1：Experimental Design and Method Selection:

The study involved the rational design of TPyzPz indicators with phenol substituents to modulate pKa via electronic effects. Synthesis was performed using mixed cyclotetramerization of substituted pyrazine-2,3-dicarbonitriles, followed by purification via column chromatography. Photophysical characterization included absorption, fluorescence, and singlet oxygen quantum yield measurements. pH sensing was tested in microemulsions and liposomes to mimic biological environments.

2：Sample Selection and Data Sources:

Compounds included a series of magnesium (II) and zinc (II) TPyzPzs with various substituents (e.g., tert-butyl, bromo, nitro) on the phenol ring. Precursors were synthesized from substituted acetophenones. Data were collected in organic solvents (THF), microemulsions (with Cremophor? EL), and liposomes (DOPC lipids).

3：List of Experimental Equipment and Materials:

Instruments: Nicolet 6700 spectrometer (IR), VNMR S500 NMR spectrometer (NMR), Vario Micro Cube Elemental Analyzer (elemental analysis), Shimadzu UV-2600 spectrophotometer (UV-Vis), FS5 Spectrofluorometer (Edinburgh Instruments, fluorescence), UHPLC system Acquity UPLC I-class with Synapt G2Si mass spectrometer (HR MS), Zetasizer nano-ZS (Malvern, dynamic light scattering), CyberScan pH510 pH meter. Materials: Magnesium turnings, butanol, pyrazine-2,3-dicarbonitriles, p-toluenesulfonic acid, zinc acetate, DOPC lipids, Cremophor? EL, medium-chain triacylglycerides, Britton-Robinson buffer, Bates and Bower buffer.

4：Experimental Procedures and Operational Workflow:

Synthesis involved cyclotetramerization with magnesium butoxide, purification by column chromatography, and metallation/demetallation as needed. Photophysical measurements were conducted in THF, microemulsions, and liposomes. pH titrations were performed by adding stock solutions to buffers and measuring absorption/fluorescence changes. Liposomes were prepared by film hydration and extrusion; microemulsions by solvent evaporation and hydration.

5：Data Analysis Methods:

pKa values were determined from fluorescence intensity vs. pH plots. Quantum yields were calculated relative to reference compounds. Spectral data were analyzed for shifts and quenching effects.