BIOSYNTHETIC PRODUCTION OF [N-2,1,3,7,9,N-15]GUANOSINE AND [1,3,7,9-N-15]INOSINE AND CONVERSION INTO [N-6,1,3,7,9-N-15]ADENOSINE FOR STRUCTURE ELUCIDATION OF RNA BY HETERONUCLEAR NMR

A procedure was developed for the biosynthetic preparation of N-15-labelled guanosine and inosine through the action of a mutant Bacillus subtilis strain. Crude [N-2,1,3,7,9-N-15]guanosine and [1,3,7,9-N-15]inosine were isolated from the culture filtrate by precipitation and anion-exchange chromatography (Scheme 1). No cell lysis and no enzymatic degradation was necessary. The per-isobutyrylated derivatives 1 and 2 were isolated from a complex mixture, purified by virtue of their different lipophilicity, and separated in three steps involving normal and reversed-phase silica-gel chromatography. One litre of complex nutrient medium yielded 8.44 mmol of guanosine derivative and 2.84 mmol of inosine derivative with high average N-15 enrichment (83.5 and 91.9 atom-%, resp.). [N-6,1,3,7,9-N-15]Adenosine (4) was obtained from 2',3',5'-tri-O-isobutyryl[1,3,7,9-N-15]inosine (1) through the ammonolysis of its 1,2,4-triazolyl derivative with aqueous (NH3)-N-15 (Scheme 2).