DOI:10.1021/ol8002755

(Figure Presented) The self-assembly of the L-alanine derived zwitterion 3 leads to the formation of soft vesicles in solution even though this surprisingly small molecule does not possess the classical amphiphilic features of other vesicle-forming monomers.

Full text of DOI:10.1021/ol8002755

ORGANIC
LETTERS
2008
Vol. 10, No. 7
1469-1472
A New Type of Soft Vesicle-Forming
Molecule: An Amino Acid Derived
Guanidiniocarbonyl Pyrrole Carboxylate
Zwitterion
Thomas Rehm,† Vladimir Stepanenko,† Xin Zhang,† Frank Wu
1
rthner,†
Franziska Gro
1
hn,‡ Katja Klein,‡ and Carsten Schmuck*,†
Institut fu¨r Organische Chemie, UniVersita¨t Wu¨rzburg, Am Hubland, 97074 Wu¨rzburg,
Germany, and Max Planck Institut fu¨r Polymerforschung, Ackermannweg 10,
55128 Mainz, Germany
schmuck@chemie.uni-wuerzburg.de
Received February 6, 2008
ABSTRACT
The self-assembly of the
L-alanine derived zwitterion 3 leads to the formation of soft vesicles in solution even though this surprisingly small
molecule does not possess the classical amphiphilic features of other vesicle-forming monomers.
The self-assembly of molecules can lead to the formation
of large aggregates such as micelles, bilayers, or vesicles.1
The size and structure of the aggregates formed are deter-
mined by the shape as well as kind of interactions between
the monomers. The classical vesicle-forming molecules are
amphiphilic lipids. Also other classes of amphiphiles,
molecules with segmented hydrophilic and hydrophobic
parts, form vesicles such as block-copolymers,2 oligopep-
tides,3 calixarenes, or cyclodextrins functionalized with
lipophilic chains or hydrophobic shape persistent macrocycles
with hydrophilic tails.4 Bolaamphiphiles are another class
of vesicle builders but also have the traditional amphiphilic
(2) Representative examples: (a) Shen, H.; Eisenberg, A. Angew. Chem.,
Int. Ed. 2000, 39, 3310. (b) Che´cot, F.; Lecommandoux, S.; Gnanou, Y.;
Klok, H.-A. Angew. Chem., Int. Ed. 2002, 41, 1339. (c) Kros, A.; Jesse,
W.; Metselaar, G.; Cornelissen, J. J. L. M. Angew. Chem., Int. Ed. 2005,
44, 4349. (d) Holowka, E. P.; Pochan, D. J.; Deming, T. J. J. Am. Chem.
Soc. 2005, 127, 12423. (e) Vriezema, D. M.; Hoogboom, J.; Velonia, K.;
Takazawa, K.; Christianen, P. C. M.; Maan, J. C.; Rowan, A. E.; Nolte, R.
J. M. Angew. Chem., Int. Ed. 2003, 42, 772. (f) Yang, M.; Wang, W.; Yuan,
F.; Zhang, X.; Li, J.; Liang, F.; He, B.; Minch, B.; Wegner, G. J. Am.
Chem. Soc. 2005, 127, 15107.
† Universita¨t Wu¨rzburg.
‡ Max Planck Institut fu¨r Polymerforschung.
(1) Reviews on vesicles: (a) Discher, D. E.; Eisenberg, A. Science 2002,
297, 967. (b) Antonietti, M.; Fo¨rster, S. AdV. Mater 2003, 15, 1323. (c)
Morishima, Y. Angew. Chem., Int. Ed. 2007, 46, 1370.
(3) (a) Lo¨wik, D. W. P. M.; van Hest, J. C. M. Chem. Soc. ReV. 2004,
33, 234. (b) Kokkoli, E.; Mardilovich, A.; Wedekind, A.; Rexeisen, E. L.;
Garg, A.; Craig, J. A. Soft Matters 2006, 2, 1015.
10.1021/ol8002755 CCC: $40.75
© 2008 American Chemical Society
Published on Web 03/01/2008

composition.5 Dipeptides such as L-Phe-L-Phe can self-
assemble into vesicles, although the kind of interaction and
the aggregation mode leading to vesicle formation are
unclear.6 We report here that a molecule as small as
zwitterion 3, which does not have a classical amphiphilic
structure with well-separated polar and unpolar segments,
self-assembles into soft vesicles in DMSO.
pairing occurs as confirmed by the concentration-dependent
shift changes in an NMR dilution study in DMSO-d6 (Figure
1).10 The rather small complexation induced shift change of
The synthesis of zwitterion 3 is shown in Scheme 1:
Scheme 1. Synthesis of Zwitterion 3
L-alanine methyl ester hydrochloride 2 was coupled with the
Boc-protected guanidinocarbonyl pyrrole carboxylic acid 17
using PyBOP in DMF as the coupling reagent (yield 63%).
The Boc group was removed with TFA, and afterward, the
methyl ester was cleaved with LiOH to yield zwitterion 3
(yield: 80%).
Zwitterion 3 is self-complementary as the cationic guani-
diniocarbonyl pyrrole8 is an efficient binding site for car-
boxylates.9 Intramolecular ion pairing of 3 is not possible
for geometric reasons; therefore, an intermolecular ion
Figure 1. Self-association of zwitterion 3 (the red arrow indicates
the NOE contact); parts of the H NMR spectrum showing the
concentration dependent shift changes in DMSO-d6 (1-100 mM
from bottom to top).
1
(4) Recent examples for vesicle formation from non-polymers: (a) Xie,
D.; Jiang, M.; Zhang, G.; Chen, D. Chem.-Eur. J. 2007, 13, 3346. (b)
Dong, D.; Baigl, D.; Cui, Y.; Sinay, P.; Sollogoub, M.; Zhang, Y.
Tetrahedron 2007, 63, 2973. (c) Schmuck, C.; Rehm, T.; Klein, K.; Gro¨hn,
F. Angew. Chem., Int. Ed. 2007, 46, 1693. (d) Seo, H. S.; Chang, J. Y.;
Tew, G. N. Angew. Chem., Int. Ed. 2006, 118, 7688; (e) Balakrishnan, K.;
Datar, A.; Zhang, W.; Yang, X.; Naddo, T.; Huang, J.; Zuo, J.; Yen, M.;
Moore, J. S.; Zang, L. J. Am. Chem. Soc. 2006, 128, 6576. (f) Hoeben, F.
J. M.; Shklyarevskiy, I. O.; Pouderoijen, M. J.; Engelkamp, H.; Schenning,
A. P. H. J.; Christianen, P. C. M.; Maan, J. C.; Meijer, E. W. Angew. Chem.,
Int. Ed. 2006, 45, 1232. (g) Holowka, E. P.; Pochan, D. J.; Deming, T. J.
J. Am. Chem. Soc. 2005, 127, 12423. (h) Lee, H.-K.; Park, K. M.; Jeon, Y.
J.; Kom, D.; Oh, D. Y.; Kim, H. S.; Park, C. K.; Kim, K. J. Am. Chem.
Soc. 2005, 127, 5006. (i) Lee, M.; Lee, S.-J.; Jiang, L.-H. J. Am. Chem.
Soc. 2004, 126, 12724. (j) Zhou, Y.; Yan, D. Angew. Chem., Int. Ed. 2004,
43, 4896.
the guanidinio amide NH proton a of ∆δ ) 0.47 is indicative
for the formation of a bidentate carboxylate-guanidinium ion
pair as shown in Figure 1.11 An NOE-contact between amide
NH proton b and pyrrole CH proton c confirms that 3 is
present in an extended conformation as shown with the amide
NH of the alanine pointing to the back.
Unexpectedly, all NMR samples of higher concentration
(c > 10 mM) showed a strong Tyndall effect, indicating the
formation of even larger aggregates than just ion-paired
dimers or smaller oligomers. Therefore, atomic force mi-
croscopy (AFM), dynamic light scattering (DLS), and
transmission electron microscopy (TEM) were performed to
further investigate the self-assembly of 3. For AFM analysis,
a 5 mM solution of 3 in DMSO was spin coated onto silica
wafers and analyzed in the tapping mode. Figure 2 shows
spherical particles with a mean diameter of ca. 25 nm
(measured at half-height of the particles) and a height of ca.
4 nm, about six times smaller than the average diameter.
The mean diameter of 25 nm of the particles is significantly
larger than the molecular dimension of zwitterion 3 (ca. 1.4
nm estimated from molecular modeling; see Supporting
Information). Therefore, it is very unlikely that the particles
(5) Fuhrhop, J.-H.; Wang, T. Chem. ReV. 2004, 104, 2901.
(6) Recent examples for the self-assembly of dipeptides: (a) Reches,
M.; Gazit, E. Science 2003, 300, 625. (b) Adler-Abramovich, L.; Reches,
M.; Sedman, V.; Allen, S.; Tendler, S.; Gazit, E. Langmuir 2006, 22, 1313.
(c) Yan, X.; He, Q.; Wang, K.; Duan, L.; Cui, Y.; Li, J. Angew. Chem.,
Int. Ed. 2007, 46, 2431. (d) Song, Y.; Challa, S.; Medforth, C.; Qui, Y.;
Watt, R.; Pena, D.; Miller, J.; van Swol, F.; Shelnutt, J. Chem. Commun.
2004, 1044. (e) Reches, M.; Gazit, E. Nano Lett. 2004, 4, 581. (f) Soldatov,
D.; Moudrakovski, I.; Grachev, E.; Ripmeester, J. J. Am. Chem. Soc. 2006,
128, 6737. (g) Soldatov, D.; Moudrakovski, I.; Ripmeester, J. Angew. Chem.,
Int. Ed. 2004, 43, 6308.
(7) Schmuck, C.; Bickert, V.; Merschky, M.; Geiger, L.; Rupprecht, D.;
Dudaczek, J.; Wich, P.; Rehm, T.; Machon, U. Eur. J. Org. Chem. 2008,
324.
(8) Schmuck, C. Coord. Chem. ReV. 2006, 250, 3053.
(9) For recent reviews on oxoanion binding by guanidinium cations
see: (a) Schug, K. A.; Lindner, W. Chem. ReV. 2005, 105, 67. (b) Houk,
R. J. T.; Tobey, S. L.; Anslyn, E. V. Top. Curr. Chem. 2005, 255, 199. (c)
Blondeau, P.; Segura, M.; Perez-Fernandez, R.; de Mendoza, J. Chem. Soc.
ReV. 2007, 36, 198.
(10) Connors, K. Binding Constants; Wiley & SonsL Chichester, 1987.
(11) Schmuck, C. Tetrahedron 2001, 57, 3063.
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Org. Lett., Vol. 10, No. 7, 2008

Figure 3. TEM image of 3 prepared from DMSO solution and
stained with uranyl acetate.
Figure 2. AFM height image (A) of 3 on silica wafer (Z scale is
12 nm) shows spherical aggregates. The cross section analysis along
the yellow line (C) provides a mean diameter of ca. 25 nm and a
height of 4 nm. The appearance of the vesicles in the phase image
(B) is typical for deformed soft hollow vesicles on a surface (D).
the TEM images compared to the DSL studies is most likely
caused by a swelling due to water from the uranyl acetate
staining solution entering the DMSO filled vesicles. There-
fore, all experimental data (DLS, AFM, and TEM) consis-
tently confirm that zwitterion 3 forms soft vesicles in solution
that however collapse upon interaction with a surface as
typical for soft particles. The differences in the particle sizes
obtained from these experiments are surprisingly small for
this type of soft vesicle.
are micelle-like aggregates, which generally have a diameter
about twice as large the molecular dimension. Instead, the
profile of the particles in the phase image (Figure 2B) clearly
identifies these particles as soft, hollow vesicles, which
typically show a lower central part surrounded by a higher
periphery in AFM experiments.2f,4a
Vesicle formation by zwitterion 3 is most likely due to a
hierarchical self-assembly as shown in Figure 4. In solution,
DLS experiments in solution ([3] ) 5.55 mM in DMSO)
also confirm the presence of vesicles in solution with a
hydrodynamic radius of ca. rH ) 20-25 nm, resulting in a
particle diameter of 40-50 nm.12 The larger particle size
seen in DLS is most likely due to the drying occurring during
sample preparation in AFM, which causes shrinking of the
particles. Also, the surface of the silica wafer can have an
effect on the aggregate size in AFM experiments, whereas
DLS measurements detect the real and original state of the
aggregates in solution.
The vesicular nature of the aggregates was further
confirmed by TEM. The TEM image (Figure 3) clearly
shows a vesicle with a diameter of ca. 60 nm and an
estimated membrane thickness of ca. 3 nm. In contrast to
hard vesicles, the electron contrast is relatively weak as
typically observed for soft vesicles in TEM studies.2f,4a
Collapsed soft vesicles have an almost constant thickness
from the periphery to the center of the particle (as seen in
the height profile of the AFM image). However, as the
particles were prepared from DMSO solution and not water,
the hollow, solvent-filled inner part of the vesicle is still
loaded with DMSO molecules. DMSO is a strongly coor-
dinating ligand for the uranyl acetate staining agent giving
rise to the even darker inner area compared to the membrane
itself. The slightly larger size of the aggregates observed in
Figure 4. Suggested membrane formation via the self-assembly
of 3.
3 forms ion-paired dimers that have a kink of ca. 38°
according to molecular modeling studies (Macromodel V 8.0,
amber* force field, GB/SA water solvation model, MC
conformational search with 50 000 steps). A similar dimer-
ization has been observed before for a more simple rigid
zwitterion.11 These dimers then aggregate into membranes.
As the dimers are tilted, a curvature is induced that gives
rise to the formation of soft vesicles.
To test this aggregation model, we have performed a
molecular mechanics calculation of a membrane segment
composed of 32 such dimers. For the membrane construction,
dimers of 3 in the conformation obtained from the Monte
Carlo conformational search were used. Structure minimiza-
tion then provided a stable curved membrane (Figure 5) with
a thickness of 2.7 nm in perfect agreement with the value of
ca. 3 nm for the membrane thickness extracted from the TEM
(12) Static light scattering to further characterize the particles in solution
was not possible due to the presence of a small amount of larger aggregates
(rh > 100 nm), which limits the analysis to the DLS part. Also, SANS
measurements to further characterize the internal structure of the vesicles
in more detail were not successful due to an insufficient scattering contrast
between sample and solvent.
Org. Lett., Vol. 10, No. 7, 2008
1471

for further electrostatic interactions of the oppositely charged
groups between the dimers and most likely π-stacking or
hydrophobic interactions between the aromatic moieties. At
least in the energy minimized structure the planar aromatic
parts of the molecules are in close contact with each other.
All methyl groups are presented at the same side, which
results in a steric bulk especially in the middle part of the
membrane. This together with the tilt of each dimer induces
the curvature. The important role of the alanine methyl group
is also supported by the observation that an analogous glycine
derived zwitterion, which lacks this methyl group, does not
form vesicles but just small linear oligomers.13 Hence, the
alanine methyl group in 3 is crucial for vesicle formation.
In conclusion, we have shown here that a hierarchical self-
assembly of zwitterion 3 causes the formation of soft vesicles
even in polar solutions. Further studies with similar zwitte-
rions derived from other amino acids with other polar and
nonpolar side chains are currently underway to further
explore the potential of this new class of vesicle-forming
molecules.
Acknowledgment. C.S. thanks the DFG and the Fonds
der Chemischen Industrie for ongoing financial support of
his work. X.Z. thanks the Alexander von Humboldt founda-
tion for a fellowship.
Supporting Information Available: Full synthesis and
characterization of compound 3, including experimental data
of the NMR, AFM, DLS, and TEM measurements. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Figure 5. Calculated structure of a membrane segment (the alanine
methyl groups needed for vesicle formation are shown in yellow):
(A) view along the rows of stacked dimers showing the vesicle
curvature; (B) side view of the stacked dimers showing their
alternating antiparallel orientation.
OL8002755
(13) (a) Schmuck, C.; Rehm, T.; Geiger, L.; Scha¨fer, M. J. Org. Chem.
2007, 72, 6162. (b) Schmuck, C.; Rehm, T.; Gro¨hn, F.; Klein, K.; Reinhold,
F. J. Am. Chem. Soc. 2006, 128, 1430.
image. The membrane is composed of dimers which stack
next to each other in an antiparallel orientation. This allows
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Org. Lett., Vol. 10, No. 7, 2008