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Abstract
Fluorescein isothiocyanate was reacted in dimethyl sulfoxide with a tenfold excess of diaminopentane, and the mono-substituted thiourea product was isolated by DEAE-cellulose chromatography, lyophilisation 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 l-ethyl-3-(31 -dimethylaminopropyl) carbodiimide hydrochloride, and the reaction products were purified by column chromatography on DEAE-cellulose. Exhaustive elution with 1 M ammonium bicarbonate removed several byproducts then finally afforded the exclusively γ-linked fluorescein - MTX derivative. After lyophilisation and acid-base precipitation the compound was obtained in good yield (> 40%), was homogeneous by reverse-phase HPLC ∊493(0 1 N NaOH) = 66,000 and was a comparable inhibitor to MTX for rat-liver dihydrofolate reductase.