Neomycin B (I) was protected with Boc2O to afford (II), which was then converted to sulfonate (III) upon treatment with 2,4,6-triisopropylbenzenesulfonyl chloride and pyridine. Displacement of the sulfonate group with 2-mercaptoethylamine (IV) produced the (aminoethyl)thioether (V). This was then condensed with 9-phenoxyacrydine (VI) to furnish, after acid deprotection of the Boc groups, the title neomycin-acridine conjugate.