Scheme 2.1. Synthetic route of anionophores
2.4. Experimental section 1. Synthesis of compounds
2.4.5. Ion transport experiments
2.4.5.1. Ion transport activity studies using fluorescence-based assay
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2.4.5.1.1. Preparation of HPTS encapsulated EYPC/CHOL-LUVHPTS
To prepare the large unilamellar vesicles (LUVs) of EYPC/CHOL-LUVHPTS, first 50 µL of EYPC (100 mg/mL in 8:2 CHCl3/MeOH) and 80 µL of cholesterol (25 mg/mL in 8:2 CHCl3/MeOH) were taken in a clean and dry glass vial so that the molar ratio of EYPC and cholesterol would be 6:4. Then, this solution was dried by continuous purging of nitrogen gas for 2-3 hours to obtain a thin film of lipids inside the glass vial.
To remove any traces of CHCl3 and MeOH solvent the transparent film was kept under reduced pressure for 4-5 hours at room temperature. The dry film was then hydrated with 800 µL of 20 mM HEPES buffer, pH 7.2 containing 100 mM NaCl and 1 mM HPTS for 2 hours with occasional vortexing. Then, this suspension was sonicated (5 times, 30 sec of sonication followed by 30 sec of incubation in ice-water) to disrupt the aggregated vesicles. The suspension was further passed through 12-13 cycles of freeze-thaw (freezing with liquid N2 and melting with lukewarm water, respectively) to break up the multilamellar vesicles. The vesicle solution was extruded through a polycarbonate membrane (using a mini-extruder from Avanti Polar Lipids) having a pore size of 200 nm (size of LUVs are > 200 nm) for 19/21-times (as it must be an odd number), to give LUVs with a mean diameter of ~200 nm. Finally, gel filtration (Sephadex G-50) column chromatography was performed with 20 mM HEPES buffer, pH 7.2, containing 100 mM NaCl as a running solution to removed free/ uncapsulated HPTS. The HPTS encapsulated LUVs (without extra vesicular HPTS dye) were collected, and the final volume was adjusted to 800 µL using 20 mM HEPES buffer, pH 7.2, containing 100 mM NaCl. The final lipid concentration obtained was 25 mM (assuming 100 % lipid regeneration).11-12 2.4.5.1.2. Ion transport activity assay across EYPC/CHOL-LUVHPTS
For the HPTS assay, first 2925 µL of 20 mM HEPES buffer, pH 7.2, containing 100 mM NaCl and 50 µL of the EYPC/CHOL-LUVHPTS were taken in a 3 mL fluorescence cuvette, and the cuvette was placed in the fluorescence spectrophotometer under slow stirring condition (inbuilt magnetic stirrer in the instrument). After that, compounds (10 µL from a 5 μM stock solution in DMSO) were added to the solution to achieve a concentration ratio of 1: 25,000 for compound and lipids. The cuvette was then kept in the fluorescence instrument under stirring conditions for 3 minutes to allow maximum incorporation of the compounds into the lipid bilayers. The HPTS fluorescence intensity was monitored (t = 0 sec) at 510 nm (λex = 450 nm). Subsequently, 15 μL of NaOH (0.5
Chapter 2 M) solution was added into the cuvette after 50 sec to create a pH gradient (∆pH = ~ 0.5) between the extra and intra-vesicular regions and to initiate the Cl─ transport kinetics.
After 350 sec, the kinetic experiment was terminated by adding 20 L of 20% Triton- X100 solution (to rupture the vesicular arrangements) into the cuvette, and the fluorescent measurements were continued for another 50 sec (i.e., up to t = 400 sec).11-12
2.4.5.1.3. Quantitative measurement of transport activity from HPTS
The fluorescence emission intensities (Y-axis) of the HPTS dye were normalized, and the intensities are appearing at t = 0 and t = 400 s were taken as 0, and 100 units, respectively, and the normalized fluorescent intensities (FI) at t = 350 s (prior to the addition of Triton X-100) were considered to measure the transport activity of the compounds.
𝑖. 𝑒. 𝑇𝑟𝑎𝑛𝑠𝑝𝑜𝑟𝑡 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦, 𝑇𝐻𝑃𝑇𝑆 =(𝐹𝐹𝑡−𝐹0
∞−𝐹0)× 100 % ………Eq. 2.2
Where, Ft = fluorescence intensity at t = 350 s (prior to the addition of Triton X-100), F0
= fluorescence intensity immediately before the addition of the NaOH (t = 0 s) and F∞ = fluorescence intensity after addition of Triton X-100 (i.e., at saturation after complete leakage at t = 400 s).11-12
2.4.5.1.4. Preparation of lucigenin encapsulated EYPC/CHOL-LUVlucigenin
To prepare the LUVs of EYPC/CHOL-LUVlucigenin, first 50 µL of EYPC (100 mg/mL in 8:2 CHCl3/MeOH) and 80 µL of cholesterol (25 mg/mL in 8:2 CHCl3/MeOH) were taken in a clean and dry glass vial so that the molar ratio of EYPC and cholesterol would be 6:4. Then, this solution was dried by continuous purging of nitrogen gas for 2-3 hours to obtain a thin film of lipids inside the glass vial. To remove any traces of CHCl3
and MeOH solvent, the transparent film was kept under reduced pressure for 4-5 hours at room temperature. The dry film was then hydrated with 800 µL of 20 mM HEPES buffer, pH 7.2 containing 100 mM NaNO3 and 1 mM lucigenin for 2 hours with occasional vortexing. Then, this suspension was sonicated (5 times, 30 sec of sonication followed by 30 sec of incubation in ice-water) to disrupt the aggregated vesicles. The suspension was further passed through 12-13 cycles of freeze-thaw (freezing with liquid N2 and melting
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solution was extruded through a polycarbonate membrane with a pore size of 200 nm for 19/21-times to give LUVs a mean diameter of ~200 nm. Finally, gel filtration (Sephadex G-50) column chromatography was performed with 20 mM HEPES buffer, pH 7.2, containing 100 mM NaNO3 as a running solution to removed free/ uncapsulated lucigenin.
The lucigenin encapsulated LUVs (without extra vesicular lucigenin dye) were collected and the final volume was adjusted to 800 µL using 20 mM HEPES buffer pH 7.2, containing 100 mM NaNO3). The final lipid concentration obtained was 25 mM (assuming 100 % lipid regeneration).11-12
2.4.5.1.5. Ion transport activity assay across EYPC/CHOL-LUVlucigenin
For the lucigenin assay, first 2890 µL of 20 mM HEPES buffer, pH 7.2, containing 100 mM NaNO3 and 50 µL of the EYPC/CHOL-LUV lucigenin were taken in a 3 mL fluorescence cuvette, and the cuvette was placed in the fluorescence spectrophotometer under slow stirring condition. After that, compounds (10 µL from a 5 μM stock solution in DMSO) were added to the solution to achieve a concentration ratio of 1: 25,000 for compound and lipids. The cuvette was then kept in the fluorescence instrument under stirring conditions for 3 minutes to allow maximum incorporation of the compounds into the lipid bilayers. The lucigenin fluorescence intensity was monitored (t = 0 sec) at 506 nm (λex = 455 nm). Subsequently, 50 μL of NaCl (2.0 M) solution was added into the cuvette after 25 sec to initiate the Cl─ transport kinetics. After 475 sec, the kinetic experiment was terminated by adding 20 µL of 20% Triton-X100 solution (to rupture the vesicular arrangements) into the cuvette, and the fluorescent measurements were continued for another 25 sec (i.e., up to t = 500 sec).11-12
2.4.5.1.6. Quantitative measurement of transport activity from lucigenin assay The fluorescence emission intensities (Y-axis) of the lucigenin dye were normalized, and the intensities are appearing at t = 0 and t = 500 s were taken as 0, and 100 units, respectively, and the normalized fluorescent intensities (FI) at t = 475 s (prior to the addition of Triton X-100) were considered to measure the transport activity of the compounds.
𝑖. 𝑒. 𝑇𝑟𝑎𝑛𝑠𝑝𝑜𝑟𝑡 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦, 𝑇𝑙𝑢𝑐𝑖𝑔𝑒𝑛𝑖𝑛=(𝐹𝐹0−𝐹𝑡
0−𝐹∞)× 100 % ……….Eq. 2.3
Chapter 2 Where, F0 = fluorescence intensity immediately before the addition of the NaCl (at t = 0 s), F∞ = Fluorescence intensity after addition of Triton X-100 (i.e., at saturation, after complete leakage at t 500 sec), Ft = Fluorescence intensity at t = 475 sec (prior to the addition of Triton X-100).11-12
2.4.5.1.7. Half-life and initial transport rate calculation from lucigenin assay
The kinetic data obtained for quenching of fluorescence intensity of lucigenin were normalized for simplicity of analysis, and the Normalized fluorescence quenching curves (F/F0) were fitted to a first-order exponential decay equation.
𝐹
𝐹0 = 𝑦 + 𝑎. 𝑒−[𝐴]𝑡 . … . … … … … . . Eq. 2.4
At, t = t½ the value of 𝐹
𝐹0 = 𝑦 + 𝑎
2
Introducing the Eq. 2.4 and we get
𝑡1 2
= ln(2)
[𝐴] … … … . . Eq. 2.5
The initial transport rates were determined from a second-order exponential decay equation
𝐹
𝐹0 = 𝑦 + 𝑎. 𝑒(−𝑏𝑡)+ 𝑐. 𝑒(−𝑑𝑡) … … … . . Eq. 2.6
Differentiating according to t gives,
∂y
∂t = a. b. e(−bt)+ c. d. e(−dt) … … … … . . Eq. 2.7
The initial transport rate ri (t = 0) gives,
ri = ∂y
∂t = a. b + c. d … … … … . . Eq. 2.8
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The initial transport rate was calculated from a second order exponential decay curve to obtain improved regression factor (R2).11-12
2.4.5.1.8. Measurement of half maximal effective concentrations (EC50) of compound from HPTS assay
The fluorescence signals (Y-axis) from the HPTS- dye were normalized between 0 to 100 units [t = 0 to t = 400 s (X-axis)]. The normalized fluorescent intensity (FI) values at t = 350 s (prior to the addition of Triton X-100) were considered the compounds' transport activity. The transport activity (T) of a compound at a particular concentration was determined by using previous Eq.2.2. To get the effective concentration (EC50) of the compound, the transport activity values (Y-axis) were plotted against concentration (X- axis) and fitted in the Hill equation (Eq.2.9).
𝑇 = 𝑇∞+ 𝑇0− 𝑇∞ [1 + ( 𝑐
𝐸𝐶50)𝑛]
… … … . . … … … … . … . Eq. 2.9
Here, T0 and T∞ correspond to the transport activity obtained in the absence and at an excess concentration of the compound, respectively. C = concentration of the compound.
EC50 value corresponds to the concentration of compound required to obtain half of the maximum transport activity. The number of molecules required for the transportation of a single ion is given by ‘n’, and it is the Hill coefficient for the transporter molecule.11-12