The ubiquity of phenols in industrial settings means that we often have to deal with the presence of these toxins in water sources. This water can be treated using H2O2 and UV light, a cheap protocol that oxidizes phenols to polyhydroxylated products such as catechol, in addition to maleic, formic and oxalic acids — species more easily degraded by microbes or by our bodies. Phenol oxidations are unselective, and along with acids one also gets toxic by-products such as benzoquinones — reactive electrophiles that sequester biological nucleophiles. But it gets worse. Writing in Proceedings of the National Academy of Sciences, David Sedlak, Carsten Prasse and colleagues have observed the generation of 2-butene-1,4-dial, which is biologically deleterious under the oxidative conditions used for purifying water.
“This approach can capture reactive electrophiles ... that are generally very difficult to detect”
2-Butene-1,4-dial is toxic because it can cleave or crosslink DNA, and can target albumin, prealbumin and transferrin proteins. Our bodies generate the dicarbonyl when cytochrome p450 encounters furan, which is present in cigarette smoke and exhaust fumes. Sedlak's team found that UV light irradiation of phenol causes it to degrade into products that include 2-butene-1,4-dial, a reaction that is faster in the presence of H2O2, when up to 2% of the reacting phenol gives 2-butene-1,4-dial. This (and potentially other polar products) had flown under our radar because they are not easily extracted from water and are present in low concentrations. The team got around this by irradiating aqueous phenol/H2O2, and then added aliquots to separate solutions of N-α-acetyl lysine and glutathione, as well as a solution of both N-α-acetyl lysine and N-acetyl cysteine. Such derivatization — an approach also used for screening drug metabolites — enables 2-butene-1,4-dial to be trapped as a stable adduct amenable to separation and quantification. “This approach can capture reactive electrophiles, especially polar, low molecular weight compounds that are generally very difficult to detect by conventional approaches such as liquid chromatography–tandem mass spectrometry”, notes Prasse. The amount of 2-butene-1,4-dial generated is indirectly determined by quantifying the N-substituted pyrroles and pyrrolin-2-ones that result from cyclization of the dicarbonyl onto the amines.
Benzene oxidation in the gas phase has been known to first afford bicyclic alkoxy radicals, which fragment into 2-butene-1,4-dial and glyoxal. The new work by the team “provides the first experimental evidence for similar reactions in the aqueous phase”, claims Prasse, with the 2-butene-1,4-dial eventually converting into carboxylic acids. Having demonstrated the reactivity of 2-butene-1,4-dial towards amines and thiols, the team sought to find out what the phenol/H2O2 photoproducts would do to proteins. Once more, they irradiated aqueous phenol/H2O2, this time adding aliquots to a mixture of cysteic proteins. After tagging unreacted thiols with rhodamine, the team used electrophoresis to identify the proteins most susceptible to the photoproducts. Among the proteins at risk are some involved in energy metabolism, and protein and steroid biosynthesis. Moreover, isotopic labelling and mass spectrometry enabled Sedlak and co-workers to not only identify the reactive proteins but also their most reactive cysteine residues, which include one at the active site of an apoptosis-regulating enzyme.
The present phenol degradation pathway appears quite general: substituted derivatives give the respective α,β-unsaturated enedials and oxoenals, and N-acetyltyrosine undergoes ring opening to give a substituted 2-butene-1,4-dial. All products were characterized as their amino acid adducts, and in the case of N-acetyltyrosine, the photoproduct crosslinks to cysteine and lysine — a model reaction for what may occur in surface waters, as well as in skin and other cellular regions experiencing oxidative stress. To find out how dangerous the products of phenol oxidation are, “we need to determine how long these compounds persist in the presence of microbes and disinfectants used during water treatment”, says Prasse. Beyond phenols, the team is now considering other contaminants and their oxidations, such that we can best evaluate the safest method to clean our water.
Prasse, C. et al. Unexpected transformation of dissolved phenols to toxic dicarbonyls by hydroxyl radicals and UV light. Proc. Natl Acad. Sci. USA https://doi.org/10.1073/pnas.1715821115 (2018).
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Green Chemistry (2020)