Reaction of diethyl pyrocarbonate with nucleic acid components: Adenosine-containing nucleotides and dinucleoside phosphates

Abstract

Reactions of diethyl pyrocarbonate with adenine-containing nucleotides and dinucleoside phosphates have been explored in order to assess both chemical and biochemical effects of this reagent upon adenosine units in nucleic acids. 5′-AMP and 3′(2′)-AMP were converted, by imidazole ring opening, to corresponding 4,5- dicarbethoxyamino-6-N- ribofuranosylamino-pyrimidine phosphates, characterized as the disodium salts. Dinucleoside phosphate model compounds A-A, A-U and U-A were converted in high yields to N- carbethoxy derivatives VII, VIII and IX, respectively, in which the imidazole ring of each adenosine unit has been opened. Ultraviolet absorption spectra of the dinucleoside phosphate products were compared with those predicted by additive combination of the spectra of the “half” molecules in each case. Modified compounds VIII and IX from A-U and U-A were digested, if at all, more slowly than the unmodified counterparts by venom phosphodiesterase, ribonuclease T 2, bovine pancreatic ribonuclease A and bovine spleen phosphodiesterase; product VII from A-A was not hydrolyzed by any of the enzymes under the conditions employed. Potential hazards to structural integrity of nucleic acids in presence of excess diethyl pyrocarbonate are discussed, as well as possible applications to structure determination.