Estudio de la degradaci on de las bases heteroc clicas del ADN en soluciones acuosas de aminas

Abstract

The present work is a study of the reactions undergone by the heterocyclic bases of DNA in aqueous amine solutions at high temperature, using as simple models 2'- deoxynucleosides or, in some cases, short oligonucleotides. It is thought that the di erential response of the various types of site in the DNA is due to di erences in the reactivity of the various heterocyclic bases with the amines, as the initial step in the cleavage of the DNA chain is the chemical labilization of the nucleobases, followed by two eliminations in rapid succession, which lead to scission of the chain at the position initially attacked. In the model reactions which were studied, the degradation of the nucleosides was monitored as a function of time, and the corresponding pseudo- rst-order rate constants for their disappearance were determined. The results obtained show a signi cant di erence from the reaction rates which had been roughly estimated for the corresponding bases in the oligonucleotide setting, under the same conditions, with the exception of adenine, where the data approximately coincide. As to the reactivity of the guanine group, a considerable di erence was found between the two situations. This di erence is probably due to the e ect of the electric eld of the phosphate groups located in the oligonucleotides, which are absent in the nucleoside case. The largest di erence was seen in the reactivity of 2'-deoxythymidine, whose rate of aminolysis was slower by about one order of magnitude compared to the estimate reported for thymine bases in oligonucleotides. Against expectation, the cytosine group in the deoxynucleoside showed an increased reaction rate compared to the rate of scission of C positions in polydeoxyribonucleotides, under comparable aminolysis conditions. 3 Based on these results, di erent reaction conditions were explored. Ammonia was used as an amine of lower basicity, obtaining in this case a greater reactivity for the guanine moiety, in line with the observations made with oligonucleotides. An increase in the concentration of the amine, pyrrolidine, resulted in a monotonous rise in reaction rate. The use of ethanol-water solvents of di erent compositions was also explored. The results showed, for 2'-deoxyadenosine, a slowing of the reaction with these solvents, which was interpreted as an e ect of the reduced dielectric constant, which should complicate the formation of the zwitterion, the likely rate-determining step of the reaction. In the case of 2'-deoxyguanosine, di erent compositions of the water-alcohol solvent produced opposite e ects on the rate of aminolysis, probably re ecting opposite e ects of the reduction of the dielectric constant: destabilization of the initial zwitterions, which should slow down the reaction, and suppression of the ionization of the guanine group, which should lead to an increase in the observed reaction rate. In the mixtures obtained by aminolysis of 2'-deoxyadenosine for several hours, the free base adenine was identi ed on the grounds of its chromatographic mobility, its UV absorbance spectrum in the elution solvent, and its UV spectrum taken in aqueous solutions at various pH values. The results are compatible with a cleavage of the N-glycosidic bond in the molecule, with formation of adenine and 2-deoxyribose as the primary products. The chromatographic trace of the reaction mixture also showed one band which, based on its UV spectrum, could be 3-acetylacrolein, a product of the reaction of 2- deoxyribose in the pyrrolidine solution. In the case of 2'-deoxyguanosine, some reaction products may have been lost due to insolubility. During aminolysis, 2'-deoxycytidine undergoes hydrolytic deamination at position C-4, producing 2'-deoxyuridine, which in turn reacts under these aminolysis conditions with formation of uracil. These products were identi ed in a manner similar to that used in the identi cation of adenine as a product in the aminolysis of 2'-deoxyguanosine.