Methodology for the regiospecific synthesis and characterization of methotrexate conjugates

An efficient one-step synthesis of a methotrexate fluorescein conjugate is described. Reaction of dried methotrexate with 5-aminofluorescein, mediated by HBTU, produced an isomerically pure methotrexate conjugate in good yield. Proof of structure was achieved by HPLC comparison of materials synthesized by development of a regiospecific pathway employing suitable protecting groups. Either strategy offers a general solution for the preparation of regiospecific methotrexate conjugates.