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

Abstract

A variety of nucleic acid components and related compounds undergo photoreaction with water to form so-called "photohydrates" (e.g. uracil forms 6-hydroxy-5,6-dihydrouracil). However, the corresponding hydrates of 5-methylcytosine (a minor nucleobase in eukaryotic DNA) and related compounds have not been characterized. We report the preparation of opened-ring forms of such products for 5-methylcytosine (m5C) and 1,5-dimethylcytosine (DMC). This was accomplished via thermal reaction of ring-opened amine adducts (e.g. N-carbamoyl-3-amino-2-methylacrylamidine (IVa) or N-(N'-methylcarbamoyl)-3-amino-2-methylacrylamidine (IVb)) produced by photo-induced reactions of m5C with ammonia or methylamine. When these adducts were treated with dilute trifluoroacetic acid, the amino group at the 3-position was replaced with a hydroxyl group; with IVa, N-carbamoyl-3-hydroxy-2-methylacrylamidine (Va) was formed, while reaction of IVb led to N-(N'-methylcarbamoyl)-3-hydroxy-2-methylacrylamidine (Vb). These compounds are ring-opened isomers of 5,6-dihydro-6-hydroxy-5-methylcytosine (Ia and IIa) and 5,6-dihydro-6-hydroxy-1,5-dimethylcytosine (Ib and IIb). Compounds Va and Vb each undergo thermal ring closure reactions to form two unstable compounds with chemical and UV spectral properties expected for Ia and IIa (or Ib and IIb). The latter compounds have been identified as minor products in UV-irradiated aqueous solutions of m5C and DMC. Evidence is also presented that the 2'-deoxycytidine photohydrates coexist with an opened-ring form, possibly similar in nature to Vb.

Mass spectrometry analysis of nucleosides and nucleotides

“…The major stable adducts were identified with HPLC, UV‐spectroscopy, and LC‐MS/MS, and this is the first study to explain the adducts of isoprene monoepoxides with dAdo. As well as reporting on cyclobutane dimers, Shetlar and Chung also reported on the preparation of opened‐ring forms of adducts of 5‐methylcytosine (m5C) and 1,5‐dimethylcytosine (DMC) (Shetlar & Chung, ). These results were achieved by the thermal reaction of ring‐opened amine adducts: N‐carbamoyl‐3‐amino‐2‐methylacrylamidine or N‐(N′‐methylcarbamoyl)‐3‐amino‐2‐methylacrylamidine, in a photo‐induced reaction of m5C with ammonia or methylamine.…”

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

“…It is known, however, that UV irradiation of polycytidylic acid in the presence of cysteine induces photobinding of this amino acid , presumably via a bond involving sulfur. It is also known that 5MeCyt photoreacts with lysine and related compounds , as well as alcohol analogs of serine and threonine .…”

Photohydrate‐Mediated Reactions of Uridine, 2′‐Deoxyuridine and 2′‐Deoxycytidine with Amines at Near Neutral pH

“…Another possible reaction that could occur at physiological pH is adduct formation via reaction of N-terminal amino groups of proteins complexed with photohydrate-containing DNA or RNA. The peptides (Gly) 2 (pK a = 8.25) and (Gly) 3 (pK a = 7.91) can be viewed as models for N-terminal amino acid regions in proteins. The observed reactivities of the amino groups of these peptides with nucleoside photohydrates suggest that, under the right circumstances, thermal adduct formation could occur in UV irradiated nucleic acid-protein complexes.…”

“…To answer this question, we examined the reaction products formed when a variety of amines were incubated at ambient temperature with a mixture of the two diastereomeric uridine hydrates at near neutral pH. In addition to EDA and GlyAm, we examined the reactivity of glycine (Gly), glycylglycine (Gly) 2 , glycylglycylglycine (Gly) 3 (DMEDA can be regarded as a model of the secondary amine moieties found in Spd and Sper.) Most of these amines have a pK a (or set of pK a values) that indicate a significant proportion of the total amine concentration exists in a partially unprotonated state in aqueous solution at pH 7.5 (see Table S1 in the Supporting Information).…”

“…For example the product formed by irradiation of uracil in water is 6‐hydroxy‐5,6‐dihydrouracil . Photohydration reactions of the pyrimidine nucleobases cytosine (Cyt) and thymine (Thy) also received early attention , while the hydrates of 5‐methylcytosine (m5Cyt) have been studied more recently . The pyrimidine nucleosides of uracil and cytosine are particularly reactive towards photohydrate formation (for general reviews, see ).…”