Although 7-deazapurines are well known and feature in the hypermodified RNA base queuosine, and in a range of nucleoside antibiotics such as toyocamycin, a mechanistic understanding of their biosynthesis is a longstanding problem. In particular, the obligatory loss of the N-7 nitrogen atom is puzzling, and in order to address this mechanistic conundrum a novel doubly labeled purine, [2-¹³C, 7-¹⁵N]-adenine, has been prepared and used as a biosynthetic precursor to toyocamycin in Streptomyces rimosus. NMR spectroscopy and mass spectrometry clearly showed incorporation of ¹³C but loss of ¹⁵N in the toyocamycin produced.