Alkylating potential of styrene oxide: reactions and factors involved in the alkylation process.

The chemical reactivity of styrene-7,8-oxide (SO), an alkylating agent with high affinity for the guanine­N7 position and a probable carcinogen for humans, with 4-(p-nitrobenzyl)pyridine (NBP), a trap for alkylating agents with nucleophilic characteristics similar to those of DNA bases, was investigated kinetically in water/dioxane media. UV­vis spectrophotometry and ultrafast liquid chromatography were used to monitor the reactions involved. It was found that in the alkylation process four reactions occur simultaneously: (a) the formation of a ß-NBP­SO adduct through an SN2 mechanism; (b) the acid-catalyzed formation of the stable α-NBP­SO adduct through an SN2' mechanism; (c) the base-catalyzed hydrolysis of the ß-adduct, and (d) the acid-catalyzed hydrolysis of SO. At 37.5 °C and pH = 7.0 (in 7:3 water/dioxane medium), the values of the respective reaction rate constants were as follows: kalkß = (2.1 ± 0.3) × 10­4 M­1 s­1, kalkα = (1.0 ± 0.1) × 10­4 M­1 s­1, khydAD = (3.06 ± 0.09) × 10­6 s­1, and khyd = (4.2 ± 0.9) × 10­6 s­1. These values show that, in order to determine the alkylating potential of SO, none of the four reactions involved can be neglected. Temperature and pH were found to exert a strong influence on the values of some parameters that may be useful to investigate possible chemicobiological correlations (e.g., in the pH 5.81­7.69 range, the fraction of total adducts formed increased from 24% to 90% of the initial SO, whereas the adduct lifetime of the unstable ß-adduct, which gives an idea of the permanence of the adduct over time, decreased from 32358 to 13313 min). A consequence of these results is that the conclusions drawn in studies addressing alkylation reactions at temperatures and/or pH far from those of biological conditions should be considered with some reserve.

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 p-nitrostyrene oxide as an alkylating agent. A kinetic approach to biomimetic conditions.

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 ß-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 ß-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.

On the Conductivity of Proton-Exchange Membranes Based on Multiblock Copolymers of Sulfonated Polysulfone and Polyphenylsulfone: An Experimental and Modeling Study.

The effect of relative humidity (RH) and degree of sulfonation (DS) on the ionic conductivity and water uptake of proton-exchange membranes based on sulfonated multiblock copolymers composed of polysulfone (PSU) and polyphenylsulfone (PPSU) is examined experimentally and numerically. Three membranes with a different DS and ion-exchange capacity are analyzed. The heterogeneous structure of the membranes shows a random distribution of sulfonated (hydrophilic) and non-sulfonated (hydrophobic) domains, whose proton conductivity is modeled based on percolation theory. The mesoscopic model solves simplified Nernst-Planck and charge conservation equations on a random cubic network. Good agreement is found between the measured ionic conductivity and water uptake and the model predictions. The ionic conductivity increases with RH due to both the growth of the hydrated volume available for conduction and the decrease of the tortuosity of ionic transport pathways. Moreover, the results show that the ionic conductivity increases nonlinearly with DS, experiencing a strong rise when the DS is varied from 0.45 to 0.70, even though the water uptake of the membranes remains nearly the same. In contrast, the increase of the ionic conductivity between DS=0.70 and DS=0.79 is significantly lower, but the water uptake increases sharply. This is explained by the lack of microphase separation of both copolymer blocks when the DS is exceedingly high. Encouragingly, the copolymer membranes demonstrate a similar performance to Nafion under well hydrated conditions, which can be further optimized by a combination of numerical modeling and experimental characterization to develop new-generation membranes with better properties.

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.

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.

Chemical reactivity and biological activity of diketene.

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.

Alkylating potential of potassium sorbate.

A kinetic study of the alkylating potential of potassium sorbate (S)-a food preservative used worldwide-in 7:3 water/dioxane medium was performed. The following conclusions were drawn: (i) Potassium sorbate shows alkylating activity on the nucleophile 4-(p-nitrobenzyl)pyridine (NBP), a trap for alkylating agents with nucleophilic characteristics similar to those of DNA bases, (ii) The NBP alkylation reaction complies with the rate equation r = k(alk)[H+][S][NBP]/(K(a) + [H+]), K(a) being the sorbic acid dissociation constant and k(alk) the rate constant of NBP alkylation by the undissociated acid. In the range of pH 5-6, the alkylation time ranges between 18 days (pH 5.2) and >1 month (pH > or = 6). (iii) NBP alkylation occurs through a reaction with deltaH# = 78 kJ mol(-1), which is much higher than those of NBP alkylation by stronger alkylating agents. (iv) The absorption coefficient of the sorbate-NBP adduct was determined to be epsilon = 204 M(-1) cm(-1) (lambda = 580 nm), this value being rationalized in terms of the adduct structure. (v) The results can help to establish suitable expiration times for products preserved with potassium sorbate.

Biocatalytic oxidation of phenolic compounds by bovine methemoglobin in the presence of H2O2: quantitative structure-activity relationships.

In the present work, 13 p-substituted phenols with different functional groups have been systematically evaluated as metHb substrates by means of HPLC analysis. Non-hyperbolic kinetics were observed and Hill coefficients in the 0.37-1.00 range were obtained. The catalytic constants and the Hill coefficients were found to be quantitatively correlated with two independent variables: the energy level of the highest-occupied molecular orbital (E(HOMO)), which describes the intrinsic redox activity of the substrates and the pK(a)-values, which are related to substrate ionization. Oxygen evolution in the presence of each phenol derivative was also measured, and good correlation between peroxidase-like and catalase-like activities of the protein was observed. It is also shown that bovine metHb, although less active than other peroxidases, may represent a good alternative from an economical point of view for phenol removal processes. The equations here obtained may serve as a basis to further explore the potential use of metHb-mediated reactions in the treatment of phenols in wastewaters and to predict which phenol will be removed most efficiently under this treatment with satisfactory reliability.

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.

Reactivity of lactones and GHB formation.

The behavior of lactones in their hydrolysis reactions is a good indicator of their reactivity as electrophilic molecules. The hydrolysis of four- to six-membered lactones was investigated in neutral (water) and slightly acid media and in water/dioxane media. The following conclusions were drawn: (i) The reactivity of beta-propiolactone in neutral water is more than four times greater than that of beta-butyrolactone, due to the flow of charge caused by the latter's methyl substituent. Reactivity is enthalpy-controlled. (ii) The reactivity of beta-lactones diminishes in water/dioxane media when the percentage of dioxane increases. The increase in the dioxane percentage relaxing the intermolecular hydrogen bonds in the ordered structure of the water reduces DeltaH# and simultaneously increases the -DeltaS# value. (iii) An inverse solvent kinetic isotope effect in the acid-catalyzed hydrolysis of gamma-butyrolactone and delta-valerolactone was observed, this being indicative of acyl cleavage. (iv) The DeltaH# and DeltaS# values permit discrimination between alkyl and acyl cleavage. (v) A correlation was found between the chemical reactivity of lactones and their carcinogenic activity. (vi) The results suggest that orally ingested gamma-butyrolactone remains largely in its nonhydrolyzed form in the stomach before passing into the blood. (vii) The concentration equilibrium constant of GHB formation at human body temperature is Keq (37 degrees C)=0.40. (viii) Study of GHB formation shows that, contrary to earlier results, this is an endothermic process, with DeltarH=3.6 kJ mol(-1).

A kinetic approach to the alkylating potential of carcinogenic lactones.

The alkylating potential of beta-propiolactone (BPL), beta-butyrolactone (BBL), gamma-butyrolactone, and delta-valerolactone, which can be formed by the in vivo nitrosation of primary amino acids, was investigated kinetically. The nucleophile NBP, 4-(p-nitrobenzyl)pyridine, a trap for alkylating agents, was used as an alkylation substrate. The alkylation reactions were performed under mimicked cellular conditions at neutral pH in water/dioxane solvent mixtures. To gain insight into the effect of the hydrolysis of lactones on their alkylating efficiency, alkylation and competing hydrolysis were studied in parallel. Conclusions were drawn as follows: (i) gamma-Butyrolactone and delta-valerolactone afford neither appreciable NBP alkylation nor hydrolysis reactions; (ii) the alkylating potential of BPL is 10-fold higher than that of BBL, the reactivity of both being essentially enthalpy-controlled; (iii) a correlation was found between the alkylating potential of lactones and their carcinogenicity; (iv) the hydrolysis of lactones is not sufficiently effective to prevent alkylation; (v) the efficiency of alkylation, expressed as the alkylation rate/hydrolysis rate ratio, decreases strongly with increasing amounts of dioxane in the reaction media; (vi) the absorption coefficients of the NBP-lactone adducts are as follows: epsilon(NBP-BPL) = 5101 +/- 111 M(-1) cm(-1) (lambda = 584 nm) and epsilon(NBP-BBL) = 462 +/- 19 M(-1) cm(-1) (lambda = 586 nm), the pronounced difference between these values being rationalized in terms of the adducts' structure; and (vii) linear correlations exist between the adducts' absorption coefficients and the water/dioxane ratio in the reaction media.