Switchable foldamer ion channels with antibacterial activity

Triazole-capped α-aminoisobutyric acid (Aib) octameric foldamers formed very active ion channels in phospholipid bilayers after the addition of copper(ii) chloride, with activity “turned off” by copper(ii) extraction.

N3AibnOR (1 eq) was dissolved in EtOH (5 mL/1 mmol) under a nitrogen atmosphere. Pd/C (10%, 10mg/1 mmol) was added and the reaction mixture stirred under an atmosphere of H2 until IR indicated complete consumption of the starting material (24 h). The mixture was washed through a pad of Celite with EtOAc and the filtrate concentrated.

General procedure 2: Synthesis of N3AibnOR
N3AibOH (2 eq) was dissolved in dry CH2Cl2 (4 mL/1 mmol). EDC*HCl (2 eq) was added in portions and stirred for 15 min. Afterwards, HOBt (0.1 eq) and Et3N (1.2 eq) was added dropwise and stirred for another 15 min. NH2AibnOR (1 eq) was added and stirred at room temperature overnight. The reaction mixture was washed with 5% aq. KHSO4 solution (2 × 20 mL), sat. NaHCO3 solution (2 × 20 mL). The aqueous layer was washed with EtOAc (2 × 10 mL). The organic layers were combined and dried over MgSO4, filtrated and the solvent was removed under reduced pressure. N3AibnO t Bu (1 eq) was dissolved in a 1:2 mixture of trifluoroacetic acid (1 mL/1 mmol) and CH2Cl2 (2 mL/1mmol). The resulting reaction mixture was stirred at room temperature for 24 h under a nitrogen atmosphere. The solvent was removed and the yellow high viscous oil was re-dissolved in Et2O (5 × 30 mL) and the solvent was removed.

General procedure 4: Formation of the active ester with EDCˑHCl
AibnOH (1 eq) in was dissolved in dry CH2Cl2 (2 mL/1mmol) and EDCˑHCl (2 eq) was added slowly. To the resulting reaction mixture was stirred at room temperature overnight under a nitrogen atmosphere. The solution was diluted with CH2Cl2 (5 mL/1mmol) and the organic phase was washed with sat. NaHCO3 (2 × 5 mL/mmol) and brine (5 mL/mmol). The organic layer was dried over MgSO4, filtered and the solvent removed.

General procedure 5: Synthesis of N3AibnO t Bu by reaction of active ester
Aibn-active ester (1 eq) was dissolved in MeCN (2 mL/1 mmol) and NEt3 (1.2 eq) was added slowly. Afterwards the appropriate amine (1.0 eq) was added. The resulting reaction mixture was heated to reflux and stirred for 48 h. The solvent was removed and the resulting residue was purified by flash column chromatography on silica (SiO2) with the appropriate solvent system.

N3AibO t Bu
Sodium azide (6.65 g, 100.8 mmol) was dissolved in dry DMF (50 mL). To the resulting white suspension tert-butyl-2-bromo-2-methylpropionate (12.5 mL, 67.2 mmol) was added and the reaction mixture was stirred at room temperature for 72 h under a nitrogen atmosphere. The resulting white suspension was diluted with H2O (40 mL) and acidified to pH = 2 with 1M HCl (30 mL) and extracted with tert-butyl methyl ether (3 × 50 mL). The organic layer was washed with 1M HCl (4 × 20 mL), dried over MgSO4, filtered and the solvent removed to yield the title compound as a pale yellow oil, which was used in the next step without further purification (12.

NH2AibO t Bu
N3AibO t Bu (5.90 g, 30.75 mmol) was hydrogenated using procedure 1 and the product was isolated as a pale yellow oil.

N3Aib2O t Bu
H2N-AibO t Bu (2.40 g, 15.1 mmol) was used following procedure 2 and the product was isolated as a pale yellow oil.

H2NAib2O t Bu
N3Aib2O t Bu (1.92 g, 7.10 mmol) was hydrogenated following procedure 1 and the product was isolated as a white solid (1.56 g, 6

NH2Aib4O(CH2)2TMS
N3Aib4OTMS (0.25 g, 0.58 mmol) was hydrogenated following procedure 1 and the product was isolated as a white solid (0.18 g, 0.43 mmol, 74 %) Spectroscopic data is consistent with the reported data in the literature. 2 S10

Aib foldamer 7
Two methods were used to synthesise this compound: Method 1: N3Aib4O t Bu (10 mg, 0.023 mmol) and N,N-bis(pyridin-2-ylmethyl)propargylamine (4 mg, 0.017 mmol) were dissolved in EtOH (1.5 mL). Sodium ascorbate (0.25 mL of 20 mM aq. solution), copper turnings (2.5 mg) and CuSO4 (0.25 mL of 10 mM aq. solution) was added. The yellow solution was stirred overnight. The resulting dark brown solution was diluted with EtOAc (10 mL) and washed with sat. EDTA solution (10 mL, pH adjusted to pH = 7 with NaOH). The organic layer was dried over MgSO4, filtered and the solvent removed under reduced pressure. The crude brown solid was purified via HPLC (as for 1 previously) resulting in a white solid (2 mg, 0.003 mmol, 13%).

Method 2:
N-Propargyl-di(2-picolyl)amine (28.5 mg, 0.12 mmol), N3Aib4O t Bu (44 mg, 0.1 mmol) and copper acetate (3 mg, 0.04 mmol) were dissolved in dry DMF (1 mL) in a round bottom flask, and the solution was heated to 80°C for 1 h under an argon atmosphere. The DMF was co-evaporated with toluene (3 ×) under reduced pressure and the resulting solid was purified by column chromatography on alumina (10% MeOH in CH2Cl2) to give a brown solid. This brown solid was purified by semi-preparative high performance liquid chromatography (HPLC) on an Agilent 1100 series HPLC equipped with a semi-preparative C18 column Agilent eclipse XDB-C18, 5 μm, 9.4 mm × 250 mm with a flow rate of 1 mL/min. The product containing fractions were combined and the organic solvent was removed under reduced pressure. The aqueous solution was freeze-dried to give the product as a white solid (32 mg, 0.47 mmol, 47%).

Conditions for complexation of CuCl with foldamer 1
Foldamer 1 (6 mg, 6 µmol) was dissolved in MeOH (3 mL). Afterwards a CuCl solution in MeOH (50 mL, 0.1 mM) was added over 1 h. The resulting solution was stirred for 1 h and the solvent was reduced to 5 mL. The resulting solution was stored in a sealed jar containing a small amount of diethylether. After three days blue crystals were formed. The crystals were stored in solution to try and prevent oxidation. Before use the crystals were filtered and dried in a ventilated oven at 60 °C to obtain 2 mg (2 µmol, 30%) of blue crystals.

Conditions for complexation of CuCl with foldamer 2
Foldamer 2 (6 mg, 6 µmol) was dissolved in MeOH (3 mL). Afterwards a CuCl solution in MeOH (50 mL, 0.1 mM) was added over 1 h. The resulting solution was stirred for 1 h and the solvent was reduced to 5 mL. The resulting solution was stored in a sealed jar containing a small amount of diethyl ether. After three days blue crystals were formed. The crystals were stored in solution to try and prevent oxidation. Before use the crystals were filtered and dried in a ventilated oven at 60°C to obtain 2 mg (

Complex Cu(II)[2]Cl2
Foldamer 2 (10 mg, 10 µmol) was dissolved in MeOH (3 mL). Afterwards a CuCl2 solution in MeOH (5 mL, 2 mM) was added. The resulting solution was stirred for 10 min and the resulting solution was stored in a sealed jar containing a small amount of diethyl ether. After two days green crystals were formed. Diethyl ether (4 mL) was carefully added on top of the MeOH solution and left for one more day to mix. The solid was filtered off and air dried for two days and afterwards further dried in a ventilated oven at 60 °C to obtain 8 mg (7 µmol [2]Cl2 dissolved in methanol were mixed with the vesicle suspensions for 60 s before adding a base pulse to change the external pH to 8.4. 6 The resulting change in HPTS fluorescence was followed for 6 min, then Triton X-100 was added at 7 min to lyse the vesicles and allow. Each assay was repeated three times on new vesicles. The resulting normalized data were fitted to pseudo first-order rate equations as an approximation (see the ESI), similar to the procedure of Regen and co-workers. 7 Although the change in fluorescence after the "burst phase" is likely to arise from multiple processes, including inter-vesicle transfer of foldamers, 8  with F0 = Ft at addition of base pulse, F∞ = Ft at saturation after complete leakage.

Procedure for the determination of first order rate constants
The normalised data (In) was fitted to first order kinetics using an equation of the general form: with t in seconds. In is the emission intensity at time t, I∞ is the final normalised fluorescence intensity at t = ∞ and Io is the estimated initial normalised fluorescence intensity at t = 0 minutes.
The fit was started from t = 0, as this is the point of compound addition.
HPTS assays were repeated three times and showed good experimental reproducibility. Repeated fitting to pseudo-first order kinetics and assessment of the goodness of fit provided an approximation of the errors inherent in the curve fitting process, estimated as ±0.001 s −1 . (c) KBr (100 mM). a Base pulse at 1 min; b CuCl2 addition (2 eq. at 120 s) switches activity "on"; c EDTA addition (2.2 eq. at 180 s) switches activity "off"; d TX-100 added at 7 min. The corresponding data for the addition of MeOH (20 μL) has been subtracted from these data.

Effect of CuCl2 addition on the HPTS assay
To confirm that neither the addition of CuCl2 solution, nor the addition of EDTA solution had an ionophoric effect, the "gating" experiment was performed in the same way but in the absence of the active compounds (1 or 2).

Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) data
Analysis of the effect of FCCP on the measurement of ion transport by 1, 2, Cu(II) [1]Cl2 and Cu(II) [2]Cl2 in the HTPS assay were performed according to the procedure of Shinde and Talukdar. 9 FCCP was added at 2 minutes, but was only observed to have a significant effect on the membrane activity of the free foldamers 1 and 2; the corresponding Cu(II)Cl2 complexes showed little change. Addition of FCCP on its own did not cause a significant change.

Preparation of large unilamellar vesicles for lucigenin assays
Egg yolk phosphatidylcholine (EYPC, 64 μmol) and cholesterol (16 μmol . Triton X-100 lysed the vesicles and quenched all the lucigenin, allowing data normalisation. Data was collected for a further minute (60 s). After normalisation, the data was fitted to first-order kinetics as an approximation (see the ESI).
Fluorescence time courses were normalised using the equation with F0 = Ft at addition of NaCl pulse, F∞ = Ft at saturation after complete leakage (Triton X-100 addition).
The normalised data (In) was fitted to first order kinetics using an equation of the general form: with t in seconds. In is the emission intensity at time t, I∞ is the final normalised fluorescence intensity at t = ∞ and Io is the estimated initial normalised fluorescence intensity at t = 0 minutes (and equal to 1).
The fit was started from t = 0, as this is the point of compound addition. Compounds added at 0 min, NaCl added at 1 min, TX-100 added at 7 min. EYPC-cholesterol (4:1) 800 nm vesicles were prepared as described above, using the above mentioned 5(6)carboxyfluorescein (CF) (50 mM) solution.

Curve fitting of lucigenin assays and data for Cu(II)[1]Cl2
Unencapsulated 5/6-CF was removed by gel permeation chromatography on PD-10 SEC columns (Sephadex G-25) as described for the procedure for LUV preparation.

U-tube metal picrate transport experiments
The used procedure was developed by modification of published methods. 2

U-tube lucigenin transport experiments
The used procedure was developed by modification of published methods. 11  incubated in a water bath at 25 °C and the chloroform phase was stirred at 300 rpm during the entire experiment, ensuring efficient diffusion of any potential carrier-ion complex to the receiving phase.
Aliquots (1 mL) were taken from the receiving phase and analysed for the presence of lucigenin by UV spectroscopy (at 455 nm). After measurement, the sample was immediately replaced back in the U-tube. Absorbance at 455 nm.

Procedure for CF assays
Samples were prepared as described for the procedure for HPTS assays. After 1 min 20 μL of the principal compound in methanol was added then after 7 min 40 μL of a 10 % v/v solution of Triton X-100 detergent in MOPS was added to lyse the vesicles. The release of 5/6-carboxyfluorescein from the prepared vesicles was measured by observing the 5/6-CF emission intensity at 517 nm following excitation at 492 nm.
For each experiment, the initial (F0) and total (FTriton, 10% Triton X-100) fluorescence was determined and used to determine the final value: (F-F0)/(FTriton-F0). Following normalization of the 5/6-CF data, the background methanol was subtracted from the raw data, and the intensity of emission (IF) plotted.

Planar bilayer conductance (PBC) studies
Alamethicin provides a useful comparison to the data reported in the manuscript and in the ESI.
Alamethicin is a mildly cation selective channel-former 12 shows non-ohmic behavior and produces discrete events with multiple conductance levels (0.02 nS to 0.15 nS, with open lifetimes in the order of 50 to 80 ms). 13,14 The values found can also be compared to an N-acetylated Aib octamer studied previously (0.34 nS at 100 mV) 15 and the conductances typically found for channels (1 to 100 pS) and pores (0.1 to 5 nS). 16

Procedure
Single channel experiments were performed in a custom built cell. The cell ( Figure S13) was formed of two Teflon blocks, each with a machine-drilled well (approx. 10 mm diameter and 1 mL volume). Each well contained a side opening, such that when the blocks were bolted together the adjacent side openings connected the two wells. Additionally, each well contained two access channels (approx. 2 mm diameter), drilled at a 45° angle, such that the channels joined the bottom of the main well. An aperture (approx. 100 mm diameter) was created in a Teflon sheet (Goodfellow, 25 mm thick) using a 30 kV spark gap generator (Ealing Spark Source). The Teflon sheet containing the aperture was clamped and sealed with silicone glue (3140 RTV coating, Dow Corning) between the two blocks, such that the aperture was positioned in the central lower half of the side opening between the wells. The buffer solution on both sides of the Teflon sheet was aspirated and dispensed using a Hamilton syringe S32 to 'paint' a phospholipid bilayer across the aperture. A ± 1 mV pulse was applied at 1333 Hz to determine when a bilayer was obtained (capacitance of 40 to 80 pF). The membrane was characterized with successive 2 second sweeps under an applied potential ranging from +100 to −100 mM. The membrane was deemed acceptable if the range of current flow across the membrane measured < 1 pA in > 10 consecutive characterization sweeps.
The appropriate foldamer (5-10 mL of a 1 mM solution in MeOH) was added to the ground well.
Characterization sweeps were continued for 2 hours, or until substantial channel-forming activity was observed.
All data were collected using the patch clamp amplifier, and digitised (Axon Instruments Digidata 1332A) at

Repeated I-V curves for EYPC lipid/cholesterol bilayers with complex Cu(II)[2]Cl2
Conductance was measured in increasing increments of 10 mV up to +100 mV, before the current was reduced to -40 mV in the following steps (+80 mV, +40 mV, 0 mV, -40 mV). This procedure provided two measurements at +80 mV, +40 mV, 0 mV and -40 mV. The current was found to increase over time ( Figure   S16), which we ascribe to slow insertion of the foldamer into the membrane, an equilibration process that is accelerated under strongly positive and especially strongly negative potential differences. 17 Under the same conditions employed for foldamer 2 and complex Cu(II) [2]Cl2, the complex Cu(II) [2]Cl.HCO3 showed a mixture of behaviours. Short-lived "flicker"-type openings could be observed ( Figure S18), which displayed conductance levels (0.07 ± 0.01 nS, 0.16 ± 0.01 nS) very similar to those observed for Cu(II) [2]Cl2.
In addition, much longer lived "square topped" openings could be observed ( Figure S19), which had very regular well-defined quantized current steps with large current levels (0.18 ± 0.01 nS) that were open for up to 20 ms at +100 mV). Although small differences in pore structure are possible between complex Cu(II) [2]Cl2 and complex Cu(II) [2]Cl.HCO3, we expect respective pore structures to be similar as the precursor complexes only differ in counterion; indeed the higher conductance level of the "blue" complex forming species in the membrane for the more soluble "blue" complex, which diffuses more rapidly into the membrane to produce more frequent, longer-lived openings of larger, higher nuclearity pores.

Estimation of channel diameter
We have used the Hille equation 18 to estimate the pore sizes that correspond to the different conductance levels observed:

Antimicrobial assays
Metal ion chelating Aib foldamers 1 and 2 bear a novel amino-terminal copper and nickel (ATCUN) binding motif; other ATCUN motifs are reported to increase antibiotic properties by generating reactive oxygen species upon metal binding. 24 Furthermore many copper-containing metallodrugs have been studied for their anticancer 25 and antibiotic properties. 26 Introducing the Cu(II)(BPTA) group onto the Aib octamer unit also increased water solubility, mitigating to some extent the solubility problems that hampered earlier antibiotic studies of long Aibn oligomers (n ≥ 10). 15

Antimicrobial assays: minimum inhibitory concentrations (MIC)
The used procedure was developed by modification of published methods. 27  The plates were shaken for about 30 seconds and the optical density (OD) was measured at 600 nm.
Afterwards the plates were incubated at 30 °C under aerobic conditions with shaking overnight. The absorbance was then measured at 600 nm using a ClarioStar plate reader. The minimum inhibitory concentration (MIC) was defined as the lowest antibiotic concentration that completely inhibited the growth of the tested bacteria. The assays were performed in three biological repeats with three technical repeats of each experiment.
The concentration of the tested compound which caused an OD equal to the OD of LB (± 5%) was considered as the MIC.

Haemolysis assays
Red blood cells were collected with full Research Ethics Committee (REC) approval for use in evaluation of new treatment strategies (REC reference 10/H1017/73). All samples were collected with full consent for research use with identity traced only through the collecting medical institution (Manchester Royal Infirmary, custodian Dr John Burthem). There are no direct or indirect medical implications for the donor.
The used procedure was developed by modification of published methods. 28 Freshly taken blood was diluted with PBS buffer (1:2). Aliquots (500 μL) of this diluted blood were placed into an Eppendorf tube.
Aliquots (5 μL where complete haemolysis was achieved by mixing the erythrocytes with 1% Triton X-100. The channel concentration required to cause 50% haemolysis (HC50) was determined directly from the graph.  Single crystals suitable for X-ray diffraction analysis were grown by slow diffusion of diethyl ether in with compound Cu [2]Cl.HCO3 saturated solution of chloroform. Data were collected on a dual source Rigaku FR-X rotating anode diffractometer using CuKα wavelength radiation (λ = 1.54184) at a temperature of 150 K.
The data were reduced using CrysAlisPro 171.39.30c and absorption correction was performed using empirical methods (SCALE3 ABSPACK) based upon symmetry-equivalent reflections combined with measurements at different azimuthal angles. 29 The structure was solved and refined against F 2 using Shelx-2018/3 implemented through Olex2 v1.2.9. 30 Crystals of the peptide Cu [2]Cl.HCO3 are orthorhombic, space group = P212121, with unit-cell dimensions of a = 9.1099(2), b = 24.1351(9), c = 34.1137(11) Å. R-factor (%) = 7.18. Single crystals suitable for X-ray diffraction analysis were grown by slow diffusion of diethyl ether in with compound Cu [2]Cl2 saturated solution of chloroform. Data were collected on a dual source Rigaku FR-X rotating anode diffractometer using CuKα wavelength radiation (λ = 1.54184) at a temperature of 150 K.
The data were reduced using CrysAlisPro 171.39.30c and absorption correction was performed using empirical methods (SCALE3 ABSPACK) based upon symmetry-equivalent reflections combined with measurements at different azimuthal angles. 29 The structure was solved and refined against

Electron paramagnetic resonance (EPR) studies
Electron paramagnetic resonance (EPR) spectroscopy was performed on an EMX with a 1.8 T electromagnet in the range from 20 K to 130 K.
The Q band (34 GHz) electron paramagnetic resonance (EPR) data (Figures 24-26) unpaired electrons were present (consistent with Cu(II)). It was consistent with a Cu(II) center with Jahn-Teller distortion, 37 and did not show significant differences upon changing the temperature from 20 K to 130 K ( Figure S24), which suggests the Cu(II)2 [2]2Cl2 unit has a relatively a rigid structure.