It has been suggested that Lys234 residue participates in -lactamase catalysis by acting as an electrostatic anchor for the C-3 carboxylate of penicillin. The aim of the present work is to test the role of the carboxylate group at C-3 in binding with the enzyme. A novel penicillin derivative, 3-aminomethyl-6-phenylacetamidopenicillanate, was prepared in which the carboxylic acid group at C-3 was replaced by an amino group. This was achieved by the reduction of a mixed anhydride of penicillin G to obtain 6-phenylacetamidopenicillanyl alcohol. The behavior of the alcoholic function in reacting with acidic components, following Mitsunobu reaction, was investigated, and 3-di-tert-butoxycarbonylaminomethyl-6-phenylacetamidopenicillanate was prepared as a crude product. After purification using column chromatography, the crude product undergoes deprotection of the amino group to produce the desired compound 3-aminomethyl-6-phenylacetamidopenicillanate. The hydrolysis of this compound by β-lactamase and the altered β-lactamase was determined and studied. The alteration in -lactamase was done by changing the lys234 residue to glutamic acid residue using site specific mutagenesis.