Table 2.

Troubleshooting table.

Step	Problem	Possible reason	Solution
34	Reaction fails	O2 and/or H2O in the reaction	Make sure the reaction is set up in air-free conditions and make sure all reagents are as dry as possible
40	Product sticks to column	Silica can deprotect the pinacol to yield the boronic acid, which is hydrophilic and is very difficult to get off of the column	Run the column as quickly as possible, keep solvent flowing throughout the purification
47	Product does not respond to H2O2	Impurities in the final preparation	Purification of MitoPY1 requires a balance between running the column fast enough so that pinacol deprotection does not occur and slow enough to purify away from possible contaminants, especially triphenylphosphonium by-products, which tend to streak through the column
54	High fluorescent background in cells	Boronate converted phenol	MitoPY1 should be stored in dry, cool conditions to maintain the boronate. Boronate deprotection will yield fluorescent product, which will in turn increase the background fluorescence of the experiment and lower the sensitivity of the assay. Care should be taken to get MitoPY1 as pure as possible
	Low fluorescent background of cells	Pinacol deprotected	The boronic acid form of MitoPY1 is less cell permeable, which leads to less accumulation of the dye and less sensitivity of the assays. Stocks of MitoPY1 can be checked by LC-MS for the ratio of boronate to boronic acid, which should be primarily boronate in the final preparations. MitoPY1 should be applied to cells immediately after addition to aqueous buffer, as the boronate will slowly convert to the boronic acid in the presence of H2O