Emilio Calle

Computational Calculation of Equilibrium Constants: Addition to Carbonyl Compounds

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.

Computational Study of the Acid Dissociation of Esters and Lactones. A Case Study of Diketene

A computational study of the aqueous pK(a) of some saturated and unsaturated cyclic and linear esters and ketones was carried out at the DFT-B3LYP 6-31++G(2df,2pd), CBS-Q, and G2 levels, with the integral equation formalism polarizable continuum model for solvation, using a proton exchange mechanism. The influence of unsaturation, position of the double bond, and cyclization were studied. The computational results show that (a) in all cases studied except that of diketene (4-methylene-2-oxetanone), the alpha-beta unsaturated isomer is 20-30 kJ mol(-1) lower in energy that the beta-gamma unsaturated one; (b) alpha-beta unsaturation lowers the pK(a) of an ester approximately 6 units, whereas beta-gamma unsaturation lowers it by approximately 10 units, and cyclization lowers the pK(a) by approximately 3 units. In order to check the predictive power of the methodology, the acid dissociation constant of diketene in water was measured via kinetic study of its base-catalyzed hydrolysis. The pK(a) value obtained (15.2 +/- 0.3) is in keeping with the expected value for a beta-gamma unsaturated beta-lactone. This low value also suggests that deprotonated diketene does not interconvert to a more stable, less acidic alpha-beta unsaturated isomer, which is also consistent with computational results.

Mechanisms of Lactone Hydrolysis in Neutral and Alkaline Conditions

The neutral and base-catalyzed hydrolysis of nine carboxylic acid esters was studied using a hybrid supermolecule-PCM approach including six explicit water molecules. The molecules studied included two linear esters, four β-lactones, two γ-lactones, and one δ-lactone: ethyl acetate and methyl formate, β-propiolactone, β-butyrolactone, β-isovalerolactone, diketene (4-methyleneoxetan-2-one), γ-butyrolactone, 2(5H)-furanone, and δ-valerolactone. DFT and ab initio methods were used to analyze the features of the various possible hydrolysis mechanisms. For all compounds, reasonable to very good qualitative and quantitative agreement with experimental work was found, and evidence is provided to support long-standing hypotheses regarding the role of solvent molecule as a base catalyst. In addition, novel evidence is presented for the existence of an elimination-addition mechanism in the basic hydrolysis of diketene. A parallel work addresses the acid-catalyzed hydrolysis of lactones.

Potential of the NBP Method for the Study of Alkylation Mechanisms: NBP as a DNA-Model

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.

DNA-damaging disinfection byproducts: alkylation mechanism of mutagenic mucohalic acids.

Hydroxyhalofuranones form a group of genotoxic disinfection byproduct (DBP) of increasing interest. Among them, mucohalic acids (3,4-dihalo-5-hydroxyfuran-2(5H)-one, MXA) are known mutagens that react with nucleotides, affording etheno, oxaloetheno, and halopropenal derivatives. Mucohalic acids have also found use in organic synthesis due to their high functionalization. In this work, the alkylation kinetics of mucochloric and mucobromic acids with model nucleophiles aniline and NBP has been studied experimentally. Also, the alkylation mechanism of nucleosides by MXA has been studied in silico. The results described allow us to reach the following conclusions: (i) based on the kinetic and computational evidence obtained, a reaction mechanism was proposed, in which MXA react directly with amino groups in nucleotides, preferentially attacking the exocyclic amino groups over the endocyclic aromatic nitrogen atoms; (ii) the suggested mechanism is in agreement with both the product distribution observed experimentally and the mutational pattern of MXA; (iii) the limiting step in the alkylation reaction is addition to the carbonyl group, subsequent steps occurring rapidly; and (iv) mucoxyhalic acids, the hydrolysis products of MXA, play no role in the alkylation reaction by MXA.

Sorbate—Nitrite Interactions: Acetonitrile Oxide as an Alkylating Agent

Because chemical species with DNA-damaging and mutagenic activity are formed in sorbate-nitrite mixtures and because sorbic acid sometimes coexists with nitrite occurring naturally or incorporated as a food additive, the study of sorbate-nitrite interactions is important. Here, the alkylating potential of the products resulting from such interactions was investigated. Drawn were the following conclusions: (i) Acetonitrile oxide (ACNO) is the compound responsible for the alkylating capacity of sorbate-nitrite mixtures; (ii) ACNO alkylates 4-(p-nitrobenzyl)pyridine (NBP), a trap for alkylating agents with nucleophilic characteristics similar to those of DNA bases, forming an adduct (AD; epsilon = 1.4 x 10(4) M(-1) cm(-1); lambda = 519 nm); (iii) the NBP alkylation reaction complies with the rate equation, r = d[AD]/dt = k(alk)(ACNO)[ACNO][NBP]-k(hyd)(AD)[AD], k(alk)(ACNO) being the NBP alkylation rate constant for ACNO and k(hyd)(AD) the rate constant for the adduct hydrolysis reaction; (iv) the small fraction of ACNO forming the adduct with NBP, as well as the small magnitude of the quotient (k(alk) (ACNO)/k(hyd)(ACNO)) as compared with those reported for other alkylating agents, such as some lactones and N-alkyl-N-nitrosoureas, reveals the ACNO effective alkylating capacity to be less significant; (v) the low value of the NBP-ACNO adduct life (defined as the total amount of adduct present along the progression of the NBP alkylation per unit of alkylating agent concentration) points to the high instability of this adduct; and (vi) the obtained results are in accordance with the low carcinogenicity of ACNO.

Reactivity of the Mutagen 1,4-Dinitro-2-methylpyrrole as an Alkylating Agent

The formation of chemical species with DNA-damaging and mutagenic activity for bacterial test systems was detected in sorbic acid-nitrite mixtures. 1,4-Dinitro-2-methylpyrrole (NMP), one the main products resulting from the reaction between sorbic acid and nitrite, has mutagenic properties, and here its alkylating capacity was investigated. The conclusions drawn are as follows: (i) In aqueous medium, after the addition of a hydroxide ion and the subsequent loss of nitrite, NMP affords 5-methyl-3-nitro-1H-pyrrol-2-ol. This species is in equilibrium with 5-methyl-3-nitro-1H-pyrrol-2(5H)-one, the effective alkylating agent responsible for the genotoxic capacity of NMP; (ii) 5-methyl-3-nitro-1H-pyrrol-2(5H)-one alkylates 4-(p-nitrobenzyl)pyridine (NBP), a molecule with nucleophilic characteristics similar to those of DNA bases, forming an adduct (AD; epsilon = 1.14 x 10(4) M(-1) cm(-1)); (iii) The calculated energy barrier for the alkylation of NBP for NMP and the value of the fraction of alkylating agent forming the adduct are consistent with the observed mutagenicity of NMP; (iv) The reactivity of NMP can be explained in terms of the instability of the N-NO(2) bond as well as the effect of this group on aromaticity.

Connecting the Chemical and Biological Reactivity of Epoxides

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.

Reactivity of mucohalic acids in water

One group of disinfection byproducts of increasing interest are the halogenated furanones, which are formed in the chlorination of drinking water. Among these halofuranones is mucochloric acid (MCA, 3,4-dichloro-5-hydroxyfuran-2(5H)-one), and mucobromic acid (MBA, 3,4-dibromo-5-hydroxyfuran-2(5H)-one). Both mucohalic acids (MXA) are direct genotoxins and potential carcinogens, with the capacity to alkylate the DNA bases guanosine, adenosine and cytosine, and they have been measured in concentrations ranging up to 700 ng/l in tap water. MCA and MBA react in basic aqueous medium to form mucoxyhalic acids (4-halo-3,5-hydroxyfuran-2(5H)-one). Since: i) this reaction may represent the first step in the abiotic decomposition of mucohalic acids, ii) mucoxyhalic acids have been proposed as possible intermediates in the reaction of MXA with DNA, a kinetic study of the reaction mechanism is of interest. Here, the following conclusions were drawn: a) At moderately basic pH, the reaction of mucohalic acids with OH(-) to form mucoxyhalic acids is kinetically significant. b) The nucleophilic attack of hydroxide ions on MXA occurs through a combination of two paths: one of them is first-order in hydroxide whereas the other is second-order and are proposed to occur through the deprotonation of the hydrate of MXA. c) The hydration constants of mucohalic acids -0.23 and 0.17 for MCA and MBA respectively - corresponds to the very significant hydrate concentrations. Since hydrates are not electrophilic, these values imply a decrease in the alkylating capacity of mucohalic acids.

Reactivity of some products formed by the reaction of sorbic acid with sodium nitrite: decomposition of 1,4-dinitro-2-methylpyrrole and ethylnitrolic acid.

Sorbic acid reacts with nitrite to yield mutagenic products such as 1,4-dinitro-2-methylpyrrole (NMP) and ethylnitrolic acid (ENA). In order to know the stability of these compounds, a kinetic study of their decomposition reactions was performed in the 6.0-9.5 pH range. The conclusions drawn are as follows: (i) The decomposition of NMP occurs through a nucleophilic attack by OH- ions, with the rate equation as follows: r = k(dec)NMP[OH-][NMP] with k(dec)NMP (37.5 degrees C) = 42 +/- 1 M(-1) s(-1). (ii) The rate law for the decomposition of ENA is as follows: r = k(dec)ENA[ENA]K(a)/(K(a) + [H+]), with K(a) being the ENA dissociation constant and k(dec)ENA (37.5 degrees C) = (7.11 +/- 0.04) x 10(-5) s(-1). (iii) The activation energies for NMP and ENA decomposition reactions are, respectively, E(a) = 94 +/- 3 and 94 +/- 1 kJ mol(-1). (iv) The observed values for the decomposition rate constants of NMP and ENA in the pH range of the stomach lining cells, into which these species can diffuse, are so slow that they could be the slow determining step of the alkylation mechanisms by some of the products resulting from NMP and ENA decomposition. Thus, the current kinetic results are consistent with the low mutagenicity of these species.