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1,4-Dicarbonyl compounds, which include 2,5-hexanedione and recently discovered endogenous 4-ketoaldehydes (levuglandins, isoketals, and neuroketals), exhibit severe toxicity. The key step in the toxicity of these compounds is their reaction with the lysyl residues of proteins to form pyrrole adducts. To screen for effective scavengers of these toxic compounds, we determined the reaction rates of pyrrole formation for a series of primary amines with a model 4-ketoaldehyde, 4-oxopentanal (OPA). We found pyridoxamine (PM) to react extremely rapidly, with a second-order rate constant at physiological pH being approximately 2300 times faster than that of Nalpha-acetyllysine. The extreme reactivity of PM was unique to 1,4-dicarbonyls, as its reactions with methylglyoxal and 4-hydroxy-2(E)-nonenal were much slower and only slightly faster than with Nalpha-acetyllysine. The phenolic group of PM was found to be essential to its high reactivity, and the rate constant for pyrrole formation with OPA exhibited a maximum at pH 7.5, close to the second pKa of PM. We therefore propose a mechanism involving transfer of the phenolic proton to the carbonyl of the initially formed hemiacetal, which facilitates subsequent nucleophilic attack and ring closure. Only 1,4-dicarbonyls are likely to participate in the proposed mechanism, thereby conferring unique sensitivity of this class of compounds to scavenging by PM.

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In order to understand the modifications of proteins produced by aldehydes of lipid peroxidation, [1-13C]-2(E)-hexenal, [1-13C]-4-oxopentanal, and a mixture of [1-13C]- and [2-13C]-4-hydroxynon-2(E)-enal were synthesized and the reaction of each of the labeled aldehydes with bovine serum albumin was analyzed by 13C NMR spectroscopy. Protein nucleophiles add to the 3-position of hexenal, and the resulting propanal moieties appear to undergo aldol condensation, form imine cross-links with lysyl residues, or lead to pyridinium rings. During the reaction of 4-oxopentanal with the lysyl residues of bovine serum albumin, only 1-alkyl-2-methylpyrrole and a possible intermediate leading to the pyrrole were observed. Hydroxypyrrolidine cross-links such as 25 could not be detected, leaving the pyrrole as the mediator of protein cross-linking. The Michael adducts are the major products in the reaction between 4-hydroxynon-2-enal and proteins. They exist almost exclusively in the cyclic hemiacetal form and do not appear to cross-link through imine formation with lysyl residues. A minor pathway involves the reaction of 4-hydroxynon-2-enal with the lysyl amino groups of protein resulting in 2-pentylpyrrole adducts that may mediate protein cross-linking. The Michael adducts appear not to be the direct source of the pyrrole, but the imine 32 and the enamine 35 are likely intermediates toward the five-membered ring.