& Ion Channels Cation-Transporting Peptides: Scaffolds for Functionalized Pores? Harekrushna Behera, Venkatachalam Ramkumar, and Nandita Madhavan* [a] 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 membranes play a vital role in cellular processes. [1] A selectivity filter inside these pores has been shown to dictate its ion pref- erence. [1, 2] Robust synthetic ion channels have been developed that mimic the activity of natural proteins. [3] Synthetic pore- forming compounds have found application as antibacterial drugs, molecular switches, catalysts, and sensors. [4] 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- cies, and DNA. [5] 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. There are few examples of synthetic peptide-based internally functionalized pores. Cyclic peptides containing a repeating llld [6] or an ld amino acid sequence with aromatic [7] or tetra- hydrofuran [8] 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 pore. [3h, 9] 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- fold. [10] In peptides 1-3, an sp 3 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 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. Results and Discussion Octapeptides 1a and 1b, containing m- and p-substituted aro- matic units, were synthesized in solution as shown in Scheme 1. [11] The (methylamino)benzoic acid derivative 5 was 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). 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  wide) lined with carbonyl groups that held water molecules inside by hydrogen-bonding (Figure 3 a). [12] Four intramolecular hydrogen bonds stabilize the folded structure of the peptide, while one intermolecular bond stabilizes the assembly (Figure 3 b). We found it interest- ing that peptide 1a crystallized with trace amounts of water Figure 1. Proposed scaffolds for functionalized pores. [a] H. Behera, V. Ramkumar, Dr. N. Madhavan Department of Chemistry Indian Institute of Technology, Madras Chennai - 600036, Tamil Nadu (India) E-mail : nanditam@iitm.ac.in Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/chem.201500881. Chem. Eur. J. 2015, 21,1–7  2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1 && These are not the final page numbers! ÞÞ Full Paper DOI: 10.1002/chem.201500881