N. Leonard, J. J. Mcdonald, R. E. Henderson
Aug 31, 1971
Citations
0
Influential Citations
68
Citations
Journal
Biochemistry
Abstract
The use of diethyl pyrocarbonate as a nuclease inhibitor in the preparation of RNA of high molecular weight has prompted a study of the possible reactions of this compound with nucleic acid components under the conditions generally employed for providing inhibition. The first substrate investigated was adenine, which has been found to undergo ring opening with the formation of 5(4)-N-carbethoxyaminoimidazole-4(5)-N'-carbethoxycarboxamidine (II). This product was converted efficiently to isoguanine by treatment with ammonia. The structure of II was established by spectroscopy. For comparisons of reactivity and of spectroscopic and chromatographic properties with the adenine-diethyl pyrocarbonate product, the compounds 9-carbethoxyadenine, 6-N-carbethoxyaminopurine (V), and 6-ethylaminopurine were made; compound V was made by employing the 1-ethoxyethyl protecting group in the synthetic sequence. Purine compounds can be converted to 9-(1-ethoxyethyl) derivatives simply by refluxing in acetal. The facile reaction of adenine with diethyl pyrocarbonate illustrates the importance of gaining information as to the fate of various nucleic acid components in the presence of diethyl pyrocarbonate. The successful preparation of high molecular weight, biologically active RNA depends on the rapid inactivation of nucleases in the early steps of the isolation. Conservation of biological activity upon prolonged storage also requires the total absence of nuclease activity in these preparations. Interest in specific nuclease inhibitors was often motivated by these considerations. Recently the inhibitory activity of diethyl pyrocarbonate (DEP) (ethoxyformic anhydride) was utilized in the isolation of high molecular weight RNA.'2 This bactericidal agent3 was shown to convert pri mary and secondary amines into carbamic acid esters (urethans). More recent investigations have demonstrated that amino acids and proteins reacted in a similar way and were converted into N-carbethoxy derivatives at either free a-amino groups or the z-aminio group of lysine.5 6 In spite of these substitutions, it was concluded that irreversible inactivationl of enzymatic activity was most probably due to conformational chainges resultiing in insolubility.5 Subsequently, it was shown by others that the insolubility of proteins after reaction with DEP may be attributed to formation of intermolecular covaleint bonds, possibly as a result of a much more complicated reaction iinvolving both amino and carboxylic groups.7'8 In addition to these findings it was also reported