Biosynthetic Production of [N2,1,3,7,9-15N]Guanosine and [1,3,7,9-15N]inosine and conversion into [N6,1,3,7,9-15N]adenosine for structure elucidation of RNA by heteronuclear NMR |
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Authors: | Annette C Niemann Mnica Meyer Thomas Engeloch Oliver Botta Alfons Hdener Peter Strazewski |
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Institution: | Annette C. Niemann,Mónica Meyer,Thomas Engeloch,Oliver Botta,Alfons Hädener,Peter Strazewski |
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Abstract: | A procedure was developed for the biosynthetic preparation of 15N-labelled guanosine and inosine through the action of a mutant Bacillus subtilis strain. Crude N2,1,3,7,9-15N]guanosine and 1,3,7,9-15N]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 15N enrichment (83.5 and 91.9 atom-%, resp.). N6,1,3,7,9-15N]Adenosine ( 4 ) was obtained from 2′,3′,5′-tri-O-isobutyryl1,3,7,9-15N]inosine ( 1 ) through the ammonolysis of its 1,2,4-triazolyl derivative with aqueous 15NH3 (Scheme 2). |
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