Fig. 2: Identification of three key on-pathway intermediates (11–13) through feeding studies.

a, Schematic illustrating the natural conversion of 6 into the corresponding alcohol congener 11, along with the structure of the synthetic isotopically labelled analogue 11b that was used to feed tissues from C. pubescens plantlets. MS/MS (20 to 50 eV) spectrum of the synthetic subtract 11b is shown. b,c, Extracted ion chromatograms (EICs) and MS/MS (20 to 50 eV) spectra evidencing the incorporation of 11b into 1, 2 and 10. Labelled compounds elute slightly faster than the non-labelled counterparts (a retention time difference of about 0.05 min). In b, the asterisk indicates an unknown compound that is not part of the cinchona alkaloid pathway as evidenced by re-feeding experiments with partially purified material of that labelled compound. Data showing incorporation of 11b into keto-quinoline intermediates 8 and 9 are provided in Supplementary Fig. 4. d–g, Identification of a previously unknown compound, 12, and cyclized cinchonaminal (13) as on-pathway intermediates. EIC and MS/MS spectra evidencing the incorporation of 11b into 12 and 13 are shown. Details on the structural characterization and synthesis of 12 and 13 are provided in the Supplementary Information. EIC and MS/MS spectra demonstrating the incorporation of 12b and 13b into downstream alkaloids 1, 2, 10 and 13 are similar to the data shown in b, c and f (Supplementary Fig. 5). Mock denotes samples fed with water instead of the labelled compound (control group).