Aminobenzoic acid incorporated octapeptides for cation transport

Bioorganic & Medicinal Chemistry 23 (2015) 1413–1420

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Aminobenzoic acid incorporated octapeptides for cation transport Bahiru P. Benke, Nandita Madhavan ⇑ Department of Chemistry, Indian Institute of Technology Madras, Chennai 600036, India

a r t i c l e

i n f o

Article history: Received 4 December 2014 Revised 16 February 2015 Accepted 17 February 2015 Available online 24 February 2015 Keywords: Peptides Pores Ion transporters Carriers Channel

a b s t r a c t Robust oligopeptides that mimic natural ion channels are attractive for use as molecular switches or model systems to study ion transport. Herein, we report octapeptides derived from aminobenzoic acid and L/D amino acids. Two of the alanine containing peptides were found to be most active and the peptide containing p-aminobenzoic acid was found to be most active (2.4 times its m-analog). Experimental studies indicate the peptides do not transport halides and transport alkali metal ions. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Proteins that selectively transport ions across cell membranes are essential for controlling cell volume/pH and regulating cellular signal transduction.1 Altered functioning of these proteins is known to be associated with diseases such as cancer, cystic fibrosis and cardiac arrhythmia.1–3 The limitations associated with the stability and characterization of large proteins, inspires researchers to develop oligopeptides capable of ion-selective membrane transport.4–6 Non-toxic low molecular weight peptidic ion transporters are potentially useful for pharmaceutical applications as well as attractive model systems for gaining mechanistic insights into membrane transport through larger proteins. A variety of the small oligopeptide ion transporters are derived from cyclic peptides comprising alternating D- and L-amino acids.7,8 These peptides have been used as sensors9,10 and antibacterial agents.11,12 Cyclic peptides with b or c amino acids have also been reported.13–15 Acyclic peptides containing –Gly3-Pro-Gly3– units which are readily accessible in comparison to cyclic peptides have been developed and reported to be anion-selective.16–18 Acyclic pentadecapeptide Gramicidin A comprising D- and L-amino acids is known to specifically transport alkali metal cations via formation of a dimeric pore in the cell membrane.19 The Gramicidin A channel does not allow transport of anions such as halides. The membrane affinity of Gramicidin A is attributed to the presence of the multiple tryptophan units in the peptide backbone. We have recently demonstrated that acyclic octapeptides synthesized from ⇑ Corresponding author. Tel.: +91 44 22574239; fax: +91 44 22574202. E-mail address: [email protected] (N. Madhavan). http://dx.doi.org/10.1016/j.bmc.2015.02.031 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

L/D

alanine and m-aminobenzoic acid (ABA) transport ions across the lipid bilayer more efficiently than their cyclic analog.20 Herein, we report the ion transport activity of octapeptides incorporated with aromatic aminobenzoic acid units (Fig. 1). Octapeptide 2 containing p-aminobenzoic acid and alanine is found to be most active. Vesicle-based assays indicate that the most active octapeptide 2 preferentially transports alkali metal ions and not halides. 2. Results and discussion Octapeptides 1–5 contain two aminobenzoic acid units in a scaffold of alternating L- and D-amino acids. The octapeptides vary in the position of the aromatic units, the substitution pattern of the aromatic units or the nature of the L-amino acids in the scaffold. Octapeptide 1 was synthesized as reported earlier.20 Peptide 2 was synthesized in solution as outlined in Scheme 1. Peptides 3–5 were also synthesized in solution, following a slightly modified synthetic route.21 To study ion transport, pH sensitive 8-hydroxypyrene-1,3, 6-trisulfonic acid, trisodium salt (HPTS) dye was encapsulated in vesicles.22,23 The vesicles were prepared in HEPES/NaCl buffer at pH 7.2 by multiple extrusions through a 0.1 lM polycarbonate membrane and external dye was removed by size exclusion chromatography.24 At the beginning of the experiment, peptide dissolved in DMSO was added to the vesicles, following which NaOH (0.5 N) was added to introduce a pH gradient of 0.6 units (Fig. 2a). HPTS has different excitation maxima in its protonated (HPTSOH) and deprotonated state (HPTSO). To gauge ion

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O BocHN

N H

Me

O

H N O

Me

O

Me

O

N H

BocHN

N H

Me

O

H N

O 1

Me

O

H N

O

Me

O

H N

N H

O

Me N H

O

Me

H N H N

O O

H N

O

Me N H

COOMe

H N

O

COOH

O

Me N H

Me H N

N H

O

NHBoc

7 O

f

d,e 82%

CH3 HN

Me COOMe

COOMe CH3

COOMe

N H

O

H N

O

b,c 55%

NHBoc

6

O

COOMe CH3

a 75%

Me N H

O

H N

N H

2

O BocHN

Me N H

NHBoc O 8 H N

O

CH3

99%

CH3 OH

N H

O

3 O BocHN

O

H N

N H

Me N H

O

H N

O

NH

NH

O

O

H N

N H

Me N H

O

O

COOMe

BocHN O

O

Me

Me N H

O

H N

Me

N H

H N O

O

Me

Me N H

CH3 N H

NHBoc

O

NHBoc O 10 + H N

h 45% O

O H N

O

COOMe

9

CH3

g 98%

Me

5

2

CH 3 N H

COOMe

CH3 HN

Figure 1. Octapeptides containing ABA units synthesized.

NH 2 O 11

transport, the emission of HPTSO was monitored over the course of the experiment. Upon adding NaOH, a gradual increase in HPTSO concentration was observed with peptides 1–5 (Fig. 2b). An increase in the concentration of HPTSO indicates an increase in the internal pH of the vesicles upon addition of NaOH. Such an increase could be attributed to peptide mediated transport of Na+ ions into the vesicles, which would lead to OH co-transport (symport) or exit of H+ ions (antiport) from vesicles to maintain charge neutrality. Alternatively an increase in the internal pH could be due to peptide mediated OH transport into vesicles, which would lead to Na+ symport or Cl antiport. The fluorescence intensity of HPTSO in Figure 2b has been normalized with the final equilibrated intensity of the dye obtained upon lysing the vesicles using the non-ionic detergent Triton X. The HPTS assay indicated that all the peptides were transporting ions across the lipid bilayer. However, the rate of transport varied depending on the nature of the peptide. The ion transport rates were quantified by fitting the curves to Eq. 1, where k, t, and I correspond to the rate constant, time and intensity of HPTSO, respectively.

I ¼ Aekt þ B

CH3 HN

COOMe

CH3 HN

O

O

CH3

NH

NH 4

H N

H N

ð1Þ

Upon comparing the average k values over multiple experiments (Table 1), we determined that the sequence, nature of amino acids and aromatic substitution pattern of the peptides play a significant role in determining its activity.25 Peptide 1 was found to be 1.5 times more active than peptide 5 which varies in the sequence of amino acids. Upon comparing peptides that vary only in the nature of the L-amino acids, it was observed that peptide 1 containing alanine was 1.2 and 1.5 times more active than its Leu and Trp analogs, respectively. Peptide 2 containing p-aminobenzoic acid was found to be the most active, its activity being 2.4 times more than its m-analog 1. A variety of natural and synthetic ion channels are known to possess Trp and Leu side chains to promote membrane insertion. It was surprising to observe that presence of these amino acids in our octapeptide scaffold lowered its activity (Table 1). Upon studying the effect of peptide on vesicle stability using dynamic light scattering (DLS), we observed an increase in vesicle size and dispersity after adding peptides 3/4 in contrast to the other

Scheme 1. Synthesis of peptide 2. Reagents and conditions: (a) L-Ala-OMe, HBTU, HOBT, CH2Cl2, DIEA, 15 h, 0 °C–rt; (b) TFA, CH2Cl2, 2 h, 0 °C–rt; (c) Boc-L-Ala-OH, HBTU, HOBT, CH2Cl2, DIEA, 24 h, 0 °C–rt; (d) LiOH, H2O, MeOH, 4 h, rt; (e) D-AlaOMe, HBTU, HOBT, CH2Cl2, DIEA, 10 h, 0 °C–rt, (f) LiOH, H2O, THF, 3 h, rt; (g) TFA, DCM, 0 °C–rt, 3 h. (h) HCTU, DIEA, THF, 8 h, 0 °C–rt.

peptides 1, 2 and 5 (Figs. 3, S7 and S8).26 The effect was more pronounced with peptide 4, indicating that these peptides, might be affecting the structural integrity of vesicles and the larger aggregates might be due to vesicle fusion (Fig. 3b and c). Therefore, the availability of peptides for ion transport might be less leading to their lower activity. Such a disruption of vesicles was not observed for peptides 1, 2 and 5 indicating that these peptides were not affecting the structural integrity of the vesicles and also not lysing the vesicles. Furthermore, the lipid solutions were found to turn turbid with peptides 3 and 4, which was not the case with peptides 1, 2 and 5. In order to gain insights into the mode of ion transport by the most active peptides 1 and 2, the HPTS assay was carried out with different concentrations of these peptides (Fig. 4a and b). The observed k values for each concentration were determined by fitting the plot to Eq. 1. The k values were plotted against peptide concentration for these peptides (Fig. 4c and d). Eq. 2 illustrates the relationship between the observed k values and peptide concentration.27 In this equation, n corresponds to the number of peptide units that interact with a single ion and K corresponds to the equilibrium for peptide aggregate–monomer dissociation. Since a background transport is observed with DMSO, the dependence of k with concentration is given by Eq. 3, where k0 corresponds to rate constant with DMSO.27

k / K½peptide

n

ð2Þ n

k ¼ kp K½peptide þ k0

ð3Þ

The plots (Fig. 4c and d) were fitted using Eq. 3 and the n values were obtained. The n values were found to be close to 1 for peptides 1 and 2.

B. P. Benke, N. Madhavan / Bioorg. Med. Chem. 23 (2015) 1413–1420

(a)

1415

(b)

1) t=0s

2) 0.5 N NaOH t = 50 s

Na+ OHsymport

+

Na

H+ antiport

Triton-X t = 250 s Vesicles lysed = HPTSO= HPTSOH Figure 2. (a) Schematic representation of HPTS assay (channel mechanism). (b) Fluorescence versus time plot comparing response of peptides 1–5 (16.6 lM, which corresponds to 4.5 mol % with respect to lipid).

Y ¼ Y 1 þ ððY 0  Y 1 Þ=ð1 þ ðconcn=EC50 Þp Þ

Table 1 Comparison of k values for all octapeptides No.

Peptidea

kb (103 s1)

1 2 3 4 5

1 2 3 4 5

18.6 ± 1.4 45.4 ± 7.5 15.1 ± 1.3 11.1 ± 1.4 12.4 ± 1.3

a

16.6 lM. Average value. k value for each experiment calculated by fitting the respective curve to Eq. 1 using Origin 8.1 software. b

The n value of 1 is often associated with ion transport through monomolecular pores, pores formed by a stable peptide assembly and a carrier mechanism. Our computational studies with peptide 120 indicated that the length of the peptide is 20 Å, which is too small to span the lipid bilayer. Therefore, formation of a monomolecular pore will be difficult. Microscopy studies with peptide 1 also showed that it self-assembles to form nanotubes.20 The TEM image of octapeptide 2 indicated that it also self-assembles to form bundles of nanotubes (Fig. 5). We believe, it would be difficult for such peptide assemblies to transport ions back and forth across the lipid bilayer like a carrier. Thermodynamically stable assemblies have been reported to show a linear dependence of rate constants on the concentration of the ion transporter, that is, an n value of 1.28,29 Based on the TEM images and our earlier computational studies20 we hypothesize that peptides 1 and 2 self-assemble to form pores that transport ions across the lipid bilayer. The CD data of peptides 1–5 show peaks that typically correspond to a b-sheet type structures which also support self-assembly. The assemblies are presumably highly stable, which is why they show a linear dependence to peptide concentration.28,29 However, more studies will be required to confirm the formation of the pore as well as to determine its precise nature. The TEM image of peptide 3 and AFM image of peptide 5 also show that these peptides also forms fibre/tubular type of structure.24 Hill analysis was also carried out for Figure 4a and b, where the final normalized intensity for each concentration (before addition of the detergent) was plotted against the concentration.28,29 The data was fit to Eq. 4, where Y corresponds to the normalized activity obtained just before addition of Triton X, Y1 value corresponds to the Y value obtained with large excess of the peptide, Y0 corresponds to the Y value obtained when no peptide was added (DMSO value). The EC50 value corresponds to the concentration to get half of the final activity and p corresponds to the Hill coefficient.24

ð4Þ

Upon fitting the data to Eq. 4, the EC50 values corroborated our earlier comparison using the pseudo first order rates that peptide 2 was more active than peptide 1 (Table 2). The Hill coefficient also indicated that these peptides might be forming pore via thermodynamically stable assemblies. In order to determine the cation-selectivity of peptide 2 the HPTS assay was modified where instead of NaOH, various alkali metal hydroxides were added (Fig. 6a).30a–c The assay indicated that the rate of ion transport did not vary with the nature of the alkali metal ions (Fig. 6b). The transport processes that could account for internal pH increase are Na+/OH (symport), Na+/H+ (antiport), OH/Cl (antiport). If the first two processes are dominant, the results from this assay would indicate that the peptide transports all alkali metal ions equally well. If the anion transport is dominant, the results would indicate that the peptide has a preference for anions. To determine whether peptide 2 allows halide transport, a fluorescence assay was carried out with vesicles entrapped with halide sensitive lucigenin dye (Fig. 7a).31,32 Lucigenin fluorescence is known to quench in the presence of halides. A solution of peptide in methanol was added to the vesicles, following which sodium halide (0.5 N) was added. The fluorescence of lucigenin was monitored over time and no change was observed upon adding peptide (Fig. 7b and c).33 Based on the lucigenin assay, we can hypothesize that the results from the HPTS assay indicate that peptide 2 transports all alkali metal ions efficiently (as the OH/Cl antiport might not be operative). To further confirm cation transport, a 23Na NMR assay was carried out with the most active peptide 2.34a In the assay, vesicles are prepared in NaCl and a shift reagent is added outside the vesicles to distinguish the external Na peaks from the internal peaks. Sodium exchange was determined by observing an increase in the line broadening of the Na peaks. We observed an increase in the line width of the internal Na peak in the presence of peptide 2 indicating that it was indeed transporting Na+ ions.24 In conclusion, octapeptides derived by incorporating aminobenzoic acid units in a scaffold of alternating L- and D-amino acids have been developed. Their ion transport ability was gauged using vesicles as cell models and fluorescence spectroscopy as a tool. It was observed that the nature of L-amino acids, sequence and the aromatic substitution pattern had a significant effect on the rate of ion transport. Peptides with alanine were found to be most active. Among the active peptides, it was found that p-substitution at the aromatic ring led to a 2.4 fold rate enhancement. Concentration

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Figure 3. DLS data showing effect of octapeptides (a) 2, (b) 3 and (c) 4 on vesicles.

dependence, CD and microscopy studies indicated that the most active octapeptides might be transporting ions via pores formed by thermodynamically stable peptide assemblies. The ion-selectivity through peptide 2 was probed using vesicle based assays. The studies indicated that peptide 2 does not transport halides and transports alkali metal ions. Efforts are in progress to gain further insights into the mode of ion transport through these octapeptides and regulate their transport properties. 3. Experimental section 3.1. General methods All reagents for synthesis were purchased from commercial suppliers and used without further purification unless stated otherwise. All air-sensitive reactions were performed using oven-dried glassware under an inert atmosphere of nitrogen. Syringe or cannula was used to transfer air-sensitive solvents and solutions. Tetrahydrofuran (THF) was distilled from sodium benzophenone ketyl; methanol was distilled from magnesium methoxide; N,N-diisopropylethylamine (DIEA) and dichloromethane were distilled from calcium hydride and N,N-dimethylformamide (DMF) was dried over 4 Å molecular sieves. All dry solvents were stored over 4 Å molecular sieves prior to use. All peptides were synthesized in solution using O-(6-Chlorobenzotriazol-1-yl)-N,N,N0 ,N0 -teramethyluronium hexafluorophosphate (HCTU) or O-(Benzotriazol-1yl)-N,N,N0 ,N0 -tetramethyluronium hexafluorophosphate (HBTU) as

coupling reagents. Hydrogenated egg yolk phosphatidylcholine (EYPC), 8-hydroxypyrene-1,3,6 trisulfonic acid, trisodium salt (HPTS) and lucigenin dye were purchased from Sigma–Aldrich and dehydrogenated EYPC in chloroform was purchased from Avanti Polar Lipids, Inc. (USA) and stored at 20 °C. All 1H and 13C NMR spectra were recorded on Bruker 400 or Bruker 500 spectrometers using CDCl3 or DMSO-d6 as solvent. Solvent peaks were used as standard reference peaks for the NMR spectra. Mass spectra (HRMS) were recorded on the MICRO-Q-TOF mass spectrometer using the ESI technique. JASCO FT/IR-4100 spectrometer was used to record the IR spectra. KBr pellets of the compounds were used for recording IR spectra. IR spectra peaks are reported in wavenumbers (cm1) as strong (s), medium (m), weak (w), and broad (br). Vesicles were prepared using the Mini extruder from Avanti Polar Lipids. Size exclusion chromatography was carried out using Sephadex (G-50) resin. Fluorescence spectra were recorded on a JASCO FP-6300 fluorescence spectrofluorometer using 3 mL quartz cuvettes. A small magnetic stir plate was placed beside the cuvette holder to ensure that the cuvette solutions could be stirred. Dynamic light scattering data was recorded on a Malvern Zetasizer ZS instrument using 3 mL quartz cells. CD spectra were obtained using a Chirascan photophysics instrument. 250 lL quartz cuvettes were used for CD measurement. Waters semi-preparative RP-HPLC (Reverse Phase High Performance Liquid Chromatography) was used for the purification of octapeptides. Water (0.1% TFA), acetonitrile (0.1% TFA) and methanol (0.1% TFA) were used as mobile phase solvents. The flow rate used for semi-preparative RP-HPLC

B. P. Benke, N. Madhavan / Bioorg. Med. Chem. 23 (2015) 1413–1420

1417

Figure 4. Intensity versus time plots for different concentrations of peptides (a) 1, (b) 2. k versus concentration plots for peptides (c) 1 and (d) 2.

Figure 5. TEM image of peptide 2.

Table 2 Comparison of EC50 and Hill coefficient values of peptides 1 and 2

a b

No.

Peptide

EC50a (lM)

pb

1 2

1 2

8.79 2.45

0.90 0.94

Obtained by Hill analysis. 2.45 lM corresponds to 0.66 mol %. Hill coefficient.

was 4.1 mL/min. The peptides were injected at a concentration of 10 mg/mL. Peptide elution was monitored at 220 nm and 280 nm with the Waters 2489 UV/visible detector.

Dipeptide 7:20,35 DIEA (2.363 g, 18.285 mmol, 3.4 equiv) was slowly added to a solution of the Boc protected p-aminobenzoic acid 6 (1.276 g, 5.378 mmol, 1 equiv), methyl ester of L-alanine (0.750 g, 5.378 mmol, 1 equiv), HOBt (0.727 g, 5.378 mmol, 1 equiv) and HBTU (2.243 g, 5.916 mmol, 1.1 equiv) in CH2Cl2 (10 mL). The reaction mixture was allowed to stir for 15 h, following which it was concentrated in vacuo. Ethyl acetate (70 mL) was added to the residue and the solution was washed sequentially with water (7  30 mL) and a saturated aqueous solution of NaHCO3 (3  25 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Purification using flash column chromatography (silica gel; 30–50% ethyl acetate in hexane, v/v) afforded 1.303 g (75%) of dipeptide 7. TLC Rf = 0.38 (hexane/ethyl acetate 2.5:1, v/v). 1 H NMR (500 MHz, DMSO-d6, 25 °C): d 9.62 (s, 1H; NHp-ABA), 8.59 (d, J = 7.0 Hz, 1H; NHAla), 7.80 (d, J = 8.5 Hz, 2H; ArH), 7.52 (d, J = 8.5 Hz, 2H; 2 ArH), 4.45 (app. quint, 1H; CHAla), 3.63 (s, 3H; OCH3), 1.48 (s, 9H; C(CH3)3), 1.39 (d, J = 7.0 Hz, 3H; CH3(Ala)); 13C NMR (125 MHz, DMSO-d6, 25 °C): d 173.3, 165.7, 152.6, 142.5, 128.3, 126.9, 117.0, 79.5, 51.8, 48.2, 28.1, 16.8; IR (KBr pellet): m 3373 (s), 3308 (s), 2983 (m), 1743 (s), 1708 (s), 1638 (s), 1515 (s), 1236 (s) cm1; HRMS (ESI+): calcd for C16H22N2O5 (MH+) 323.1607, found 323.1596. Tripeptide 8:20,35 Trifluoroacetic acid (3.015 g, 26.450 mmol, 10 equiv) was slowly added to a solution of dipeptide 7 (0.855 g, 2.645 mmol, 1 equiv) in dichloromethane (3 mL) maintained at 0 °C. The reaction mixture was allowed to warm to room

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(a)

(b)

1) t=0s M+

+

2) 0.5 N MOH (M = Li, Na, K, Rb, Cs) t = 50 s OHsymport

H+ antiport

M = HPTSO-

= HPTSOH

Figure 6. (a) Schematic representation of HPTS assay for cation selectivity. (b) Fluorescence versus time plot comparing response of peptide 2 (3.33 lM, 0.9 mol % with respect to lipid). Legend indicates k values determined by fitting the curves to Eq. 1.

(a)

X-

Me N

1) t=0s

=

2 NO3

quenching 2) 2 N NaX

N Me

(X = Cl, Br)

(b)

(c)

Figure 7. (a) Schematic representation of the lucigenin assay for detecting halide transport (channel mechanism). Fluorescence versus time plot showing response of peptide 2 with (b) NaCl and (c) NaBr.

temperature and stirred for 2 h, following which excess TFA was removed in vacuo as an azeotropic mixture with water (11 mL) to give the deprotected dipeptide. The product was dissolved in water (2 mL) and lyophilized to give 0.850 g (95%) of the free amine of dipeptide 7 as a white solid. The free amine was used for the next step without further purification. DIEA (1.159 g, 8.969 mmol, 3.4 equiv) was slowly added to a solution of the free amine of dipeptide 7 (0.887 g, 2.638 mmol, 1 equiv), Boc-L-Ala-OH (0.499 g, 2.638 mmol, 1 equiv), HOBt (0.356 g, 2.638 mmol, 1 equiv) and HBTU (1.100 g, 2.902 mmol, 1.1 equiv) in CH2Cl2 (4 mL). The reaction mixture was allowed to stir for 24 h, following which CH2Cl2 was removed in vacuo. The residue was dissolved in ethyl acetate (60 mL) and sequentially washed with water (7  20 mL) and a saturated aqueous solution

of NaHCO3 (3  20 mL). The organic layer was concentrated in vacuo and purified by column chromatography (silica gel; 50–70% ethyl acetate in hexane, v/v) to afford 0.623 g (59%) of tripeptide 8. TLC Rf = 0.31 (hexane/ethyl acetate 1:1, v/v). 1 H NMR (400 MHz, DMSO-d6, 25 °C): d 10.13 (s, 1H; NHp-ABA), 8.64 (d, J = 6.8 Hz, 1H; NHAla), 7.85 (d, J = 8.8 Hz, 2H; ArH), 7.68 (d, J = 8.8 Hz, 2H; ArH), 7.08 (d, J = 7.2 Hz, 1H; NHBoc), 4.47 (app. quint, 1H; CHAla), 4.13 (app. quint, 1H; CHAla), 3.64 (s, 3H; OCH3), 1.40–1.36 (12H; CH3(Ala) and C(CH3)3), 1.26 (d, J = 7.2 Hz, 3H; CH3(Ala)); 13C NMR (100 MHz, DMSO-d6, 25 °C): d 173.2, 172.2, 165.6, 155.2, 141.9, 128.3, 128.1, 118.2, 78.1, 51.8, 50.5, 48.2, 28.2, 17.8, 16.7; IR (KBr pellet): m 3330 (s, br), 3313 (s, br), 2986 (s), 1733 (s), 1670 (s), 1515 (s, br), 1250 (s) cm1; HRMS (ESI+): calcd for C19H27N3O6 (MNa+) 416.1798, found 416.1802.

B. P. Benke, N. Madhavan / Bioorg. Med. Chem. 23 (2015) 1413–1420

Tetrapeptide 9: To a solution of tripeptide 8 (0.335 g, 0.851 mmol, 1 equiv) in 2:1 methanol/water (3 mL) was added LiOH (0.071 g, 1.702 mmol, 2 equiv) with continuous stirring. After completion of reaction, methanol was concentrated in vacuo and the residual solution was acidified with HCl (2%). The regenerated acid was extracted with ethyl acetate (4  15 mL). The combined organic layers were dried over sodium sulphate, filtered and concentrated in vacuo to give 0.294 g (91%) of the free acid of tripeptide 8. The free acid was used for next step without further purification. DIEA (0.052 g, 0.405 mmol, 3.4 equiv) was slowly added to a solution of the free acid of tripeptide 8 (0.294 g, 0.775 mmol, 0.9 equiv), methyl ester of D-alanine (0.119 g, 0.852 mmol, 1 equiv), HOBt (0.115 g, 0.852 mmol, 1 equiv) and HBTU (0.355 g, 0.937 mmol, 1.1 equiv) in CH2Cl2 (4 mL). The reaction mixture was allowed to stir for 10 h, following which CH2Cl2 was removed in vacuo. The residual reaction mixture was dissolved in ethyl acetate (40 mL) and sequentially washed with water (5  20 mL) and a saturated aqueous solution of NaHCO3 (2  20 mL). The organic layer was concentrated in vacuo and purified by column chromatography (silica gel; 60–100% ethyl acetate in hexane, v/v) to afford 0.322 g (90%) of tetrapeptide 9. TLC Rf = 0.34 (hexane/ethyl acetate 1:2.5, v/v). 1 H NMR (400 MHz, DMSO-d6, 25 °C): (mixture of rotamers) d 10.11 (s, 1H; NHp-ABA), 8.35–8.25 (2H, NHAla), 7.9–7.8 (m, 2H; ArH), 7.68 (dd, J = 8.8, 2.0 Hz, 2 H; ArH), 7.06 (d, J = 6.8 Hz, 1H; NHBoc), 4.6–4.45 (m, 1H; CHAla), 4.29 (app. quint, 1H; CHAla), 4.13 (app. quint, 1H; CHAla), 3.62 (s, 3H; OCH3), 1.38 (s, 9H; C(CH3)3), 1.35–1.22 (9H; 3 CH3(Ala); 13C NMR (100 MHz, DMSO-d6, 25 °C): (mixture of rotamers) d 172.7, 172.3, 172.2, 165.3, 155.0, 141.6, 128.5, 128.2, 118.2, 78.0, 51.6, 50.4, 48.5, 48.4, 47.4, 28.0, 18.0, 17.8, 17.0, 16.8; IR (KBr pellet): m 3324 (s), 3291 (s) 2987 (w), 1737 (s), 1670 (s), 1630 (m) 1530 (s) cm1; HRMS (ESI+): calcd for C22H32N4O7 (MH+) 465. 2349, found 465.2360. Octapeptide 2: To a solution of tetrapeptide 9 (0.104 g, 0.224 mmol, 1 equiv) in 2:1 THF/water (1.5 mL) was added LiOH (0.019 g, 0.448 mmol, 2 equiv) with continuous stirring. After completion of reaction, methanol was concentrated in vacuo and the residual solution was acidified with HCl (2%). The regenerated acid was extracted with ethyl acetate (4  15 mL). The combined organic layers were dried over sodium sulphate, filtered and concentrated in vacuo to give 0.102 g (99%) of the free acid 10. The free acid was used for the next step without further purification. TFA (0.255 g, 2.240 mmol, 10 equiv) was slowly added to a solution of tetrapeptide 9 (0.104 g, 0.224 mmol, 1 equiv) in CH2Cl2 (0.5 mL) cooled at 0 °C. The reaction mixture was allowed to stir at room temperature following which excess TFA was removed in vacuo as an azeotropic mixture with water. The product was dissolved in water (2 mL) and lyophilized to give 0.104 g (98%) of the free amine 11. The tetrapeptide 11 was used for the next step without further purification. DIEA (0.097 g, 0.748 mmol, 3.4 equiv) was slowly added to a solution of tetrapeptide 10 (0.099 g, 0.220 mmol, 1 equiv), tetrapeptide 11 (0.105 g, 0.220 mmol, 1 equiv) and HCTU (0.100 g, 0.242 mmol, 1.1 equiv) in THF (1.5 mL). The reaction mixture was allowed to stir for 8 h, following which THF was removed in vacuo. The residual reaction mixture was dissolved in ethyl acetate (25 mL) and sequentially washed with water (3  20 mL) and a saturated aqueous solution of NaHCO3 (2  10 mL). The organic layer was concentrated in vacuo and purified by RP-HPLC (C18 column; 10–70% water in methanol (v/v) in 50 min) to afford 0.079 g (45%) of octapeptide 2. TLC Rf = 0.41 (methanol/dichloromethane 1:12, v/v). 1 H NMR (500 MHz, DMSO-d6, 25 °C) (mixture of rotamers) d 10.2–9.9 (2H; NHP-ABA), 8.52–8.4 (1H; NHAla), 8.38–8.05 (4H; NHAla), 7.9–7.82 (4H; ArH), 7.73–7.58 (4H; ArH), 7.1 (d,

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J = 5.0 Hz, 1H; NHBoc), 4.57–4.35 (3H; CHAla), 4.32–4.2 (2H; CHAla), 4.12 (app. quint, 1H; CHAla), 3.61 (s, 3H; OCH3), 1.38 (s, 9H; C(CH3)3), 1.36–1.22 (18H; 6 CH3(Ala)); IR (KBr pellet): m 3305 (s, br), 2921 (s), 1739 (w), 1666 (s), 1536 (s) 1253 (m) cm1; HRMS (ESI+): calcd for C38H52N8O11 (MNa+) 819.3653, found 819.3685. 3.2. Ion transport studies with octapeptides using the HPTS assay 3.2.1. Preparation of vesicles36a,b In a 10 mL round bottom flask, cholesterol (1.6 mg, 4.1 lmol, 1 equiv) was added to a solution of EYPC lipids (28.4 mg, 36.9 lmol, 9 equiv) in chloroform (0.284 mL). The solvent was removed under a stream of nitrogen and further in vacuo for 3 h at 0–4 °C to give a thin lipid film. The lipid film was hydrated with 1 mL of buffer solution containing HPTS dye (0.1 mM HPTS, 100 mM NaCl, 10 mM, HEPES) at pH 7.2. The resulting suspension was allowed to stir at 30 °C for 30 min and then subjected to eight freeze-thaw (liq N2 and 40 °C water) cycles. The vesicle solution was placed in a water bath and subsequently sonicated in a bath sonicator at 30 °C for a total time of 2 min (30 s on and 30 s off in degas mode). The vesicles were allowed to anneal for 12 h in the refrigerator, following which they were extruded 10 times through 0.1 lM 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 milky white solution containing vesicles was collected and it was diluted to 2.5 mL with HEPES/ NaCl (10:100 mM) buffer. 3.2.2. HPTS assay to study the comparison of ion transport of Peptides 1–5 The lipid suspension (75 lL) from the stock solution, HEPES/ NaCl (10:100 mM) buffer (2.925 mL) and an appropriate concentration of peptide in DMSO (10 lL) were added to a cuvette equipped with a magnetic stir bar. The solution in the cuvette was stirred for 1.5 min before the fluorescence experiment was started. 50 s after the experiment started, a solution of 0.5 N NaOH (20 lL) was rapidly added to the vesicle solution. 5% Triton X (50 lL) solution was added at 250 s to get the final emission intensity of the dye. Fluorescence measurements were done at an emission wavelength of 510 nm and an excitation wavelength of 460 nm. 3.2.3. HPTS assay to study the cation transport of peptide 2 The cation selectivity experiments were performed following the procedure described above for the HPTS assay. The experiments were performed by varying the cations in the salts MCl (M = Li, Na, K, Rb, Cs) used to prepare the extravesicular buffer and the base MOH (M = Li, Na, K, Rb, Cs) added at 50 s. 3.3. Anion transport studies for peptide 2 using the lucigenin assay 3.3.1. Preparation of vesicles36a,b Dehydrogenated EYPC (28.4 mg, 36.9 lmol, 9 equiv) in chloroform and cholesterol (1.6 mg, 4.1 lmol, 1 equiv) were taken in a 10 mL round bottom flask. The chloroform was evaporated using a stream of N2 to afford a thin film which was dried in vacuo for 3–4 h at 0 °C. The lipid cake was hydrated with 1 mL lucigenin dye (1.0 mM dye in 225 mM NaNO3 solution). The lipid suspension was swirled for 5–10 min at room temperature. The resulting suspension was sonicated at 30 °C in a bath sonicator for 2 min (30 s on and 30 s off in degas mode). The vesicle solution was subjected

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to 8 freeze thaw cycles (liq N2 and 40 °C). The extravesicular dye was removed by size exclusion chromatography using Sephadex G-50 (eluent: 225 mM NaNO3). The vesicle solution was diluted to 2.5 mL with 225 mM NaNO3 and used for the lucigenin assay. 3.3.2. Fluorescence experiments to determine anion transport through peptides The vesicle suspension (75 lL), aqueous NaNO3 (2.925 mL, 225 mM) and an appropriate concentration of peptide in methanol were placed in a cuvette equipped with a magnetic stir bar. The solution in the cuvette was stirred for 250 s and 50 lL of 2 N NaX (X = Cl, Br) was added at 251 s to set up the chloride gradient before the fluorescence experiment was started. Total concentration of NaCl in the cuvette was 33 mM. The fluorescence experiment was started at 270 s and at 520 s a 5% aqueous solution of Triton X (50 lL) solution was added to get the final fluorescence intensity of the dye. The fluorescence data was normalised from 270 s to 570 s. The fluorescence measurements were done at an emission wavelength of 505 nm and an excitation wavelength of 455 nm. The control experiment was carried out using methanol. 3.3.3. Dynamic light scattering studies20 The vesicle stock solution was prepared as described in the HPTS assay The vesicles suspension (75 lL) was diluted to 3.0 mL with HEPES buffer and allowed to stir with an appropriate concentration of peptide in DMSO (30 lL). For control experiments, DMSO (30 lL) was added. Acknowledgments This research was supported by DST (SR/S1/OC-41/2009), New Delhi, India. B.P.B acknowledges CSIR, India for his research fellowship. We thank Dr. E. Prasad for the use of his dynamic light scattering instrument. We thank Mr. Karthikeyan for help with the 23 Na NMR experiments. Supplementary data Supplementary data (synthesis of octapeptides 3–5, raw plots for the vesicle assays, Hill plots for peptide 1 and 2, 23Na NMR data for peptide 2, DLS, CD data and NMR spectra) associated with this article can be found, in the online version, at http://dx.doi.org/10. 1016/j.bmc.2015.02.031.

References and notes 1. Hille, B. Ion Channels of Excitable Membranes, 3rd ed.; Sinauer: Sunderland, MA, 2001. 2. Alfonso, I.; Quesada, R. Chem. Sci. 2013, 4, 3009. 3. Gale, P. A.; Pérez-Tomás, R.; Quesada, R. Acc. Chem. Res. 2013, 46, 2801. 4. Chui, J. K. W.; Fyles, T. M. Chem. Soc. Rev. 2012, 41, 148. 5. Reiß, P.; Koert, U. Acc. Chem. Res. 2013, 46, 2773. 6. Otis, F. O.; Auger, M. L.; Voyer, N. Acc. Chem. Res. 2013, 46, 2934. 7. Montenegro, J.; Ghadiri, M. R.; Granja, J. R. Acc. Chem. Res. 2013, 46, 2955. 8. Bong, D. T.; Clark, T. D.; Granja, J. R.; Ghadiri, M. R. Angew. Chem., Int. Ed. 2001, 40, 988. 9. Sanchez-Quesada, J.; Ghadiri, M. R.; Bayley, H.; Braha, O. J. Am. Chem. Soc. 2000, 122, 11757. 10. Hartgerink, J. D.; Clark, T. D.; Ghadiri, M. R. Chem. Eur. J. 1998, 4, 1367. 11. Dartois, V.; Sanchez-Quesada, J.; Cabezas, E.; Chi, E.; Dubbelde, C.; Dunn, C.; Granja, J.; Gritzen, C.; Weinberger, D.; Ghadiri, M. R. Antimicrob. Agents Chemother. 2005, 49, 3302. 12. Fernandez-Lopez, S.; Kim, H.-S.; Choi, E. C.; Delgado, M.; Granja, J. R.; Khasanov, A.; Kraehenbuehl, K.; Long, G.; Weinberger, D. A.; Wilcoxen, K. M. Nature 2001, 412, 452. 13. García-Fandiño, R.; Amorín, M.; Castedo, L.; Granja, J. R. Chem. Sci. 2012, 3, 3280. 14. Clark, T. D.; Buehler, L. K.; Ghadiri, M. R. J. Am. Chem. Soc. 1998, 120, 651. 15. Reiriz, C.; Amorín, M.; García-Fandiño, R.; Castedo, L.; Granja, J. R. Org. Biomol. Chem. 2009, 7, 4358. 16. Gokel, G. W.; Negin, S. Acc. Chem. Res. 2013. 17. Ferdani, R.; Gokel, G. W. Org. Biomol. Chem. 2006, 4, 3746. 18. Pajewski, R.; Ferdani, R.; Pajewska, J.; Djedovicˇ, N.; Schlesinger, P. H.; Gokel, G. W. Org. Biomol. Chem. 2005, 3, 619. 19. Wallace, B. A. Prog. Biophys. Mol. Biol. 1992, 57, 59. 20. Benke, B. P.; Madhavan, N. Chem. Commun. 2013, 7340. 21. See Supporting information for synthesis and characterization of peptides. 22. Kano, K.; Fendler, J. H. Biochim. Biophys. Acta 1978, 509, 289. 23. Clement, N. R.; Gould, J. M. Biochemistry 1981, 20, 1534. 24. See Supporting information for details. 25. The k value with a single batch of peptide did not vary significantly indicating that the HPTS assay was reproducible. 26. The DLS data of peptides 1 and 5 shown in Supporting information. 27. Merritt, M.; Lanier, M.; Deng, G.; Regen, S. L. J. Am. Chem. Soc. 1998, 120, 8494. 28. Bhosale, S.; Matile, S. Chirality 2006, 18, 849. 29. Vargas Jentzsch, A.; Matile, S. J. Am. Chem. Soc. 2013, 135, 5302. 30. (a) De Cola, C.; Licen, S.; Comegna, D.; Cafaro, E.; Bifulco, G.; Izzo, I.; Tecilla, P.; De Riccardis, F. Org. Biomol. Chem. 2009, 7, 2851; (b) Saha, T.; Dasari, S.; Tewari, D.; Prathap, A.; Sureshan, K. M.; Bera, A. K.; Mukherjee, A.; Talukdar, P. J. Am. Chem. Soc. 2014, 136(40), 14128; (c) Sakai, N.; Matile, S. J. Phys. Org. Chem. 2006, 19, 452. 31. McNally, B. A.; Koulov, A. V.; Smith, B. D.; Joos, J.-B.; Davis, A. P. Chem. Commun. 2005, 1087. 32. Milano, D.; Benedetti, B.; Boccalon, M.; Brugnara, A.; Iengo, E.; Tecilla, P. Chem. Commun. 2014, 9157. 33. There was a large background transport observed with I. Therefore, the studies with NaI were found to be inconclusive. 34. Madhavan, N.; Robert, E. C.; Gin, M. S. Angew. Chem., Int. Ed. 2005, 44, 7584. 35. Kubik, S.; Goddard, R. J. Org. Chem. 1999, 64, 9475. 36. (a) Jeon, Y. J.; Kim, H.; Jon, S.; Selvapalam, N.; Oh, D. H.; Seo, I.; Park, C.-S.; Jung, S. R.; Koh, D.-S.; Kim, K. J. Am. Chem. Soc. 2004, 126, 15944; (b) Fyles, T. M.; Hu, C.-W.; Luong, H. J. Org. Chem. 2006, 71, 8545.