Studies on the nucleotide arrangement in deoxyribonucleic acids IX. Selective degradation of pyrimidine deoxyribonucleotides

Abstract

The study of the sequence of purine nucleotides in deoxyribonucleic acids requires the availability of suitable preparations of apyrimidinic acid, i.e., of polydeoxyribophosphate chains retaining the purines of the parent DNA in unchanged sequence and proportions. For an understanding of the degradation of DNA a detailed knowledge and a complete balance of the hydrazinolysis of pyrimidine deoxyribomononucleotides were required. The course and the rate of the degradation by hydrazine of thymidylic and deoxycytidylic acids were studied under a variety of conditions. These nucleotides, when treated with anhydrous hydrazine, give rise to stoichiometric amounts of (a) urea; (b) the hydrazone of 2-deoxyribophosphate; and (c) a heterocyclic compound. The latter is 4-methyl-5-pyrazolone in the case of thymidylic acid, and 3(5)-amino-pyrazole in the case of deoxycytidylic acid. The hydrazone of the sugar phosphate is cleaved quantitatively with benzaldehyde to yield 2-deoxyribophosphate and benzalazine. The various breakdown products were identified by comparison with authentic preparations. The first-order reaction of the degradation with anhydrous hydrazine is completed at 60° within 1.5 h for deoxycytidylic acid and within 4 h for thymidylic acid. At 40°, the time required for completion is about 4 times as long. Other orienting experiments demonstrated that purine deoxyribonucleotides withstand the treatment with hydrazine; that aqueous hydrazine solutions at various pH values effect a slower and irregular degradation; and that the replacement of hydrazine by monosubstituted hydrazine derivatives, such as phenyl- or methylhydrazine does not yield the desired results.