Sulfo-SMCC (1 eq.)(Pierce Chemical Co., Rockford, Ill.) is dissolved in sodium acetate buffer (pH 7) and ethylenediamine dihydrochloride (5 eq.) is added. The reaction is monitored by TLC visualized with fluorescamine. When reaction is complete the reaction mixture is applied directly to a C18 reverse-phase HPLC column and eluted with a gradient of acetonitrile in triethanolamine/water, pH 7. The excess ethylene diamine elutes at the solvent front, followed by the desired product. A small amount of double coupled material formed by the condensation of one molecule of ethylenediamine with two molecules of sulfo-SMCC elutes last. The desired material is characterized by 1H NMR and IR spectroscopy, mass spectrometry, and elemental analysis.
