Synthesis of 4-[2-aminoethyl(nitrosamino)]-1-pyridin-3-yl-butan-1-one, a new NNK hapten for the induction of N-nitrosamine-specific antibodies

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the most abundant and potent procarcinogens in tobacco smoke. In order to induce a strong and substained antibody response against NNK, we developed a functionalized derivative that closely mimicked its structural features, in particular, the pyridyloxobutyl moiety, the adjacent ketone, and the N-nitrosamino group. This hapten was conjugated via a C₂ linker to the highly immunogenic diphteria toxoid licensed as a vaccine in humans to induce polyclonal and monoclonal antibodies. Two monoclonal antibodies were obtained with K _d values of 45.8 nM (P9D5) and 37.6 nM (P7H3), respectively, for NNK-C₂. Both the monoclonal (P9D5 and P7H3) and polyclonal antibodies reacted strongly with NNK (IC₅₀ = 80 μM or 160 μM) and NNAL (IC₅₀ = 29 μM or 93 μM;) and to a lesser extent with some of the metabolites of NNK. Interestingly, the mAbs did not react with the metabolites of the detoxification pathways such as NNK-N-Oxide and NNAL-N-Oxide (IC₅₀ > 512 μM). Therefore, such antibodies detect NNK and NNAL and may have the potential to modulate their redistribution in vivo, perhaps reducing some detrimental effects of smoking.