Stereoselective self-sorting in the self-assembly of a Phe-Phe extended guanidiniocarbonyl pyrrole carboxylate zwitterion: Formation of two diastereomeric dimers with significantly different stabilities

Article abstract of DOI:10.1039/c1cc12520f

The 'dipeptide extended' guanidiniocarbonyl pyrrole carboxylate zwitterion GCP-Phe-Phe 1 forms stable dimers in DMSO. However, dimerization is highly stereoselective. Only homochiral dimers are formed and the (L,L)·(L,L) dimer (K_dim > 10⁵ M^-1) is significantly more stable by a factor of 10³ than the diastereomeric (D,L)·(D,L) dimer (K_dim = 120 M^-1).

Full text of DOI:10.1039/c1cc12520f

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COMMUNICATION
Stereoselective self-sorting in the self-assembly of a Phe–Phe extended
guanidiniocarbonyl pyrrole carboxylate zwitterion: formation of two
diastereomeric dimers with significantly different stabilitiesw
Fabian Rodler, Wilhelm Sicking and Carsten Schmuck*
Received 29th April 2011, Accepted 31st May 2011
DOI: 10.1039/c1cc12520f
The ‘dipeptide extended’ guanidiniocarbonyl pyrrole carboxylate
zwitterion GCP–Phe–Phe 1 forms stable dimers in DMSO. How-
ever, dimerization is highly stereoselective. Only homochiral dimers
of this ‘dipeptide extended’ guanidiniocarbonyl pyrrole carboxy-
late is highly stereoselective. Only homochiral dimers are formed
and their stability differs by a factor of at least 4103 for the two
diastereomeric dimers, the (L,L)Á(L,L) and the (D,L)Á(D,L) dimer,
respectively.
are formed and the (L,L)Á(L,L) dimer (Kdim 4 105
M
À1) is
significantly more stable by a factor of 103 than the diastereomeric
(D,L)Á(D,L) dimer (Kdim = 120 MÀ1).
The synthesis of the four stereoisomers of GCP–Phe–Phe–
OH 1a–d followed a standard protocol (SuppInfow). At
millimolar concentrations in DMSO none of the four stereo-
isomers gave any experimental indication for the formation of
larger nanostructures either in DLS nor AFM (data not
shown). Instead only formation of discrete and well defined
dimers was observed (vide infra).
Self-assembly of self-complementary building blocks in polar
solutions is still difficult to achieve.1 Hydrogen bonds which are
often used for self-assembly due to their directionality and
complementarity are not strong enough in competing solvents
such as DMSO, alcohols or even water.2 Additional noncovalent
interactions such as electrostatic and hydrophobic interactions or
p-stacking are needed to ensure stable complexation in these
solvents. We have a long standing expertise in zwitterion induced
self-assembly in polar solutions.3 About ten years ago we reported
However, the two diastereomers 1a/b (L,L and D,D) and 1c/d
(D,L and L,D) self-assemble very differently as immediately
evident from the NMR spectra (Fig. 1). The shifts in both spectra
are characteristic for ionpair formation between the GCP cation
and the carboxylate.4 For example, in the spectrum of the L,L
stereoisomer 1a the guanidinio amide NH gives a signal at
d = 14.5 and the four guanidinium NH protons give two signals
at d = 10.1 and d = 8.0, respectively. These shifts are similar to
those observed previously in related zwitterions which dimerize
in solution4 and they are diagnostic for ionpair formation.
Stereosiomer 1c also shows similar shifts, however, in contrast
to 1a the signals for the D,L stereoisomer 1c are rather broad and
appear at higher field. For example, the guanidinio amide NH
gives a broad signal at d = 13.5 for 1c, a shift of nearly 1 ppm
to higher field compared to the sharp signal at d = 14.5 for 1a.
a
rigid guanidiniocarbonyl pyrrole carboxylate zwitterion
which forms dimers held together by two H-bond assisted
ionpairs (Scheme 1). The stability of the dimers is very high
(Kdim 4 108 MÀ1 in DMSO and 102–103 MÀ1 in water).4
Recently, we found that more flexible ‘amino acid extended’
guanidiniocarbonyl pyrrole carboxylate zwitterions in which the
carboxylate and the guanidiniocarbonyl pyrrole cation (GCP) are
separated by one amino acid also self-assemble in DMSO but not
into dimers but into vesicles instead.5 This finding was in line with
other reports on self-assembly of small peptides, which often form
large nanostructures such as tubes, fibers or spherical particles.6
For example the zwitterionic dipeptide H–Phe–Phe–OH self-
assembles into nanotubes which are several micrometers in length
and ca. 50–300 nm in diameter.7,8 The cationic dipeptide
H–Phe–Phe–NH2 (as hydrochloride salt) forms nanotubes which
upon dilution then rearrange into vesicles.9 We were therefore
quite surprised when we now discovered that the dipeptide
Phe–Phe when derivatized with our GCP cation at the
N-terminus again self-assembles only into discrete dimers with
no formation of any larger aggregates. Furthermore, dimerization
University of Duisburg-Essen, Faculty of Chemistry,
Universitaetsstraße 7, 45141 Essen, Germany.
E-mail: carsten.schmuck@uni-due.de; Fax: +492211834256;
Tel: +492211833097
w Electronic supplementary information (ESI) available: Details for
the synthesis of 1, NMR spectra, UV/Vis dilution study and details on
the DFT calculations. See DOI: 10.1039/c1cc12520f
Scheme 1 The self-assembly of guanidiniocarbonyl pyrrole carboxylate
zwitterions depends on the linker between the carboxylate and the cation.
c
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Chem. Commun., 2011, 47, 7953–7955 7953

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of the dimers formed from 1a requires much lower concentrations
than accessible in a NMR experiment. Concentration dependent
UV/Vis studies in the concentration range from 0.8 to 0.005 mM
allowed to follow this dissociation. The molar absorptivity e of the
pyrrole moiety at around l = 300 nm increases with decreasing
concentrations. We know from other studies that binding of a
carboxylate to the guanidiniocarbonyl pyrrole cation causes a
decrease in this absorption band.11 Therefore, the increase of e in
more dilute solutions reflects the dissociation of the dimers into
non ionpaired monomers. A quantitative analysis of this concen-
tration dependent UV change provides a dimerization constant of
Fig. 1 1H NMR spectra of the two diastereomers of the GCP–Phe–Phe
zwitterions 1a (15 mM, top) and 1c (80 mM, bottom) in DMSO-d6.
K
dim = 3 Â 105 MÀ1
.
Also the signals for the pyrrole NH and the two signals for the
four guanidinium NH protons appear at higher field. A NMR
dilution-study of 1d in DMSO (Fig. 2) showed concentration-
dependent shift-changes in the range from 0.5 to 100 mM, which
indicated an intermolecular aggregation. Formation of a dimer
1dÁ1d was confirmed in a DOSY-NMR experiment (c = 30 mM
in DMSO-d6). A hydrodynamic radius rh = 0.87 nm and hence a
particle diameter of 1.74 nm was obtained, which compares well
with the molecular dimensions of a dimer as estimated from
molecular modeling studies (the calculated molecular surface of
the dimer of 725 A2 corresponds to a sphere with ca. 1.5 nm
diameter). A quantitative analysis of the NMR shift changes
provides a dimerisation constant of Kdim = 120 MÀ1. Hence, the
stability of the dimers is similar to other flexible zwitterions in
such a highly polar solvent.10
Hence, the enantiomers 1a,b (L,L and D,D) form significantly
more stable dimers than their diastereomers 1c,d (D,L and L,D).
The dimer stabilities differ by a factor of 103. Dimerization is also
highly stereoselective. When we mixed any of the four stereo-
isomers 1a–d no indication for the formation of mixed dimers was
observed in the NMR spectra (SuppInfow). Even though in
principle 10 different dimers could form, each stereoisomer 1a–d
in a self-sorting process exclusively interacts with itself forming
only the four homochiral dimers
The calculated energy minimized structures of the two dimers of
1a and 1c are shown in Fig. 3. In both dimers the typical H-bond
assisted ion pairing motif between the carboxylate of the one
dipeptide and the guanidiniocarbonyl pyrrole cation of the other is
seen (in agreement with the observed downfield shifts in the
NMR). Furthermore, the adjacent pyrrole carbonyl oxygen forms
an additional H-bond to the C-terminal amide NH next to the
carboxylate. In this respect both dimers are similar. However, the
change in stereochemistry of one of the Ca within the dipeptide
has a profound effect on how these two ion pairs are arranged
within the dimer. In the dimer 1aÁ1a the two molecules are facing
each other and are aligned side by side. One benzyl group of each
monomer points upwards and the other downwards from the
plane of the two ion pairs. In the diastereomeric dimer 1cÁ1c the
two ion pairs are on top of each other and all four benzyl groups
point to one side of the dimer.
Quite different results were obtained for the two enantiomeric
zwitterions 1a,b (L,L and D,D stereoisomer). As explained above
the NMR spectrum also clearly confirms the formation of ionpairs
(Fig. 1). However, at least in the range from 0.1–100 mM in
DMSO no concentration dependent shift changes are observed.
DOSY NMR (c = 50 mM) provided a diameter of 1.82 nm
confirming again the presence of dimers in solution of similar size
as the dimers formed from 1c. The lack of concentration
dependent shift changes in the NMR spectrum indicates a very
high stability of the dimers (Kdim 4 104 MÀ1) in sharp contrast to
the stability of the dimers formed from the diastereomer 1c
(Kdim = 120 MÀ1). Due to their high stability the dissociation
A DFT calculation (m052x/6-311+G**) confirmed the
observed difference in stabilities of the two dimers. The geometry
optimized structure of dimer 1aÁ1a is 5.7 kcal molÀ1 more stable
than dimer 1cÁ1c and the calculated dimerization energies
going from the optimized monomers to the dimers differ by
2.7 kcal/mol. These values are in good agreement with the
observed experimental difference in stability of DK Z 103, which
corresponds to an energy difference of DE Z 4.2 kcal molÀ1. The
difference in stability of the two dimers stems most likely from a
combination of two major effects: First, a more compact structure
which allows probably more extensive aromatic interactions and a
more favorable solvation and second a significantly more efficient
charge interaction within dimer 1aÁ1a as evident from the changes
of the molecular dipoles. Both monomers 1a and 1c have a
molecular dipole of ca. m = 13 D. However, whereas the side-
by-side alignment of the two monomers within dimer 1aÁ1a nearly
completely cancels the dipole (m = 1.8 D), the on-top orientation
within dimer 1cÁ1c actually causes an increase to m = 19 D.
Hence, relative to the monomers the charge interaction is more
favorable within dimer 1aÁ1a than within dimer 1cÁ1c.
According to the calculated structure of dimer 1aÁ1a the two
amide NH protons of the dipeptide moiety are bound in different
Fig. 2 1H NMR dilution study of zwitterion 1d in DMSO-d6.
7954 Chem. Commun., 2011, 47, 7953–7955
c
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Fig. 4 H/D exchange experiment of zwitterion 1a in DMSO-d6
(10 mM). The two amino acid amide NH protons show significantly
different exchange rates. The C-terminal amide NHe (circled green) which
is involved in the dimerization does not exchange even within 3.5 h. The
N-terminal amide NHd (circled orange) which is exposed to the solvent
does show significant H/D exchange within this time. The red arrows
indicate a diagnostic nOe which also confirms this structure.
formed which furthermore differ significantly in their stability
by at least a factor of 103. Even the switch of the configuration
of one stereocentre can thus have a tremendous effect on the
self-assembling properties of the molecule.
Fig. 3 Energy minimized structures of the dimer of 1a (top) and 1c
(bottom) as obtained from force field calculations. Nonpolar
hydrogens are omitted for clarity.
We thank the DFG (Deutsche Forschungsgemeinschaft) for
funding.
chemical environments. Whereas the N-terminal amide NHd is
solvent exposed, the C-terminal amide NHe forms an inter-
molecular H-bond to the pyrrole carbonyl oxygen of the second
molecule. This difference in the binding situation of the amide NH
protons could be confirmed by a H/D exchange experiment
(Fig. 4). After addition of 10 ml D2O to a 10 mM solution of 1a
in DMSO-d6, the intensity of the NMR signal of the amide NHd
proton decreases very fast within minutes. The signal of the amide
NHe proton which is involved in the intermolecular H-bond to the
second zwitterion remains however unchanged. Even after several
hours only very little H/D exchange is observed for this proton.
This experiment therefore confirms the proposed structure and
also the extraordinary high stability of this dimer. No significant
amount of monomer is present because otherwise exchange of
both amide NH protons would occur, which is the case for the
dimer of 1c. Both amide NH protons exchange with similar rates
and within one hour both signals have disappeared. Even though
the two amide NH protons also have a different binding environ-
ment within dimer 1cÁ1c, the significantly lower stability of this
dimer means that always a substantial fraction of monomers is
present in which both amide NH protons can exchange with the
solvent with equal rates.
Notes and references
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In conclusion, we have shown here that dimerization of
zwitterion 1 is highly stereospecific as of all possible ten
stereoisomeric dimers only the four homochiral dimers are
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 7953–7955 7955