Abstract Several routes for the regiospecific synthesis of lipophilic γ-conjugates of methotrexate are described. Coupling of methotrexate-α-tert-butyl ester with glycyl-TRIS- (mono-/di-/tri-) palmitate followed by trifluoroacetic acid cleavage of the α-protection afforded the target methotrexate-γ-glycyl-TRIS-(mono-/di-/tri-) palmitate derivatives. Methotrexate-α- benzyl-γ-glycyl-TRIS-tripalmitate was prepared but no method was found to selectively ...