Identification of Fluorescent 2‘-Deoxyadenosine Adducts Formed in Reactions of Conjugates of Malonaldehyde and Acetaldehyde, and of Malonaldehyde and Formaldehyde

Abstract

2'-Deoxyadenosine was reacted with malonaldehyde in the presence of formaldehyde or acetaldehyde. The reactions were carried out at 37 degrees C in aqueous solution at acidic conditions. The reaction mixtures were analyzed by HPLC. In both reactions, two major products were formed. The reaction products were isolated and purified by C18 chromatography, and their structures were characterized by UV absorbance, fluorescence emission, (1)H and (13)C NMR spectroscopy, and mass spectrometry. The reaction products isolated from the mixture containing formaldehyde, malonaldehyde, and deoxyadenosine were identified as 3-(2'-deoxy-beta-D-ribofuranosyl)-7H-8-formyl[2,1-i]pyrimidopurine (M(1)FA-dA) and 9-(2'-deoxy-beta-D-ribofuranosyl)-6-(3,5-diformyl-1, 4-dihydro-1-pyridyl)purine (M(2)FA-dA). In the reaction mixture consisting of acetaldehyde, malonaldehyde, and deoxyadenosine, the identities of the products were determined to be 3-(2'-deoxy-beta-D-ribofuranosyl)-7-methyl-8-formyl[2, 1-i]pyrimidopurine (M(1)AA-dA) and 9-(2'-deoxy-beta-D-ribofuranosyl)-6-(3,5-diformyl-4-methyl-1, 4-dihydro-1-pyridyl)purine (M(2)AA-dA). The yields of the compounds were 1.8 and 0.7% for M(1)FA-dA and M(2)FA-dA, respectively, and 6.8 and 10% for M(1)AA-dA and M(2)AA-dA, respectively. All compounds exhibited marked fluorescent properties. These findings show that in addition to direct reaction of a specific aldehyde with 2'-deoxyadenosine, aldehyde conjugates also may react with the base. Although three of the adducts (M(1)FA-dA, M(2)FA-dA, and M(1)AA-dA) could not be detected in reactions carried out under neutral conditions, the possibility that trace amounts of the adducts may be formed under physiological conditions cannot be ruled out. Therefore, conjugate adducts must be considered in work that aims at clarifying the mechanism of aldehyde genotoxicity.

Quantitative Analysis of Histone Modifications: Formaldehyde Is a Source of Pathological N6-Formyllysine That Is Refractory to Histone Deacetylases

“…Considering that formaldehyde reacts with amines to give a carbinolamine intermediate that is only one oxidation state away from a formamide functional group (Figure 1), we hypothesized that endogenous formaldehyde could serve as a source of N 6 -formyllysine residues in histone and other proteins. In addition to environmental and occupational sources [19]–[21], formaldehyde arises from cellular processes such as oxidative demethylation of nucleic acid and histone proteins, as well as biosynthesis of purines, thymidine, and some amino acids [20], [22], [23], making it a relatively abundant metabolite at concentrations ranging from 13 to 97 µM in human plasma [20]. To test this hypothesis and to clarify the cellular locations and quantities of N 6 -formyllysine relative to other major histone modifications, we developed a novel liquid chromatography-coupled electrospray tandem mass spectrometry (LC-MS/MS) method to quantify all N 6 -methyl-, -acetyl-, and -formyl-lysine modifications.…”

Formation of Malonaldehyde−Acetaldehyde Conjugate Adducts in Calf Thymus DNA

“…Previous studies have shown that malonaldehyde undergoes condensation reactions with acetaldehyde, and the products may consist of one or two units of malonaldehyde and one unit of acetaldehyde (M 1 AA and M 2 AA, Scheme ) (). The conjugates are highly reactive electrophiles, and we have shown that they react with nucleophilic positions in the nucleobases ( − ). Studies of the formation of malonaldehyde−acetaldehyde conjugate adducts in DNA have not been conducted.…”

“…Reactions of the mixture of malonaldehyde and acetaldehyde with 2‘-deoxyadenosine, 2‘-deoxyguanosine, and 2‘-deoxycytidine were performed at 37 °C in aqueous buffered solutions using methods described in our previous papers ( − ). Pure malonaldehyde sodium salt was used instead of 1,1,3,3-tetraethoxypropane (TEP) or 1,1,3,3-tetramethoxypropane (TMP) hydrolysate.…”

“…General Procedure for Preparation of the 2 ‘ -Deoxynucleoside Adducts. The adducts were prepared using a procedure described in our previous studies ( − ). The 2‘-deoxynucleosides (0.04 mmol) were dissolved in 2.5 mL of a 0.5 M phosphate buffer at pH 4.6 (in the case of 2‘-deoxyadenosine and 2‘-deoxycytidine) or pH 7.4 (in the case of 2‘-deoxyguanosine).…”

“…The fluorescence spectrum, λ ex,max 324 nm, λ em,max 450 nm. More detailed NMR data for the adducts were reported in our previous publications ( − ).…”