Identification of Adducts Formed in the Reactions of Malonaldehyde–glyoxal and Malonaldehyde–methylglyoxal with Adenosine and Calf Thymus DNA

Abstract

The reactions of adenosine with malonaldehyde and glyoxal, and with malonaldehyde and methylglyoxal resulted in the formation of one malonaldehyde-glyoxal and one malonaldehyde-methylglyoxal conjugate adduct, respectively. These adducts were isolated and purified by reversed-phase liquid chromatography, and structurally characterized by UV, (1)H- and (13)C-NMR spectroscopy, and mass spectrometry. The malonaldehyde-glyoxal adduct was identified as 8-(diformylmethyl)-3-(beta-D-ribofuranosyl)imidazo[2,1-i]purine (M(1)Gx-A), while the malonaldehyde-methylglyoxal one as 8-(diformylmethyl)-7-methyl-3-(beta-D-ribofuranosyl)imidazo[2,1-i]purine (M(1)MGx-A). Both adducts were also observed in calf thymus DNA when incubated in the respective aldehydes under physiological pH and temperature. Moreover, in the reaction of methylglyoxal and malonaldehyde with adenosine, an additional adduct was formed. This adduct was found to consist of one unit derived from methylglyoxal and one unit from formaldehyde. The adduct was identified as N(6)-(2,3-dihydroxy-2-methylpropanoyl)-9-(beta-D-ribofuranosyl)purine (MGxFA-A). Formaldehyde was found to originate from the commercial methylglyoxal in which it was present as an impurity.

Reactivity of the Malonaldehyde‐Glyoxal and Malonaldehyde‐Methylglyoxal Adducts of Adenine Nucleosides toward Amino Acid Cross‐Link Formation

“…M 1 Gx‐dA, M 1 MGx‐dA and M 1 Gx‐A were prepared according to previously described methods 14. Since the malonaldehyde‐glyoxal adduct of 2′‐deoxyadenosine, M 1 Gx‐dA, is formed in the relatively low yield, M 1 Gx‐A, the adenosine analogue of this compound was synthesised and used in the reaction with N α ‐acetyllysine carried out in preparative scale.…”

“…Such structures help to determine the mechanism responsible for the formation of these compounds. According to this mechanism, after Michael addition of the exocyclic amino group of the adenine nucleobase to the initially formed malonaldehyde‐glyoxal and malonaldehyde‐methylglyoxal conjugate respectively, the endocyclic N‐1 atom of the nucleobase attacks the carbonyl group of the conjugate that is derived from glyoxal and methylglyoxal, respectively 14. A similar situation likely takes place also in the case of formation of 3 ; the ϵ amino group of N α ‐acetyllysine attacks the carbonyl group of the opened form of hydrated M 1 Gx‐A that is derived from glyoxal but not from the α,β‐unsaturated aldehydic functionality derived from the malonaldehyde sodium salt.…”

“…16. Syntheses of the modified nucleosides: M 1 Gx‐A, its 2′‐deoxynucleoside analogue M 1 Gx‐dA and M 1 MGx‐dA were accomplished according to the methods published previously 14. All other reagents were purchased from Sigma–Aldrich or Fluka.…”

“…Recently we identified glyoxal and methylglyoxal adducts of adenine nucleosides generated in the presence of malonaldehyde,14 (Figure 1). M 1 Gx‐dA and M 1 MGx‐dA are derived from the malonaldehyde‐glyoxal and malonaldehyde‐methylglyoxal condensation products, respectively.…”

Synthesis, structural studies and stability of model cysteine containing DNA–protein cross-links

“…Recently, we found that aldehydic adducts of adenine nucleosides 28,29 (Fig. 1) exhibit potent cross-linking activity.…”