Scheme 3.1. Synthetic routes to the modular anion transporters
3.4. Experiential section
3.4.4. Ion transport activity studies of guanidine-based macrocyclic compounds
3.4.4.8. Evaluation of transport of phosphate using Tb(III) fluorescent chemosensor 16
3.4.4.8.1. Preparation of liposomes encapsulating Tb(III) fluorescent chemosensor
For this experiment the LUVs were prepared encapsulating the Tb(III) complex.
Briefly in a clean and dry sample vial, EYPC (in deacidified CHCl3) and cholesterol (in deacidified CHCl3) was taken in 8:2 molar ratio and a thin lipid film was formed by evaporating the solvent under reduced pressure. The lipid film was then rehydrated using 20 mM HEPES buffer containing 225 mM NaCl and 4 mM Tb (III), pH 7.2. The resulting suspension was vortexed for atleast 6-7 times. After 1 hour, the suspension was subjected to 17 to 19 freeze-thaw cycles, which was followed by immediate vortexing for minimum 15 minutes. Next the liposomes was extruded using a mini extruder (a polycarbonate membrane from Avanti Polar Lipids) to obtain a uniform liposome size of 200 nm. Finally, the liposome suspension was dialyzed using 20 mM HEPES buffer containing 225 mM NaCl, pH 7.2.
3.4.4.8.2. Ion transport activity across the Tb(III) complex entrapped LUVs through fluorescence
For the fluorescence-based experiment, 2940 µL of 20 mM HEPES buffer containing 225 mM NaH2PO4, pH 7.2 and 50 µL of LUVs were taken in a clean and dry fluorescence cuvette and the cuvette was placed in a gentle stirring condition in the fluorescence spectrophotometer. After 3 minutes of stirring, fluorescence of the Tb(III) was monitored at t = 0 s, λem = 550 nm, λex = 293 nm. At t = 50 s, the kinetics was initiated by addition of the anionophore 3.2. At t = 450 s, the vesicles were finally
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lysed by addition of 20 µL 20 % Triton X-100. The Tb(III) fluorescent measurement was carried for a further 50 s (total t = 500 s).
Figure S3.9. Structure of H3L.Tb (A). Assessment of phosphate ion transport of 3.2 in the presence of H3L.Tb (1 mM) (B).
3.4.4.9. Transport of phosphate ion through GUVs (Giant Unilamellar Vesicles) 3.4.4.9.1. Preparation of GUVs encapsulating 100 mM HEPES, 200 mM sucrose, and 225 mM NaH2PO4
The GUVs were prepared similarly according to a previously reported procedure 17. Concisely, EYPC, CHOL, and DPPS were taken respectively in the molar ratio 8:1.5:0.5 in a dry and clean glass vial. The resulting solution was dried for 4 hours under reduced pressure to form a thin lipid film. To the lipid film, 200 µL of liquid paraffin oil was added and sonicated until the film dissolved completely, followed by the addition of 20 µL of the upper buffer (100 mM HEPES containing 200 mM sucrose. 100 mM NaH2PO4, and 1 mM bis-N-methylacridinium nitrate (Lucigenin) in H2O, pH 7.2) and the solution was mixed nicely to form a white-colored emulsion. The emulsion was then slowly added to 500 µL of lower buffer (100 mM HEPES containing 200 mM glucose and 1 mM bis-N-methylacridinium nitrate (Lucigenin) in H2O, pH 7.2) and pipetted up and down thoroughly in a centrifuge tube to mix everything. The emulsion was centrifuged for 15 minutes at 10000 to remove the excess unencapsulated dye and the oil. The precipitate of liposomes was washed 3 times with 200 µL of lower buffer to eliminate excess oil and the unencapsulated lucigenin dye. After the final washing, the precipitate of GUVs was mixed with 200 µL
Chapter 3 of lower buffer, 10 µL of 100 µM Texas Red DHPE (Invitrogen, CA) solution in MeOH was added for membrane labeling, following which the solution was kept undisturbed for 1 hour. After that, the excess Texas Red dye was washed with the lower buffer solution. The final precipitate of liposomes was mixed with 100 µL of lower buffer (final vesicle concentration of 15 mM). The microscopic images were collected using these GUVs.
3.4.4.9.2. Ion transport measurements using GUVs coated on the glass surface The coating on the glass surface was done according to the procedure as mentioned in 3.4.4.11.2. The freshly prepared GUVs (30 µL) were kept undisturbed for 5-6 minutes on the coated glass surface. After 5-6 minutes, a solution of NaCl and transporter 3.2 (45 µM) were added carefully to the solution of the GUVs and the decrease in the lucigenin fluorescence intensity was recorded with time with the help of images both in the bright field and in the green field (λex = 458 nm).
3.4.4.9.3. Preparation of GUVs encapsulating 100 mM HEPES, 200 mM sucrose, and 225 mM NaCl
The GUVs were prepared according to the procedure mentioned in section 3.4.4.11.1. The GUVs were prepared using 100 mM HEPES, 200 mM sucrose, 225 mM NaCl, 1 mM bis-N-methylacridinium nitrate (Lucigenin) in H2O and the GUVs were dispersed in 100 mM HEPES buffer containing 200 mM glucose and 225 mM NaCl.
3.4.4.9.4. Ion transport measurements using GUVs coated on the glass surface The GUVs were prepared according to the procedure mentioned 3.4.4.11.1. To 100 µL of the GUV solution, 10 µL of Texas Red-PE was added and the solution was mixed thoroughly. After 15 minutes, the GUV suspension was centrifuged 2-3 times with the upper buffer to remove the excess Texas Red-PE dye. The freshly centrifuged GUVs (30 µL) were kept undisturbed for 5-6 minutes on the coated glass surface.
After 5-6 minutes, a solution of Na2HPO4 and transporter 3.2 (45 µM) were added carefully to the solution of the GUVs and the increase in the lucigenin fluorescence intensity was recorded with time with the help of images both in the red field and in the green field (λex = 458 nm). The increase in lucigenin fluorescence intensity
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signifies transport phosphate ion from the extravesicular solution to the intravesicular solution.
Figure S3.10. 31P NMR experiment showed that DMSO is not capable of phosphate transport via the Cl─/H2PO4─ antiport pathway.
3.4.4.10. Direct evidence for phosphate transport by 31P NMR measurements