Figure 3

Incorporation of deuterated l-tyrosine into major alkaloids in sacred lotus folded leaves. (A) Labeling scheme from l-tyrosine-d4 to nuciferine-d4. Deuterated dopamine-d3 and 4-HPAA-d4 condensed to form norcoclaurine-d6. Subsequent 6-O-methylation and N-methylation yielded coclaurine-d6 and N-methylcoclaurine-d6, respectively. Sequential proaporphine formation and 7-O-methylation produced pronuciferine-d5, which was converted to nuciferine-d4 after reduction, dehydration, and aromatic ring rearrangement. The red deuterium (D) atoms show the labeled positions in each structure. Dashed arrows indicate more than one reaction. Other possible intermediates resulting from diverse combinations of labeled and non-labeled dopamine and 4-HPAA have been omitted for clarity (e.g., norcoclaurine-d2 resulting from dopamine-d3 and native 4-HPAA condensation or norcoclaurine-d4 resulting from native dopamine and 4-HPAA-d4 condensation). Graphs represent the relative abundance of (B) naturally occurring alkaloids in the negative control and in L-tyrosine-d4 fed plants and (C) deuterated alkaloids derived from (i) dopamine-d3 and native 4-HPAA, (ii) 4-HPAA-d4 and native dopamine, or (iii) dopamine-d3 and 4-HPAA-d4. Values represent the mean ± standard deviation of three independent measurements. Abbreviations: 4-HPAA, 4-hydroxyphenylacetaldehyde; CNMT, coclaurine N-methyltransferase; CYP80, cytochrome P450 monooxygenase; NCS, norcoclaurine synthase; OMT, O-methyltransferase.