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

Abstract

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 C 2 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 Kd values of 45.8 nM (P9D5) and 37.6 nM (P7H3), respectively, for NNK-C 2. Both the monoclonal (P9D5 and P7H3) and polyclonal antibodies reacted strongly with NNK (IC 50 = 80 microM or 160 microM) and NNAL (IC 50 = 29 microM or 93 microM) 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 (IC 50 > 512 microM). 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.

Effects of antibodies induced by a conjugate vaccine on 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone absorptive transport, metabolism, and proliferation of human lung cells

“…As NNK diffuses quickly through all layers of the thick tracheobroncheal epithelium,34 extravascular NNK‐specific antibodies have multiple chances to interrupt first‐pass metabolism by sequestration of NNK in the tissue interstitium. Moreover, as the antibody crossreacts with NNAL,22 a decrease of its reuptake and subsequent metabolic activation to DNA‐adducting species can be expected, thereby providing a second chance to interrupt the multistep metabolic activation of NNK., Moreover, although binding of NNAL is only transient, the antibody‐controlled slow release of NNAL may kinetically favor a more complete phase II conjugation in individuals with a low genetic conjugation capacity, which is believed to be a major risk of lung cancer 1. Our previous studies showed that the P9D5 antibody did not bind to the detoxified metabolites resulting from pyridine N ‐oxidation 22.…”

“…The NNK‐derived hapten (NNK‐C2, Fig. 1) was synthesized and coupled to diphtheria toxoid (NNK‐C2‐DT) or ovalbumin (NNK‐C2‐OVA) as carrier proteins 22. Mice were immunized and monoclonal antibodies were produced as described earlier 22.…”

“…1) was synthesized and coupled to diphtheria toxoid (NNK‐C2‐DT) or ovalbumin (NNK‐C2‐OVA) as carrier proteins 22. Mice were immunized and monoclonal antibodies were produced as described earlier 22. The monoclonal antibody P9D5 was purified on a Protein G Sepharose 4 Fast Flow column (Pharmacia, Roosdaal, NL) equilibrated with 0.01 M sodium phosphate buffer containing 0.01 M EDTA at pH 7.0.…”

“…1). 22 Importantly, in contrast to previous NNK conjugates, the NNK‐C2 preserved most of the structural characteristics of this hapten, in particular the unaltered aromatic pyridine ring, the pyridyloxobutyl function, and the methyl nitrosamino group mimicked as part of the C2‐linker. Upon immunization, the conjugate was found to induce high titers of NNK‐specific antibodies, indicating the potential of immunoconjugates to induce immunity to this carcinogen.…”

“Click” chemistry toward bis(DOTA-derived) heterometallic complexes: potential bimodal MRI/PET(SPECT) molecular imaging probes

“…It was first decided to alkylate DO3A-OtBu (8) with an electrophile possessing a protected terminal OH group, which was expected to be converted to the azide functionality at the end of the synthetic sequence. The primary amino group of 2-ethanolamine (10) was protected with a Boc group, 15 while the OH was benzylated. 16 Literature procedures were used, 15,16 the protected 2-ethanolamine derivative 11 was obtained in 95% overall yield (Scheme 3).…”