Synthesis and efficient isolation procedure for gamma-linked fluorescein methotrexate.
Fluorescein isothiocyanate was reacted in dimethyl sulfoxide with a ten-fold excess of diaminopentane, and the mono-substituted thiourea product was isolated by DEAE-cellulose chromatography, lyophilization and acid precipitation from aqueous base. The dried product was then condensed in dry dimethyl sulfoxide with Methotrexate (MTX) activated by prior incubation (30 min) with 1-ethyl-3-(3'-dimethylaminopropyl) carbodiimide hydrochloride, and the reaction products were purified by column chromatography on DEAE-cellulose. Exhaustive elution with 1 M ammonium bicarbonate removed several by-products then finally afforded the exclusively gamma-linked fluorescein--MTX derivative. After lyophilization and acid-base precipitation the compound was obtained in good yield (greater than 40%), was homogeneous by reverse-phase HPLC epsilon 493 (0.1 N NaOH) = 66,000 and was a comparable inhibitor to MTX for rat-liver dihydrofolate reductase.
D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005493:Folic Acid Antagonists; D008099:Liver / Q000201:enzymology; D008727:Methotrexate / Q000031:analogs & derivatives / Q000494:pharmacology; D051381:Rats; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D013861:Thiocyanates
D005452:Fluoresceins; D005493:Folic Acid Antagonists; D013861:Thiocyanates; D016650:Fluorescein-5-isothiocyanate; D008727:Methotrexate