Alkylating agents are considered to be archetypal carcinogens. One suitable technique to evaluate the activity of alkylating compounds is the NBP assay. This method is based on the formation of a chromophore in the reaction between the alkylating agent and the nucleophile 4-(p-nitrobenzyl)­pyridine (NBP), a trap for alkylating agents with nucleophilic characteristics similar to those of DNA bases. NBP is known to react with strong and weak alkylating agents, and much insight into such alkylation mechanisms in vivo can be gained from kinetic study of some alkylation reactions in vitro. Since 1925, the NBP assay has evolved from being a qualitative, analytical tool to becoming a useful physicochemical method that not only allows the rules of chemical reactivity that govern electrophilicity and nucleophilicity to be applied to the reaction of DNA with alkylating agents but also helps to understand some significant relationships between the structure of many alkylation substrates (including DNA) and their chemical and biological responses. Given that advances in this area have the potential to yield both fundamental and practical advances in chemistry, biology, predictive toxicology, and anticancer drug development, this review is designed to provide an overview of the evolution of the NBP method from its early inception until its recent kinetic–mechanistic approach, which allows the pros and cons of NBP as a DNA-model to be analyzed. The validity of NBP as a nucleophilicity model for DNA in general and the position of guanosine at N7 in particular are discussed.

Hydration reactions are relevant for understanding many organic mechanisms. Since the experimental determination of hydration and hemiacetalization equilibrium constants is fairly complex, computational calculations now offer a useful alternative to experimental measurements. In this work, carbonyl hydration and hemiacetalization constants were calculated from the free energy differences between compounds in solution, using absolute and relative approaches. The following conclusions can be drawn: (i) The use of a relative approach in the calculation of hydration and hemiacetalization constants allows compensation of systematic errors in the solvation energies. (ii) On average, the methodology proposed here can predict hydration constants within +/- 0.5 log K(hyd) units for aldehydes. (iii) Hydration constants can be calculated for ketones and carboxylic acid derivatives within less than +/- 1.0 log K(hyd), on average, at the CBS-Q level of theory. (iv) The proposed methodology can predict hemiacetal formation constants accurately at the MP2 6-31++G(d,p) level using a common reference. If group references are used, the results obtained using the much cheaper DFT-B3LYP 6-31++G(d,p) level are almost as accurate. (v) In general, the best results are obtained if a common reference for all compounds is used. The use of group references improves the results at the lower levels of theory, but at higher levels, this becomes unnecessary.

The alkylating potential of p-nitrostyrene oxide (pNSO)--a compound used as a substrate to study the activity of epoxide hydrolases as well as in polymer production and in the pharmaceutical industry--was investigated kinetically. The molecule 4-(p-nitrobenzyl)pyridine (NBP), as a model nucleophile for DNA bases, was used as an alkylation substrate. In order to gain insight into the effect of the hydrolysis of pNSO, as well as the hydrolysis of the NBP-pNSO adduct on the pNSO alkylating efficiency, these two competing reactions were studied in parallel with the main NBP-alkylation reaction. The following conclusions were drawn: (i) pNSO reacts through an S(N)2 mechanism, with NBP to form an adduct, pNSO-NBP (AD). The rate equation for the adduct formation is: r = d[AD]/dt = k(alk)[NBP][pNSO]-k(hyd)(AD) [AD] (k(alk), and k(hyd)(AD) being the alkylation rate constant and the NBP-pNSO adduct hydrolysis rate constant, respectively); (ii) the alkylating capacity of pNSO, defined as the fraction of initial alkylating agent that forms the adduct, is similar to that of mutagenic agents as effective as β-propiolactone. The instability of the pNSO-NBP adduct formed could be invoked to explain the lower mutagenicity shown by pNSO; (iii) the different stabilities of the α and β-adducts formed between NBP and styrene oxides show that the alkylating capacity f = k(alk)[NBP]/(k(alk)[NBP] + k(hyd)) (k(hyd) being the pNSO hydrolysis rate constant) as well as the alkylating effectiveness, AL = f/k(hyd)(AD), are useful tools for correlating the chemical reactivity and mutagenicity of styrene oxides; (iv) a pNSO-guanosine adduct was detected.

The alkylating potential of diketene (4-methylene-2-oxetanone), the basic unit of many derivatives of pesticides, chemicals, pharmaceuticals, and dyestuffs, was investigated kinetically. The nucleophile 4-( p-nitrobenzyl)pyridine (NBP), a trap for alkylating agents with nucleophilic characteristics similar to DNA bases, was used as an alkylation substrate. The alkylation reactions were performed in water/dioxane solvent mixtures. To gain insight into the effect of the hydrolysis of diketene on its alkylating efficiency, alkylation and competing hydrolysis were studied in parallel. Conclusions were drawn as follows: (i) Although diketene, unlike other four-membered ring lactones, is inactive as a carcinogen in experimental animals, it shows an alkylating potential of about 2 orders of magnitude higher than beta-propiolactone or beta-butyrolactone, which are classified as possibly carcinogenic to humans by the IARC. (ii) The reactivity of diketene as an alkylating agent is enthalpy-controlled. (iii) The fact that the hydrolysis reaction of diketene is slightly faster than those of other four-membered ring lactones shows that diketene is more efficient than beta-propiolactone or beta-butyrolactone as an alkylating agent, since the hydrolysis of this species poses less competition to the alkylation reaction. (iv) Diketene undergoes acyl fission in the alkylation reaction, which results in an amide bond in the NBP-diketene adduct. The lability of the amide bond as opposed to the amine bonds formed by beta-propiolactone and beta-butyrolactone could be one of the differential factors responsible for the lack of carcinogenicity of diketene. (v) Ab initio calculations of the energy barriers help to understand the unusual reactivity of diketene.

The chemical reactivity of the mutagenic epoxides (EP) propylene oxide (PO), 1,2-epoxybutane (1,2-EB), and cis- and trans-2,3-epoxybutane (cis- and trans-2,3-EB) with 4-(p-nitrobenzyl)pyridine (NBP), a bionucleophile model for S(N)2 alkylating agents with high affinity for the guanine-N7 position, was investigated kinetically. It was found that three reactions are involved simultaneously: the alkylation reaction of NBP by EP, which yields the corresponding NBP-EP adducts through an S(N)2 mechanism, and EP and NBP-EP hydrolysis reactions. PO and 1,2-EB were seen to exhibit a higher alkylating potential than cis- and trans-2,3-EB. From a study of the correlations between the chemical reactivity (kinetic parameters) and the biological effectiveness of oxiranes, the following conclusions can be drawn: (i) the hydrolysis reactions of epoxides must be taken into account to understand their bioactivity. (ii) The fraction (f) of the alkylating oxirane that forms the adduct and the adduct life (AL) permit the potential of epoxides as bioactive molecules to be rationalized even semiquantitatively; and (iii) alkylation of DNA by epoxides and the O(6)-/N7-guanine adduct ratio are directly related to their mutagenicity in vitro.