Synthesis of a fluorescent derivative of amethopterin,

Abstract

Fluorescein isothiocyanate was treated with excess diaminopentane and the remaining unsubstituted amino group of the product was condensed, via a carbodiimide-promoted reaction, with a carboxyl group of amethopterin. The final product, a fluorescent derivative of amethopterin, was isolated by chromatography on AE-cellulose and preparative electrophoresis on polyacrylamide. It was shown to be homogeneous by analytical polyacrylamide electrophoresis and thin-layer chromatography. Proof of structure was provided by elemental analysis, absorbance spectra (at pH 7.0, lambdamax at 495 nm; fluorescence emission at 520 nm), and 1H NMR measurements. The fluorescent derivative of amethopterin inhibited transport of amethopterin into Lactobacillus casei and L1210 cells. It was also a good inhibitor of the L. casei and L1210 dihydrofolate reductases and could be used to provide a fluorescent label for the enzymes during polyacrylamide electrophoresis.