103408-45-3

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The reaction of malonaldehyde under acidic conditions (pH 4.5) with guanosine 5'-monophosphate resulted in the formation of fluorescent 3-(β-ribofuranosyl)pyrimidopurin-10(3H)-one 5'-phosphate (3a).The adduct was also isolated from malonaldehyde-reacted RNA.The amount of 3a in the modified RNA was estimated to be 0.4 per cent by weight.The fluorescence spectrum of 3a (Ex max. 360 nm, Em max. 500 nm) was similar to that of guanine- and guanosine-malonaldehyde adducts which have the same type base structure.On the other hand, another type of fluorophore (Ex max. 390 nm, Em max. 460 nm) was also formed in the reaction of malonaldehyde with a nucleic acid polymer.Thus, at least two different types of fluorophores are present in malonaldehyde-reacted nucleic acid.

A novel method for the measurement of pyrimido[1,2-α]purin-10(3H)one (M1G) has been developed. Previous methods for analysis of M 1G have been confounded by the fact that this lesion exists in equilibrium between a ring-closed form and a ring-opened aldehyde form. Poor detection sensitivity of the aldehydic form can result from loss of the adduct during analysis by its reaction with amines or proteins. We utilized the aldehyde reactive probe (ARP) to produce a stable ARP-M1G-deoxyribose (ARP-M1G-dR) conjugate to minimize adduct loss. This conjugate has increased the hydrophobicity that enhances separation of ARP-M1G-dR from unmodified DNA nucleosides by using solid phase extraction. In addition, measuring ARP-M1G-dR by selective reaction monitoring (SRM) of the [ARP-M1G-dR + H]+ (635) → [M1G + H] + (188) transition increases the detection sensitivity by nearly an order of magnitude relative to the measurement of M1G-dR by SRM. For accurate measurement, analytical standard (AS) DNA and internal standard (IS) DNA were used. High purity 15N-labeled DNA was isolated from Escherichia coli that had been grown in minimum salt medium containing ( 15NH4)SO4. The 15N-DNA and calf thymus DNA were treated with malondialdehyde to induce a high number of M 1G adducts to prepare the IS and AS DNA, respectively. A consistent calibration curve was established from the analysis of 200 μg of blank DNA, 23 ng of IS DNA (400 fmol of 15N5-M1G-dR), and AS DNA containing 0-810 fmol of M1G-dR. With the use of this novel IS DNA and selective labeling, this assay is a useful tool for monitoring oxidative stress-induced DNA damage from small amounts of DNA without the need of a specific antibody or laborious procedures. By this assay, two M 1G adducts/108 guanines can readily be detected. Furthermore, this approach should be applicable to the analysis of other aldehydic DNA adducts as well as the measurement of an array of DNA lesions.

An improved method for the synthesis of oligodeoxyribonucleotides containing the endogenous adduct, pyrimido[1,2-a]purin-10(3H)-one (M1G), is reported. The key features of the methodology include improved synthesis of the deoxynucleoside of M1G by transribosylation with deoxycytidine catalyzed by nucleoside 2'-deoxyribosyltransferase and the use of commercially available 4-tert-butylphenoxyacetyl protecting groups for normal nucleotides. Facile deprotection and removal of the M1G-containing oligomers from the solid support were achieved by treatment with a solution of potassium carbonate in methanol. NMR studies were performed to determine the stability of the oligonucleotides at different pHs.