Active ion transporters from readily accessible acyclic octapeptides containing 3-aminobenzoic acid and alanine

Article abstract of DOI:10.1039/c3cc44224a

Ion transporters have been developed from acyclic octapeptides comprising l/d alanine and m-aminobenzoic acid. These octapeptides transport cations up to 3 times faster than their cyclic analog through lipid vesicles. Preliminary CD, XRD and computational

Full text of DOI:10.1039/c3cc44224a

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Active ion transporters from readily accessible acyclic
octapeptides containing 3-aminobenzoic acid
and alanine†
Cite this: Chem. Commun., 2013,
49, 7340
Received 5th June 2013,
Accepted 24th June 2013
Bahiru P. Benke and Nandita Madhavan*
DOI: 10.1039/c3cc44224a
www.rsc.org/chemcomm
Ion transporters have been developed from acyclic octapeptides com-
prising L/D alanine and m-aminobenzoic acid. These octapeptides
transport cations up to 3 times faster than their cyclic analog through
lipid vesicles. Preliminary CD, XRD and computational studies allude to
a 6–9 Å wide tetrameric ion channel as the active ion transporter.
Transmembrane ion transporters play a key role in maintaining
the cell volume, regulating ion flow across cell membranes and
establishing a resting membrane potential in the body.1 Synthetic
ion transporters that mimic nature’s efficient as well as selective ion
conductance are attractive for use as molecular machines2 and
sensors.3–5 For in vivo and in vitro applications it is desirable to
derive ion transporters from non-toxic oligopeptides. Several peptide
derived ion transporters have been developed that exploit
the amphiphilicity, intra- or intermolecular hydrogen bonding in
peptides.6–12 A simplistic channel design utilizing small oligo-
peptides derived from alternating D and L-amino acids, similar to
the highly active natural ion channel Gramicidin A,13 remains largely
unexplored. Oligopeptides that mimic the structure and/or activity of
Gramicidin have been developed by Koert14–16 and Inoue.17,18
Among the smallest and most active oligopeptide ion transporters
comprising alternating D and L-amino acids are the cyclic
octapeptides initially reported by Ghadiri and co-workers.19,20
However, the cyclization and purification of these peptides is
non-trivial. Herein, we present ion transporters from acyclic
octapeptides 1 and 2 containing L and D alanine and 3-aminobenzoic
acid units that can be readily synthesized and transport cations even
more efficiently than their cyclic analog 3 (Chart 1).
Chart 1 Octapeptide ion transporters.
m-aminobenzoic acid and L-alanine. Boc protected m-aminobenzoic
acid 4 and L-Ala-OMe were treated with HCTU and diisopropylethyl-
amine to form the dipeptide 5 in 94% yield (Scheme 1).21 Dipeptide 5
was deprotected using trifluoroacetic acid and reacted with Boc-L-
Ala-OH to give tripeptide 6 in 83% yield. Hydrolysis of tripeptide 6 and
subsequent reaction with D-Ala-OMe afforded tetrapeptide 7 in 89%
yield. Selective deprotection of tetrapeptide 7 resulted in tetrapeptides
8 and 9, which were coupled to give octapeptide 1 in 59% yield.
Octapeptide 2wasobtainedin89%yieldbyhydrolyzingoctapeptide1.
Cyclic peptide 3 was synthesized to compare the ion transport
The acyclic octapeptides comprise hydrophobic residues
and aromatic units in order to facilitate membrane insertion. The
aromatic groups also serve as turn inducing elements and could
potentially aid transmembrane ion transport similar to the tryptophan
units in Gramicidin. Octapeptide 1 was synthesized starting from
Department of Chemistry, Indian Institute of Technology Madras, Chennai – 600036,
Tamil Nadu, India. E-mail: nanditam@iitm.ac.in; Fax: +91-44-22574202
† Electronic supplementary information (ESI) available: Detailed experimental proce-
dures, characterization of compounds, analysis and fitting of fluorescence data, compu-
tational details, crystallographic data (CIF) of tetrapeptide 7 (CCDC 923387). For ESI and
crystallographic data in CIF or other electronic format see DOI: 10.1039/c3cc44224a
Scheme 1 Synthesis of octapeptides 1–3.
c
7340 Chem. Commun., 2013, 49, 7340--7342
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Fig. 1 TEM images of (a) cyclic peptide 3; (b) acyclic peptide 1.
efficiency of the acyclic octapeptide 1 with its cyclic analog. Peptide 3
was obtained in 22% yield by deprotecting octapeptide 1 and
cyclizing the resulting peptide in the presence of T3P.
Cyclic peptides containing a single 3-amino-2-methylbenzoic unit
with alternating D- and L-amino acids have been reported to form
pores.22 The pore forming ability of cyclic peptides containing two
3-aminobenzoic acid units has not been experimentally deter-
mined.23 Transmission electron microscopy (TEM) and atomic force
microscopy (AFM) images of cyclic peptide 3 showed nanotube
bundles similar to those obtained for the cyclic peptide containing
Fig. 2 (a) Schematic representation of the HPTS assay (channel mechanism);
(b) change in fluorescence intensity of the deprotonated HPTS dye upon adding
peptides 1–3; (c) DLS data for vesicles with peptide 1.
a single aromatic unit (Fig. 1a and ESI†). The presence of an extra Table 1 Comparison of cation transport rates through peptides 1–3
aromatic unit did not hamper self-assembly of 3 and gave 5–15 nm
S. no.
Peptide
MOH
k1 (sꢀ1
c
)
wide bundles, probably via the aggregation of 10–15 cyclic peptide 3
units.22 TEM images of the acyclic octapeptide 1 were similar to
those obtained for the cyclic peptide 3 excluding the fact that the
nanotube bundles were 10–20 nm wide. The wider nanotube
bundles could be attributed to either nanotubes with bigger dia-
meters or larger number of peptides in the aggregates (Fig. 1b).
The ion transport efficiency of octapeptide 1 was gauged using
fluorescence spectroscopy.24,25 Lipid vesicles of ca. 100 nm diameter
encapsulated by the pH sensitive 8-hydroxypyrene-1,3,6-trisulfonic
acid trisodium salt (HPTS) dye were prepared for the studies via
extrusion.26 A solution of the peptide in DMSO was added to the
vesicles, following which aqueous NaOH (0.5 N) was added to create a
pH gradient of 0.6 units (Fig. 2a). The ion transport activity of the
peptides was determined by measuring the steady increase in
fluorescence intensity of the deprotonated HPTS dye upon addition
of NaOH.27 This increase in fluorescence is attributed to an increase
in the concentration of the deprotonated dye inside vesicles due to
Na+/OHꢀ symport or Na+/H+ antiport via the peptides. Finally,
detergent Triton X was added to ensure equilibration of the HPTS
dye. Fluorescence studies showed an increase in the concentration of
deprotonated HPTS when peptides 3 and 1 were added (Fig. 2b),
indicating that the peptides were transporting ions. Control experi-
ments showed background ion transport, which was accelerated with
DMSO (k = 0.0157 sꢀ1). Therefore, actual transport rates through
peptides were determined by fitting the curves to eqn (1),21,28
1
2
3
4
5
6
7
8
3a
1a
1a
1a
2a
2b
2b
2b
NaOH
NaOH
KOH
LiOH
NaOH
NaOH
KOH
0.032d
0.064d
0.067d
0.067d
0.096d
0.084e
0.083e
0.084e
LiOH
a
b
c
27 mol%. 13.5 mol% with respect to lipid. Obtained by fitting the
curve to eqn (1) with Origin 8.5. k2 = 0.0157 sꢀ1
. .
k2 = 0.02 sꢀ1
d
e
be most active (3 times more active than cyclic peptide 3). To check
whether the acyclic peptides transported other cations, the counter
ion for OHꢀ was varied in the experiment. Peptides 1 and 2 were
found to transport a variety of alkali metal ions efficiently (entries 2–8
Table 1). Dynamic light scattering (DLS) studies were carried out on
the vesicles containing peptides to rule out the possibility of the
peptides acting as detergents. The DLS data showed that the vesicles
retained their integrity after addition of the peptide, indicating that
the peptides were not lysing the vesicles (Fig. 2c).
Circular dichroism (CD) was used to determine the solution
conformation of peptide 1. The CD spectrum showed a positive
peak at 196 nm and a negative peak at 213 nm (Fig. 3a). Analysis
of the spectrum indicated a majority of b-sheet character for
peptide 1.29 Furthermore, the CD spectrum of acyclic peptide 1
was similar to cyclic peptide 3 which is presumed to have b-sheet
character. Obtaining the solid state conformation of peptide 1
was challenging as it aggregated to form micro crystals. Hence,
the crystal structure of tetrapeptide 7 was used to gain insights
into the structure of peptide 1 (Fig. 3b).30 The average c and j
dihedral angles of the amino acids C5 and C16 in peptide 7 are
1421 and ꢀ1001, respectively, characteristic of b-sheets.21
Basic computational studies were carried out to gain insights into
the conformation of peptide 1. BasedontheCDandtheXRDdata, the
beta sheet dihedral angles of amino acids were used as a starting point
to obtain the equilibrium geometry of peptide 1 using MMFF/AM1
Rate = Aeꢀk t + Beꢀk t + C
(1)
1
2
where k1 corresponds to the peptide transport rate and k2 corre-
sponds to DMSO transport. The k2 value in eqn (1) was determined by
the control experiment and kept fixed while fitting the data.21 Acyclic
peptide 1 was found to be twice as active as cyclic peptide 3 (Table 1),
making it an attractive new scaffold for ion channels. However, high
concentrations of peptides (27 mol%) were required to observe
activity due to the large background transport. The deprotected
acyclic peptide 2 containing the free carboxyl group was found to
c
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could also be explained by this model as the pore size is wider than
that for cyclic peptides (B7 Å). However, a carrier mechanism, though
unlikely cannot be ruled out.
In conclusion, a new class of readily accessible ion transpor-
ters has been derived from acyclic octapeptides containing
L/D alanine and m-aminobenzoic acid. These peptides are more
efficient than their cyclic analog in terms of synthesis and ion
transport activity, making them extremely attractive for develop-
ment of novel materials. CD, XRD and computational studies
allude to ion mediation via a tetrameric pore. This model
explains the higher transport activity, wider nanotube bundle
formation and lower cation selectivity by the acyclic peptides as
compared to their cyclic analog. Efforts are underway for enhan-
cing and modulating ion transport through these peptides.
This research was supported by DST (SR/S1/OC-41/2009),
New Delhi, India. B.P.B acknowledges CSIR, India for a research
fellowship. We thank Mr V. Ramkumar for X-Ray crystallo-
graphic analysis and Dr E. Prasad for the use of his DLS
instrument. We thank SAIF and the Dept. of Metallurgical and
Materials Engineering I.I.T. Madras for SEM and TEM images.
Fig. 3 (a) CD spectrum of peptide 1 (0.25 mM in methanol); (b) ORTEP diagram
of tetrapeptide 7.
computations.21 Frequency calculations were carried out subse-
quently to confirm minima.31 Peptide 1 was found to be S-shaped
(C-shaped from top), indicating that two peptides could align sideways
to form a pore. Such a dimer would present the alanine methyl groups
on the surface and facilitate membrane insertion as well as stabilize
the channel within the membrane. The most active peptide 2 has
polar carboxy groups which would presumably face the water–channel
interface. Therefore, to get basic insights into the self-assembly of
peptide 1, the equilibrium geometry of the laterally aligned peptide
dimer having the carboxy termini onthesamesidewasdeterminedby
AM1 calculations (Fig. 4a and b).21 The optimization resulted in a
dimeric pore which was 6–9 Å wide and B20 Å long. One can envision
two such dimers aligning in a head to head fashion to span the lipid
bilayer which has a thickness of B40 Å (Fig. 4c). The assembly would
place 8 aromatic units in the channel scaffold similar to Gramicidin A.
The model explains the enhanced activity of the acyclic peptides as
compared to the cyclic peptide as the self-assembly is curtailed to half
of that required for the cyclic peptides.19 Such an assembly could also
explain the enhanced activity of peptide 2 where all four carboxy
groups would be at the peptide–water interface. The non-selective ion
transport in the HPTS assay and thicker bundles seen in TEM images
Notes and references
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27 The steady increase in fluorescence is preceded by an initial rapid
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29 Deconvoluted using the CDNN 2.1 software.
30 The structure was solved in the space group P21 with an R factor of
8.36%. The unit cell parameters are a = 12.639 (2)0, b = 10.4737(16)0,
c = 18.814(3) Å and b = 90.117(1). Z = 20, V = 2490.54(68) Å3. CCDC
923387 contains crystallographic data for peptide 7.
31 Geometry optimization was done using the PC SPARTAN PRO Semi-
empirical program 6.0.6.
Fig. 4 Equilibrium geometry of peptide 1 dimer (a) front view; (b) top view;
(c) schematic representation of the ion channel derived from peptide 1.
c
7342 Chem. Commun., 2013, 49, 7340--7342
This journal is The Royal Society of Chemistry 2013