Cation-Transporting Peptides: Scaffolds for Functionalized Pores?

Article abstract of DOI:10.1002/chem.201500881

Protein pores that selectively transport ions across membranes are among nature's most efficient machines. The selectivity of these pores can be exploited for ion sensing and water purification. Since it is difficult to reconstitute membrane proteins in their active form for practical applications it is desirable to develop robust synthetic compounds that selectively transport ions across cell membranes. One can envision tuning the selectivity of pores by incorporating functional groups inside the pore. Readily accessible octapeptides containing (aminomethyl)benzoic acid and alanine are reported here that preferentially transport cations over halides across the lipid bilayer. Ion transport is hypothesized through pores formed by stable assemblies of the peptides. The aromatic ring(s) appear to be proximal to the pore and could be potentially utilized for functionalizing the pore interior.

Full text of DOI:10.1002/chem.201500881

DOI: 10.1002/chem.201500881
Full Paper
&
Ion Channels
Cation-Transporting Peptides: Scaffolds for Functionalized Pores?
[
a]
Harekrushna Behera, Venkatachalam Ramkumar, and Nandita Madhavan*
Abstract: Protein pores that selectively transport ions across
membranes are among nature’s most efficient machines.
The selectivity of these pores can be exploited for ion sens-
ing and water purification. Since it is difficult to reconstitute
membrane proteins in their active form for practical applica-
tions it is desirable to develop robust synthetic compounds
that selectively transport ions across cell membranes. One
can envision tuning the selectivity of pores by incorporating
functional groups inside the pore. Readily accessible octa-
peptides containing (aminomethyl)benzoic acid and alanine
are reported here that preferentially transport cations over
halides across the lipid bilayer. Ion transport is hypothesized
through pores formed by stable assemblies of the peptides.
The aromatic ring(s) appear to be proximal to the pore and
could be potentially utilized for functionalizing the pore in-
terior.
Introduction
Pore-forming proteins that selectively transport ions across
[
1]
membranes play a vital role in cellular processes. A selectivity
filter inside these pores has been shown to dictate its ion pref-
[
1,2]
Figure 1. Proposed scaffolds for functionalized pores.
erence.
Robust synthetic ion channels have been developed
[
3]
that mimic the activity of natural proteins. Synthetic pore-
forming compounds have found application as antibacterial
ions across the lipid bilayer. The most active peptide 3 trans-
ports cations and not halides across the lipid bilayer. The pep-
tides form stable assemblies and appear to form pores with
the aromatic rings proximal to the pore. These aromatic rings
could be potentially useful sites for internally functionalizing
the pore.
[
4]
drugs, molecular switches, catalysts, and sensors. Placement
of functional groups inside the pore is highly desirable for the
aforementioned applications. The pore a-Hemolysin has been
used for stochastic sensing of small molecules, charged spe-
[
5]
cies, and DNA. The selectivity of the Hemolysin pore for ana-
lytes has been tuned by placement of functional groups inside
the pore through genetic engineering or incorporation of mac-
rocyclic adapters inside the pore.
Results and Discussion
There are few examples of synthetic peptide-based internally
functionalized pores. Cyclic peptides containing a repeating
Octapeptides 1a and 1b, containing m- and p-substituted aro-
matic units, were synthesized in solution as shown in
[
6]
[7]
[11]
llld or an ld amino acid sequence with aromatic or tetra-
Scheme 1. The (methylamino)benzoic acid derivative 5 was
[
8]
hydrofuran units have been shown to give functionalized
pores. Pores obtained through the assembly of peptides ap-
pended to octaphenyl rods also place functional groups in the
synthesized, starting from the corresponding toluic acid isomer
4 in four steps. Sequential coupling and deprotection steps
were subsequently carried out to afford peptides 1a and b.
Transmission electron microscopy (TEM) images obtained
after incubating the peptides in solution for 12 h, indicated
that peptides 1a and b aggregate to form bundles of nanofib-
ers (Figure 2). The bundles with peptide 1b were found to be
slightly wider (28–38 nm) than those with peptide 1a (15–
30 nm).
[
3h,9]
pore.
Herein, we report octapeptides 1–3, containing (ami-
nomethyl)benzoic acid groups (Figure 1). We had previously in-
corporated aminobenzoic acid units into the peptide scaf-
[
10]
3
fold. In peptides 1-3, an sp center is incorporated in addi-
tion to the turn-inducing aminobenzoic acid unit to provide
conformational flexibility to the peptides. The sequence in
peptides 2 and 3 is found to be most active for transporting
The peptide 1a was also found to form a stable three-di-
mensional assembly in the solid state. The crystal packing of
peptide 1a showed a pore (4–5 Š wide) lined with carbonyl
[a] H. Behera, V. Ramkumar, Dr. N. Madhavan
Department of Chemistry
groups that held water molecules inside by hydrogen-bonding
Indian Institute of Technology, Madras
Chennai - 600036, Tamil Nadu (India)
E-mail: nanditam@iitm.ac.in
[12]
(
Figure 3a).
Four intramolecular hydrogen bonds stabilize
the folded structure of the peptide, while one intermolecular
bond stabilizes the assembly (Figure 3b). We found it interest-
ing that peptide 1a crystallized with trace amounts of water
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201500881.
Chem. Eur. J. 2015, 21, 10179 – 10184
10179
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
present in methanol to give such assemblies containing water-
filled pores. If the peptide structure in the lipid bilayer is similar
to its crystal structure, at least eight peptide units would be re-
quired to span the approximately 40 Š thick membrane (Fig-
ure 3c). While the crystal structure cannot be directly correlat-
ed to the peptide structure in the bilayer, a viable conclusion
from the microscopy as well as the crystal structure is that the
peptides have a tendency to self-assemble to form tubelike
structures.
Ion transport through the peptides was assessed using vesi-
cles entrapped with pH-sensitive 8-hydroxypyrene-1,3,6-trisul-
[13]
fonic acid trisodium salt (HPTS) dye at pH 7.2. Based on the
microscopy studies, peptide solutions were allowed to stand in
solution (to ensure self-assembly) for at least 12 h before the
experiment was conducted. The vesicles were incubated with
peptides for 1–2 min, following which 0.5n NaOH was added
to introduce a pH gradient of 0.6 units (Figure 4a). The fluores-
Scheme 1. General procedure for synthesis of peptide 1. a) SOCl
8C to RT, 5 h; b) NBS, AIBN, CH CN, 708C, 16 h; c) NaN , DMF, 808C, 14 h;
d) PPh , THF, H O, 18 h; e) BocNHdAlalAlaOH, HCTU, DIEA, CH Cl , 08C to
RT, 12 h; f) LiOH, MeOH, H O, 4 h; g) l-Ala-OMe, HCTU, DIEA, THF, DMF, 08C
to RT, 15 h; h) LiOH, MeOH, H O, 3 h; i) TFA, CH Cl , 08C to RT, 4 h; j) HCTU,
2
, MeOH,
0
3
3
3
2
2
2
2
2
2
2
DIEA, DMF, 08C to RT, 15 h (AIBN=azobisisobutyronitrile, Boc=tert-butoxy-
carbonyl, Ala=alanine, HCTU=O-(6-chlorobenzotriazol-1-yl)-N,N,N’,N’-tetra-
methyluronium hexafluorophosphate, DIEA=N,N-diisopropylethylamine,
TFA=trifluoroacetic acid).
À1
Figure 2. TEM images of peptides (0.5 mgmL ); left) 1a; right) 1b.
Figure 4. The HPTS assay for ion transport. Left) Schematic representation
À
(
channel mechanism). Right) Comparison of rate of change of HPTS con-
centration in the presence of peptides 1 (100 mm, 27 mol%) and DMSO.
À
cence intensity of deprotonated HPTS (HPTS ) was monitored
after addition of NaOH to determine transport activity of the
À
peptides. A gradual increase in HPTS concentration was ob-
served in the presence of peptides 1a and b. (Figure 4b). Be-
cause the experiment is carried out with a concentration gradi-
+
À
ent of Na and OH , this pH increase might be attributed to
+
À
Na entry into the vesicles, coupled with OH entry (symport),
+
À
À
or H exit (antiport) from the vesicles. An OH /Cl antiport
mechanism could also be operative. At the end of the experi-
ment a detergent Triton X was added to lyse the vesicles and
À
the final intensity obtained was used to normalise the HPTS
intensities (Figure 4b). The assay indicated that peptides 1a
and b were not that active. Dynamic light scattering (DLS)
studies were carried out to ascertain that peptides 1a and 1b
[11]
did not lyse the vesicles similar to Triton X.
Because peptides 1 were not very active, peptides 2a and
b that contain the sequence of our previously reported ami-
2
Figure 3. Top) Crystal packing for peptide 1a indicating water-filled pores.
Bottom left) Magnified image of peptide dimer illustrating the stabilizing in-
teractions. Bottom right) Model for pore formation by peptide 1a. Hydro-
gens omitted for clarity. Oxygens shown in black.
nobenzoic incorporated peptides were synthesized. Peptide 3,
which is the pyridyl analog of peptide 2a was also synthe-
[11]
sized. TEM and SEM were used to compare the aggregation
Chem. Eur. J. 2015, 21, 10179 – 10184
www.chemeurj.org
10180 ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
Figure 5. Left) TEM image of peptide 2a. Right) SEM image of peptide 3.
of peptides 2a and 3 (Figure 5). Bundles of nanofibers were
seen in both cases. Interestingly a twisting of the fibers to give
helical-type assemblies was observed for peptide 3 (Fig-
[11]
ure 5b).
The HPTS assay was carried out with peptides 2 and 3 and
the ion-transport rates were obtained by fitting the curves to
a first-order exponential equation. As seen from Table 1, pep-
tide 2a was 1.4 times more active than 1a and peptide 2b,
containing the p-substituted aromatic unit, was twice as active
as peptide 1b. Peptide 3 was found to be more active than its
benzyl analog (entry 5).
Figure 6. Lucigenin assay for halide transport: Top) Schematic representa-
tion. b) Fluorescence versus time plots with peptide 3 (50 mm, 13.5 mol%) in
the presence of bottom left) NaCl, bottom right) NaBr. The time at which
peptide was added has been considered as the beginning of the experiment
(
i.e., t=0 sec in the x axis).
Table 1. Comparison of activities of peptides 1–3 using the HPTS assay.
À3 À1 [c]
Entry
Peptide
k [”10 s ]
[
a]
1
2
3
4
5
1a
11.5Æ2.0
11.3Æ2.1
15.6Æ2.5
23.2Æ4.7
22.0Æ1.2
[
a]
1b
2a
2b
[
a]
[
a]
[
b]
3
[
a] 100 mm, 27 mol%. [b] 50 mm, 13.5 mol%. [c] Avg. value. Individual k
values calculated by fitting the curve using Origin 8.5.
To determine the anion selectivity of the most active pep-
tide 3, vesicles entrapped with halide sensitive lucigenin dye
[
14]
were prepared. The fluorescence of lucigenin dye is reported
to quench in the presence of halides. Aqueous solutions of
sodium chloride or bromide (2n) were added to the vesicles,
following which peptide solution was added (Figure 6a). The
fluorescence intensity of the dye was normalized based on the
final intensity obtained with Triton X. Quenching of lucigenin
fluorescence upon adding peptide 3 was found to be similar
to background transport (Figure 6b and c) indicating that pep-
tide-mediated halide transport was minimal.
2
3
Figure 7. The Na NMR assay. Top) Schematic representation. Bottom) k
versus concentration plot.
À
À
The lucigenin assay rules out an OH /Cl antiport mecha-
The k values were found to be directly proportional to the
concentration of peptide 3 (Figure 7b). In the assay, the pep-
tides were added after an incubation period of at least 12 h in
solution, to ensure peptide self-assembly. The microscopy stud-
ies show that the peptides assemble in this time period and
the crystal structure also shows water-filled pores. Therefore,
the linear correlation between the rate constant and the pep-
tide concentration can be explained by the formation of
a pore by a thermodynamically stable self-assembly of the
+
nism in the HPTS assay indicating that M transport might be
23
dominant (Figure 4a). To confirm cation transport, a Na NMR
[
15]
assay was carried out with peptide 3 (Figure 7a). A shift re-
agent was added to large vesicles (prepared in aqueous NaCl)
so that distinct peaks could be seen for the internal and exter-
+
+
nal Na ions. A broadening of the Na peaks was observed
+
upon adding peptide, which indicated Na exchange (Fig-
+
ure 7b). The rate constant for Na exchange was determined
[16]
using Equation (1), where np and n0 correspond to the line
peptides.
A monomolecular pore is ruled out because it
+
width of the internal Na peak in the presence and absence of
would be difficult for a single peptide to efficiently span the
lipid bilayer. A carrier mechanism is also unlikely as it would
not be very feasible for a peptide assembly to ferry back and
forth the lipid bilayer as a carrier.
peptide, respectively.
k ¼ pðn Àn Þ
ð1Þ
10181
p
0
Chem. Eur. J. 2015, 21, 10179 – 10184
www.chemeurj.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
2
3
The HPTS, Na NMR, and lucigenin experiments show that
our peptides prefer to transport cations over halides. To see
how the ion-transport activity varied with the concentration of
peptides in the HPTS assay (Figure 4a), the experiment was
carried out with varying concentrations of peptide 3 (Fig-
tides 2a and 3 indicated no difference in the emission intensi-
ty of HPTS in the presence of 2a and 3 (Figure S12 and S13
À
in the Supporting Information). The main difference between
peptides 2a and 3 is the presence of nitrogen atoms at the 2-
positions of the aromatic rings. Because these pores prefer to
transport cations over halides, the higher activity of peptide 3
alludes to the proximity of at least one of these electron-rich
nitrogens (i.e., aromatic rings) to the pore interior. Hence, the
aromatic ring(s) in peptide 2a can potentially be used for the
easy introduction of functional groups inside the pore.
[16a,c]
ure 8a).
The graph of fluorescence intensity (I) just before
Conclusion
In conclusion, acyclic octapeptides 1 containing (aminome-
thyl)benzoic acid units and alanine were developed and found
to self-assemble to form nanotubes stabilized by hydrogen-
bonding interactions. Ion-transport studies indicated that they
were not very active. Therefore second generation peptides 2
and 3 were synthesized. Peptides 2 and 3 were found to self-
assemble to form nanotubes and ion-transport studies showed
that they were more active than the first-generation peptides.
Figure 8. Hill analysis of peptide 3 Left) Fluorescence versus time plot with
variable concentrations of peptide 3. Right) Hill plot for peptide 3.
23
The HPTS, Na NMR, and lucigenin assays indicated that the
most active peptide 3 prefers to transport cations over halides.
Na NMR/HPTS assays with variable concentrations of peptide
addition of Triton X versus peptide concentration (c) was fitted
23
to Equation (2), that is, the Hill equation (Figure 8b, Table 2).
and microscopy studies indicate that these peptides form
pores through highly stable assemblies. Hill analysis also indi-
cated that peptide 3 (EC =28 mm) was the most active pep-
tide and was more active than its benzyl analog, that is, pep-
n
50
I ¼ I1 þ ððI ÀI Þ=ð1 þ ðc=EC Þ ÞÞ
ð2Þ
0
1
50
tide 2a (EC =51 mm). The higher activity of peptide 3 is at-
50
In Equation (2), the intensities “I ” and “I ” correspond to the
1
0
tributed to an increased pore electron density due to the pres-
ence of nitrogen atom(s) close to the pore. If indeed even
a single aromatic ring is proximal to the pore, one can envision
using it to functionalize the pore. Current efforts are focused
on determining the nature of the ion-transporting pore and
improving the activity of these pores through functionalization
of the aromatic units.
normalized fluorescence intensity with excess of peptide and
no peptide, respectively. The EC50 value corresponds to the
peptide concentration required to obtain half of the maximum
fluorescence intensity and “n” corresponds to the Hill coeffi-
cient or the number of peptides that come together to interact
with a single ion. The Hill coefficient was found to be close to
1
for peptide 3 (Table 2), corroborating the conclusion from
the NMR experiment that the pore was formed by a stable
[
16]
peptide assembly. The Hill analysis was carried out with pep-
tides 2a and 2b as well and, in all cases, the Hill coefficient
was found to be close to 1 (Table 2).
Experimental Section
Ion transport studies with peptides using the HPTS assay
The EC50 values obtained from the Hill analyses of peptides
[
17,13c]
Preparation of vesicles:
To a solution of EYPC lipids (28.4 mg,
2
a, 2b, and 3 (Table 2), show that peptide 2b is 1.4 times
more active than 2a, whereas peptide 3 is most active, that is,
.8 times more active than its benzyl analog 2a. The pyridyl
ring could, in principle, act as a base and deprotonate the
HPTS dye. Control experiments with the HPTS dye and pep-
3
4
5
6.9 mmol, 9 equiv) in chloroform (0.284 mL), cholesterol (1.6 mg,
.1 mmol, 1 equiv) was added and the solution was incubated for
min at 08C. Chloroform was removed by applying a stream of ni-
trogen gas. The resultant thin film was kept in vacuo for 5 h at
8C, following which 1 mL of HEPES buffer at pH 7.2 with HPTS
1
0
dye (0.1 mm HPTS, 100 mm NaCl, 10 mm HEPES) was added. The
resulting suspension was allowed to stir for 1 h at RT and then sub-
jected to eight freeze-thaw (liq. N and 408C) cycles. The vesicle
2
Table 2. Comparison of EC50 and n values for peptides 2 and 3
mixture was sonicated in a bath sonicator at 0–58C for a total time
of 2 min (30 s on and 30 s off in degass mode). The mixture was
extruded 40 times through 100 nm polycarbonate membrane
using a mini-extruder. The extra-vesicular dye was removed by
size-exclusion chromatography using Sephadex G-50 (eluent:
HEPES buffer at pH 7.2, 100 mm NaCl, 10 mm HEPES). The vesicle
solution was collected and the total volume was made up to
[
a,b]
[b]
Entry
Peptide
EC50 [mm]
n
1
2
3
2a
2b
3
51
36
28
1.3
1.41
1.04
[18]
[
a] 50 mm, 13.5 mol%. [b] Hill coefficient, obtained by fitting the Hill plots
to Equation (2) using Origin 8.5.
2
.5 mL with HEPES buffer at pH 7.2.
Chem. Eur. J. 2015, 21, 10179 – 10184
www.chemeurj.org
10182
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
[13c]
The HPTS assay:
Vesicle solution (75 mL) and HEPES buffer
Additional Information
(
2.9 mL) were placed in a cuvette. An appropriate amount of linear
octapeptide in DMSO (10 mL) was added to the cuvette and the so-
lution was allowed to stir for 30 s prior to the fluorescence experi-
ment. After 50 s from the start of the experiment, NaOH (20 mL, 0.5
N) was added and at 250 s aqueous Triton X (TX; 5%, 50 mL) was
added.
Crystal structure information: CCDC-1008786 (1a) contains the
supplementary crystallographic data for this paper. These data can
be obtained free of charge from The Cambridge Crystallographic
Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
Supplementary information: detailed procedures, characterization
of compounds, raw plots for assays, DLS data, and CD data has
been provided in the Supporting Information.
Lucigenin assay for halide transport
Preparation of vesicles: To a solution of dehydrogenated EYPC
Acknowledgements
(
18.8 mg) in chloroform was added cholesterol (1.2 mg). The lipid
solution was incubated for 5 min at 08C. Chloroform was removed
under a stream of nitrogen gas. The resultant thin film was kept in
vacuo for 4 h at 08C and rehydrated with 1 mL of lucigenin dye
This research was supported by DST (SR/S1/OC-41/2009), New
Delhi, India. H.B. thanks IIT Madras for fellowship. We thank Dr.
E. Prasad for the use of his DLS instrument and J. Mahita for
her contribution towards synthesis of peptide 1b. We thank
(
1.0 mm dye in 225 mm NaNO3 solution). The suspension was
swirled for 5–10 min and subsequently sonicated at 08C in a bath
sonicator for 2 min (30 s on and 30 s off in degass mode). The vesi-
23
Mr. Karthikeyan for help with the Na NMR experiments.
cle solution was subjected to eight freeze-thaw cycles (liq N and
2
4
08C) and extruded ten times through a 0.1 mm polycarbonate
membrane. The extravesicular dye was removed by size exclusion
chromatography using a G-50 sephadex column. The total collect-
ed volume was 2.0 mL.
Keywords: cation transport · ion transport · nanotube ·
peptides · pores
[
[
[
1] B. Hille, Ion Channels of Excitable Membranes, 3rd ed., Sinauer, Sunder-
land, MA, 2001.
2] D. A. Doyle, J. M. Cabral, R. A. Pfuetzner, A. Kuo, J. M. Gulbis, S. L. Cohen,
B. T. Chait, R. MacKinnon, Science 1998, 280, 69–77.
3] a) P. Reiß, U. Koert, Acc. Chem. Res. 2013, 46, 2773–2780; b) F. De Riccar-
dis, I. Izzo, D. Montesarchio, P. Tecilla, Acc. Chem. Res. 2013, 46, 2781–
2790; c) A. Vargas Jentzsch, A. Hennig, J. Mareda, S. Matile, Acc. Chem.
Res. 2013, 46, 2791–2800; d) G. W. Gokel, S. Negin, Acc. Chem. Res.
Lucigenin assay with peptide 3: Vesicle solution (100 mL) and
2
3
.9 mL of 225 mm NaNO buffer were placed in a cuvette. At 91 s,
3
6 mL of 2n NaX (X=Cl , Br ) and at 145 s an appropriate amount
À
À
of linear octapeptide 3 in 2.5% DMSO/MeOH (10 mL) was added to
the vesicle solution. Finally, at 450 s, aqueous Triton X (5%, 50 mL)
was added to the cuvette.
2
2
013, 46, 2824–2833; e) T. M. Fyles, Acc. Chem. Res. 2013, 46, 2847–
855; f) J. K. W. Chui, T. M. Fyles, Chem. Soc. Rev. 2012, 41, 148–175;
g) S. Matile, A. V. Jentzsch, J. Montenegro, A. Fin, Chem. Soc. Rev. 2011,
Ion transport study with peptide 3 using sodium NMR
4
6
0, 2453–2474; h) N. Sakai, J. Mareda, S. Matile, Mol. BioSyst. 2007, 3,
58–666; i) G. W. Gokel, I. A. Carasel, Chem. Soc. Rev. 2007, 36, 378–
[13c,17a]
Preparation of vesicles:
To
a
solution of EYPC lipids
389; j) U. Koert, J. R. Pfeifer, Synthesis 2004, 2004, 1129–1146.
(
(
28.4 mg, 36.9 mmol, 9 equiv) in chloroform (0.284 mL), cholesterol
1.6 mg, 4.1 mmol, 1 equiv) was added and the solution was incu-
[4] a) F. Xia, W. Guo, Y. Mao, X. Hou, J. Xue, H. Xia, L. Wang, Y. Song, H. Ji, Q.
Ouyang, Y. Wang, L. Jiang, J. Am. Chem. Soc. 2008, 130, 8345–8350;
b) E. C. Yusko, J. M. Johnson, S. Majd, P. Prangkio, R. C. Rollings, J. Li, J.
Yang, M. Mayer, Nat. Nanotechnol. 2011, 6, 253–260; c) V. Dartois, J.
Sanchez-Quesada, E. Cabezas, E. Chi, C. Dubbelde, C. Dunn, J. Granja, C.
Gritzen, D. Weinberger, M. R. Ghadiri, Antimicrob. Agents Chemother.
bated for 5 min at 08C. Chloroform was removed by applying
a stream of argon gas. The resultant thin film was kept in vacuo
for 5 h at 08C, following which it was rehydrated with 1 mL of
2
4
4
00 mm NaCl. The resulting suspension was allowed to stir for
2
005, 49, 3302–3310; d) R. S. Hector, M. S. Gin, Supramol. Chem. 2005,
5 min at RT and then subjected to eight freeze-thaw (liq. N and
2
17, 129–134; e) Q. H. Nguyen, M. Ali, R. Neumann, W. Ensinger, Sens. Ac-
tuators B 2012, 162, 216–222; f) N. Sorde, G. Das, S. Matile, Proc. Natl.
Acad. Sci. USA 2003, 100, 11964–11969.
08C) cycles. The vesicle mixture was sonicated in a bath sonicator
at 0–58C for a total time of 2 min (30 s on and 30 s off in degass
mode). The mixture was extruded 15 times through a 400 nm
membrane to give the large unilamellar vesicle (LUV) solution.
The vesicle solution and sample prepared for NMR study were
stored in 1.5 mL Eppendorf vials.
[5] a) H. Bayley, P. S. Cremer, Nature 2001, 413, 226–230; b) J. Clarke, H.-C.
Wu, L. Jayasinghe, A. Patel, S. Reid, H. Bayley, Nat. Nanotechnol. 2009, 4,
[18]
2
65–270; c) N. Ashkenasy, J. Sµnchez-Quesada, H. Bayley, M. R. Ghadiri,
Angew. Chem. Int. Ed. 2005, 44, 1401–1404; Angew. Chem. 2005, 117,
425–1428.
1
[
6] D. Wang, L. Guo, J. Zhang, L. R. Jones, Z. Chen, C. Pritchard, R. W.
Roeske, J. Pept. Res. 2001, 57, 301–306.
Preparation of shift reagent: The shift reagent was prepared by
mixing aqueous DyCl (1 mL, 0.1m) with aqueous sodium-tris(poly-
[7] R. Hourani, C. Zhang, R. van der Weegen, L. Ruiz, C. Li, S. Keten, B. A.
3
phosphate) solution (2 mL, 0.2m).
Helms, T. Xu, J. Am. Chem. Soc. 2011, 133, 15296–15299.
[
[
8] C. Reiriz, M. Amorín, R. García-FandiÇo, L. Castedo, J. R. Granja, Org.
Biomol. Chem. 2009, 7, 4358–4361.
9] a) A. Som, S. Matile, Eur. J. Org. Chem. 2002, 2002, 3874–3883; b) B.
Baumeister, N. Sakai, S. Matile, Angew. Chem. Int. Ed. 2000, 39, 1955–
NMR experiment: The LUV Vesicle solution (180 mL) was placed in
a vial, following which D O (100 mL) and shift reagent (100 mL)
2
were added. The solution was incubated at room temperature for
1958; Angew. Chem. 2000, 112, 2031–2034; c) S. Matile, Chem. Soc. Rev.
4
0 min. An appropriate amount of octapeptide 3 in DMSO (10 mL)
2
001, 30, 158–167.
was added to the vial and the solution was transferred to the NMR
tubes. For the control experiment, the same procedure was used
except DMSO (10 mL) was added instead of peptide 3.
[
10] a) B. P. Benke, N. Madhavan, Chem. Commun. 2013, 49, 7340–7342;
b) B. P. Benke, N. Madhavan, Bioorg. Med. Chem. 2015, 23, 1413–1420.
[11] See the Supporting Information for details.
10183 ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2015, 21, 10179 – 10184
www.chemeurj.org

Full Paper
[
12] The structure was solved in the space group C2 with R factor of 4.09%.
[16] a) S. Bhosale, S. Matile, Chirality 2006, 18, 849–856; b) T. Saha, S. Dasari,
D. Tewari, A. Prathap, K. M. Sureshan, A. K. Bera, A. Mukherjee, P. Taluk-
dar, J. Am. Chem. Soc. 2014, 136, 14128–14135; c) A. Vargas Jentzsch, S.
Matile, J. Am. Chem. Soc. 2013, 135, 5302–5303.
[17] a) C. De Cola, S. Licen, D. Comegna, E. Cafaro, G. Bifulco, I. Izzo, P. Tecilla,
F. De Riccardis, Org. Biomol. Chem. 2009, 7, 2851–2854; b) T. M. Fyles,
C.-W. Hu, H. Luong, J. Org. Chem. 2006, 71, 8545–8551.
The unit cell parameters are: a 26.576(4), b 9.7095(11), c 18.779(4). Z=
3
2
, Z’=0, V=4503.67 Š . CCDC-1008786 (1a) contains the supplementa-
ry crystallographic data for this paper. These data can be obtained free
of charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
[
13] a) K. Kano, J. H. Fendler, Biochim. Biophys. Acta Biomembr. 1978, 509,
2
1
89–299; b) N. R. Clement, J. M. Gould, Biochemistry 1981, 20, 1534–
538; c) N. Madhavan, E. C. Robert, M. S. Gin, Angew. Chem. Int. Ed.
[18] http://www.avantilipids.com/.
2005, 44, 7584–7587; Angew. Chem. 2005, 117, 7756–7759.
[14] B. A. McNally, A. V. Koulov, B. D. Smith, J.-B. Joos, A. P. Davis, Chem.
Commun. 2005, 1087–1089.
[15] a) F. G. Riddell, M. K. Hayer, Biochim. Biophys. Acta Biomembr. 1985, 817,
313–317; b) D. Milano, B. Benedetti, M. Boccalon, A. Brugnara, E. Iengo,
Received: March 5, 2015
P. Tecilla, Chem. Commun. 2014, 50, 9157–9160.
Published online on June 3, 2015
Chem. Eur. J. 2015, 21, 10179 – 10184
www.chemeurj.org
10184
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim