Scheme 3.2. Synthesis of the control compounds
3.2.6. Mechanism for transport of chloride ion of the potent compounds
The transport of Cl─ ion by the compounds 3.9b and 3.10b may follow either antiport (OH─/A─) or symport (H+/A─) mechanism.4 In this regard, the transport activity was first measured with intravesicular NaCl and an isotonic solution of NaNO3, NaI, NaBr, and NaClO4 in the extravesicular region by HPTS assay in the absence of pH gradient (i.e., pHin = pHout = 7.2). 4 Even though no pH gradient was applied still a notable change in HPTS fluorescence intensity in the presence of compounds suggests the possibility of an influx of H+ across the membrane bilayer. It is also expected that for the charge neutrality, the influx of X─ ion would be accompanied. However, in the presence of SO42─ ion, an increase in the HPTS fluorescence intensity was observed. The higher hydration energy restricts the SO42─ ion (hydration energy = 1080 kJmol-1) to cross the lipid bilayer, but the enhancement in the HPTS fluorescence signal indicates the efflux of the H+/Cl─ ion pair as symport mechanism (Figure 3.6A-B).
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Figure 3.6.Anion selectivity of compounds 3.9b (A) and 3.10b (B) was measured across EYPC/CHOL-LUVHPTS by varying the extravesicular anions without applying pH gradient. NMDG-Cl assay for compound 3.9b (C) and 3.10b (D) in the absence and presence of proton channel gramicidin D (Gra). The H+/Cl− transport efficacy of compound 3.9b (E) 3.10b (F) was measured across a U-tube by applying the HCl gradient, using chloride ion-selective electrode and pH meter. The concentration of compound 3.9b and 3.10b were 0.56 µM = 0.13 mol %, and 2.29 µM = 0.55 mol % with respect to the
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The HPTS assay was performed in the absence and presence of 4- (trifluoromethoxy)phenylhydrazone (FCCP; a protonophore) in 20 mM HEPES buffer, pH 7.2, containing 100 mM NaCl for further investigation of the Cl─ ion transport properties of compounds. The compounds showed a similar increase in the Cl─ ion transport activity both in the absence and presence of the FCCP (Figure 3.7A and 3.7C).
The rate of dissipation of the pH gradient in the FCCP experiment is not affected by the protonophore, which suggests that the involvement of Cl─ ion in the rate-limiting step of the H+/Cl─ symport transportation. We also performed the HPTS assay in the absence and presence of valinomycin (a potassium ionophore) in 20 mM HEPES buffer, pH 7.2, containing 100 mM KCl. Comparable enhancement in the Cl─ ion transport rate in the absence and presence of the valinomycin suggests preferential transport of Cl─ ion over OH─ ion (Figure 3.7B and 3.7D). As a consequence, the cotransport of H+/Cl─ by the ionophore was the operating mechanism for the transport of Cl─ ion. Besides, the NMDG- Cl (N-methyl-D-glucamine chloride) assay was performed in the absence and presence of gramicidin-D in 100 mM NMDG-Cl buffer, pH 7.0. The NMDG (5 mM) was used in the extravesicular region to create the pH gradient. The compound exhibited a comparable change in the Cl─ ion transport activity both in the absence and presence of the gramicidin- D, suggesting that Cl─ transport is the rate-limiting step (Figure 3.6C-D). Hence, these HPTS-based assays propose that the transport rate of H+/OH─ is faster than Cl─ ion, and H+/Cl─ symport is the operating mechanism.
To confirm the H+/Cl─ co-transport mechanism, we performed the classical U-tube assay. The left-arm of the U-tube was filled with the aqueous HCl (100 mM) solution (pH 1.05), while the right-arm was filled with aqueous NaNO3 (100 mM) solution, and these two arms were separated by the chloroform solution, which mimics the membrane environment. A steady decrease in the pH and a sharp increase in Cl─ ion concentration directly confirmed its H+/Cl─ co-transport mechanism (Figure 3.6E-F). The U-tube assay was also performed in the presence of bromothymol-blue (a pH-sensitive dye) in the right- arm of the U-tube along with the NaNO3 solution. The time-dependent change in the color of bromothymol-blue visually confirmed the difference in the pH of the solution in the presence of the compound (Figure 3.65).
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Figure 3.7. Assessment of Cl─ ion transport properties of compound 3.9b and 3.10b (2.29 µM = 0.55 mol% with respect to lipid) in the absence and presence of FCCP (A, C) or valinomycin (B, D) in 20 mM HEPES buffer, pH 7.2, containing 100 mM NaCl/KCl.
To investigate whether the compound-mediated transport of Cl─ ion follows carrier or channel mechanism, the cholesterol concentration dependency assay was performed (Figure 3.8). The outcome of the experiment revealed that the transport of Cl─ ion depends on the concentration of the cholesterol present in the membrane, confirming the carrier- mediated Cl─ ion transport pathway of the compounds. The classical U-tube assay also confirmed that the potent compounds 3.9b and 3.10b transport Cl─ ion through carrier mechanism. Meanwhile, the
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Figure 3.8. Effect of the concentration of cholesterol in the EYPC/CHOL membrane on Cl─ ion transport activity of compound 3.9a (A) and 3.10b (B), respectively. Leaching experiment of compounds 3.9b (C) and 3.10b (D). The extent of calcein leakage from the EYPC/CHOL-LUVcalcein in the presence of compounds 3.9b (E) and 3.10b (F).The concentration of compound 3.9b and 3.10b were 0.56 µM = 0.13 mol%, and 2.29 µM = 0.55 mol% with respect to the lipid, respectively.
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leaching test demonstrated the non-leaching behaviors of the compounds from the lipid bilayers (Figure 3.8C-D). The non-leaking ability of calcein from the LUVs in the presence of compound also supports carrier mechanism and dismisses the probability of pores or channels formations (Figure 3.8E-F). Further, the stability of the potent compounds in different buffer solutions was also performed (pH 5.5-8.0) after 72 hours of incubation. The HPLC analyses clearly showed that the structural integrity of the compounds 3.9b and 3.10b are intact (Figure 3.66).