Use of γ-(4-Nitrobenzyl)pyridine as Analytical Reagent for Ethylenimines and Alkylating Agents

Aziridine‐2‐carboxylic acid A reactive amino acid unit for a new class of cysteine proteinase inhibitors

“…The enzyme-specificity of this thiol alkylating agent in presence of the large excess of cysteine, used as an additive in the assay medium, relies mainly on the different pK, values of the active site and cysteine thiol group and thus on their markedly different nucleophilicity. This was confirmed in seperate experiments where we have analyzed the reaction rate of aziridine-2-carboxylic acid with an excess of cysteine under conditions of the enzyme assay (22°C, pH 6.5) by colorimetric monitoring of the aziridine-2-carboxylic acid consumption using 4-(4-nitrobenzyl)-pyridine as reagent according to the method of Epstein et al [16]. Within the limits of error of this assay system no appreciable reaction rate was observed at pH 6.5.…”

The use of monoclonal antibody conjugates for the diagnosis and treatment of cancer

“…Dye exclusion assays (25) and, less frequently, the sensitive but cumbersome clonogenic assays (26) have been used. In certain cases the activity of the drug can be tested in special assays, e.g., with chlorambucil, where the ability to alkylate 4-nitrobcnzylpyridine can be used to test drug activity (27), After appropriate assays to test drug and antibody activity, the specificity of the conjugate on cell lines needs to be tested and several different assays can be used. In the first, two different cell lines (one antibodyreactive, the other not) are separately incubated with a drug-antibody conjugate for 30 min, washed to remove unbound conjugate and then subjected to a 24 h incubation.…”

Aggregation of chlorambucil in vitro may cause misinterpretation of protein binding data

“…The aggregate (peak A) increases with the duration of incubation. Peaks B, C and D probably represent the formation of fully hydrolysed chlorambucil (B) from dichloro chlorambucil (D) by way of half-hydrolysed chlorambucil (C), as was suggested by Ross (1974 chlorambucil monomer, as measured by its reaction with 4-(p-nitrobenzyl) pyridine (Epstein et al, 1955) in ethanolic, but not aqueous, solution. Thus, the aggregate does not appear to be a covalent polymer involving reaction of one or both chloro groups with another portion of the molecule.…”

“…However, the data reported below indicate that an artefact was probably responsible for previous conclusions that active chlorambucil can bind non-covalently to immunoglobulin G; thus it will be necessary to re-evaluate the various studies in which the cytotoxicity of free chlorambucil in the presence of cell specific antibodies was compared with that of what now appears to be a non-existent conjugate of chlorambucil with antibody. Ghose and Nigam (1972) and Davies and O'Neill (1973) reported that significant quantities of chemically active chlorambucil, measured by a colorimetric assay for alkylating compounds (Epstein, Rosenthal and Ess, 1955), remained with the y-globulin which had been incubated under various conditions. In a more Accepted 10 MIarch 1975 extensive series of experiments, Blakeslee and Kennedy (1974) showed that, under a wide variety of incubation conditions, large amounts of chlorambucil eluted from a G-25 column together with the IgG with which it had been incubated.…”