The Photochemistry of 5‐methylcytosine and 5‐methyl‐2′‐deoxycytidine in Aqueous Solution

Abstract

The nucleobase 5-methylcytosine (I) is a minor component of eukaryotic DNA thought to be important in regulation of gene expression. The photochemical reactions of this nucleobase and its 2'-deoxyribonucleoside, 5-methyl-2'-deoxycytidine (II), in water have been studied. These reactions lead, respectively, to 3-amino-2-methylacrylamidine (Ib) and 3-(2-erythro-D-pentopyranos-1-yl)amino-2-methylacrylamidine (IIb) as the main photoproducts. The structure of the photoproducts was established by spectroscopic methods (1H and 13C NMR, UV spectroscopy, electron impact and liquid secondary ion mass spectrometry); in the case of Ib, confirmatory evidence was obtained by chemical methods (photolysis of 5-methyl[2-13C]cytosine, hydrolysis of N-carbomethoxy-3-amino-2-methylacrylamidine and reaction of Ib with 1,1'-carbonyldiimidazole to give I). The quantum yield for formation of Ib was determined to be 1.8 x 10(-3) at pH 7.5 while the quantum yield for formation of IIb has a lower value of 0.2 x 10(-3) at pH 7.5. These quantum yields depend strongly on pH and reach maximum values of 2.0 x 10(-3) at pH 7.0 (Ib) and 0.6 x 10(-3) at pH 5.0 (IIb). The mechanism of formation of Ib (or IIb) is proposed to involve nucleophilic attack of water on the C-2 position of photoexcited I (or II), followed by ring opening and decarboxylation of an intermediate carbamic acid.

Opened‐Ring Adducts of 5‐Methylcytosine and 1,5‐Dimethylcytosine with Amines and Water and Evidence for an Opened‐Ring Hydrate of 2′‐Deoxycytidine

“…The solution was then diluted with 350 mL of HPLC grade acetonitrile; the measured pH of this final solution, containing both water and acetonitrile, was above 7 in all cases. (Use of this mixed solvent medium significantly increases the yield of the desired amine adducts relative to the yield of 3-amino-2-acrylamidine (III), which is the dominant product when m5C is irradiated in pure water [20].) Stock solutions of aqueous DMC (20 mM M) were similarly used for preparation of solutions containing DMC (1 mM M) and either ammonia (100 mM M) or methylamine (100 mM M).…”

Ring‐Opening Photoreactions of 5‐Methylcytosine with 3‐Mercaptopropionic Acid and Other Thiols

“…This reaction scheme takes into account the instability of carboxyl groups attached to nitrogen (e.g. in Vb); for a discussion, see (21). The appearance of IId in solutions of Ia incubated either in the presence or the absence of 3MP can be accounted for by direct hydrolysis of the thioester group in Ia, followed by loss of CO 2 (21).…”

Human DNA-demethylating activity: A glycosylase associated with RNA and PCNA

“…The specific activity was 75,000 cpm/g DNA. 14 Clabeled-5-meC was prepared by condensation of 14 C-labeled urea (U 8629; Sigma, St. Louis, MO) and 3-bromo-2-methyl-acrylonitrile according to Celewicz and Shetlar [1992]. Purification of 14 C-5-meC was by repeated isocratic high performance liquid chromatography (HPLC) and the product identity confirmed by NMR analysis.…”