Dicyclohexylcarbodiimide → Desethyl-N-acetylprocainamide

To 1.79 g of para-acetamidobenzoic acid and 1.15 g of N-hydroxysuccinimide dissolved in 15 ml of pyridine was added 2.3 g of N,N'-dicyclohexylcarbodiimide. The reaction mixture was cooled at 4 C.° for two hours and then filtered to remove crystals which had formed. The crystals were washed with approximately 2 ml of acetone and the pyridine filtrate and acetone washings were then combined. To the combined mixture was added 0.88 g of N-ethylethylenediamine. The resulting mixture was stirred for two hours and then cooled at 4 C.° for about twenty-four hours to yield a second crop of crystals. The crystals were removed from the mixture by filtration and then rinsed with acetone. The two crops of crystals (approximately 2.0 g) were combined and then dissolved in 50 ml of distilled water. The pH of the resulting mixture was adjusted to pH 10 using a 6 N sodium hydroxide solution. A white precipitate, desethyl-N-acetylprocainamide formed and was removed by filtration and dried in a dessicator. To 10 mg of the desethyl-N-acetyl-procainamide was added 10 mg of 4-[(4,6-dichlorotriazin-2-yl)-amino]fluorescein dissolved in 1 ml of methanol. The mixture was allowed to react for ten minutes, and the crude product which had formed was purified using silica gel thin-layer chromatographic techniques employing a developing solvent comprising a 1:1 mixture of chloroform:acetone to yield a desethyl-N-acetyl-procainamide-DTAF conjugate of the formula: ##STR8##