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Supporting Text
Synthesis of l-[1,2,3-¹³C₃]dopa.
l-[1,2,3-¹³C₃]dopa was synthesized from [U-¹³C₆]glucose based on published methods of Yamada et al. (1, 2) and Lee et al. (3). Pure l-[1,2,3-¹³C₃]dopa hydrochloride was obtained in 30% yield. Synthesis of (R,S)-[1,3-¹³C₂,6-O-¹³CH₃]Coclaurine.
The synthesis of (R,S)-[1,2-¹³C₂,6-O-¹³CH₃]coclaurine was achieved according to published methods (5–9) with slight modifications, yielding pure (R,S)-[1,3-¹³C₂,6-O-¹³CH₃]coclaurine) (88%). Mass spectra (25 eV) m/z 289 [M + H]⁺, 272 [M + H – NH₃]⁺, 178 [a fragment ion of isoquinoline moiety]; isotopic distribution of ¹³C-labeled coclaurine: 0.1% [¹³C₀], 0.6% [¹³C₁], 0.3% [¹³C₂], and 99.0% [¹³C₃]. Synthesis of (R)- and (S)-[1,3,4-D₃]Norlaudanosoline.
(R)- and (S)-[1,3,4-D₃]norlaudanosoline were synthesized from papaverine hydrochloride (500 mg; 1.4 mmol) according to the methods of Rice and Brossi (10), Schönenberger and Brossi (11), and Clezy et al. (12), yielding optically pure (S)-[1,3,4-D₃]norlaudanosoline (49%) and (R)-[1,3,4-D₃]norlaudanosoline (43%). Mass spectra (25 eV) m/z 391 [M + H]⁺, 274 [M + H – NH₃]⁺, 167 [a fragment ion of isoquinoline moiety]; isotopic distribution of ²H-labeled (S)-norlaudanosoline, 2.0% [²H₀], 0.3% [²H₁], 8.3% [²H₂], 85.7% [²H₃], and 3.6% [²H₄]; and of ²H-labeled (R)-norlaudanosoline, 4.6% [²H₀], 0.8% [²H₁], 12.5% [²H₂], 81.1% [²H₃], and 0.9% [²H₄]. Synthesis of (R)- and (S)-[N-C²H₃]Reticuline.
(R)- and (S)-[N-C²H₃]reticuline were enzymatically synthesized from unlabeled (R)- and (S)-norreticuline, respectively, by action of S-adenosylmethionine synthetase (from Escherichia coli DM 25 pkA8) and N-methyltransferase (from His-tagged recombinant E. coli) (13). Optically pure (R)-[N-C²H₃]reticuline and (S)-[N-C²H₃]reticuline were obtained in 93% and 97% yield, respectively. Mass spectra (25 eV) m/z 333 [M + H]⁺, 299 [M + H – C²H₃NH₂]⁺; isotopic distribution of ²H-labeled (R)-reticuline, 0.1% [²H₀], 0.2% [²H₁], 0.5% [²H₂], 99.0% [²H₃], and 0.3% [²H₄]; and of ²H-labeled (S)-reticuline, 0.4% [²H₀], 0.5% [²H₁], 0.5% [²H₂], 98.0% [²H₃], and 0.6% [²H₄]. Synthesis of [N-C²H₃]1,2-Dehydroreticulinium Ion.
[N-C²H₃]1,2-dehydroreticuline was synthesized according to the method of Borkowski et al. (9) starting from 3-benzyloxy-N-[b -(4-benzyloxy-3-methoxyphenyl)ethyl]-4-methoxyphenylacetamide. Pure [N-C²H₃]1,2-dehydroreticulinium ion was obtained in 42% yield. Mass spectra (40 eV) m/z 331 [M + H]⁺, 312 [M + H – C²H₃]⁺, 284 [M + H – C²H₃NH₂]⁺; and isotopic distribution of ²H-labeled 1,2-dehydroreticulinium ion, 1.0% [²H₀], 0.6% [²H₁], 2.0% [²H₂], 96.0% [²H₃], and 0.4% [²H₄]. Synthesis of [7-²H]Salutaridinol and [7-²H]Episalutaridinol.
[7-²H]Salutaridinol and 7-[7-²H]episalutaridinol were synthesized from salutaridine (1 g; 3 mmol) according to the published method of Lotter et al. (14). The diastereomeric products (a mixture of salutaridinol and episalutaridinol) were obtained in 96%. The [7-²H]salutaridinol was further separated from its isomer by column chromatography [silica gel G60; 15 ´ 3 cm inside diameter, solvent system chloroform/acetone/diethylamine, 5/4/1 (vol/vol/vol)]. The [7-²H]salutaridinol was eluted first, followed by [7-²H]episalutaridinol. The crystalline [7-²H]salutaridinol was obtained in 40% yield, whereas the oily [7-²H]episalutaridinol was obtained in 38%. Mass spectra (30 eV) m/z 331 [M + H]⁺, 242 [M + H –CH₂CHN(H)CH₃-CH₃OH]⁺; and isotopic distribution of ²H-labeled [7-²H]salutaridinol, 2.5% [²H₀], 94% [²H₁], and 3.5% [²H₂]; and of [7-²H]episalutaridinol, 1.5% [²H₀], 95% [²H₁], and 3.5% [²H₂]. Synthesis of [7-²H,N-C²H₃]Oripavine.
[7-²H,N-C²H₃]Oripavine was synthesized from [7-²H]northebaine. [7-²H]Northebaine (200 mg; 0.67 mmol) was N-methylated by using the published standard method (15). [7-²H,N-C²H₃]Thebaine was further converted to [7-²H,N-C²H₃]oripavine by action of an NADPH-dependent enzyme from pig liver microsomes (16, 17). Pure [7-²H,N-C²H₃]oripavine was obtained in 16% yield. Mass spectra m/z 302 [M + H]⁺, 268 [M + H – C²H₃NH₂]⁺, and 61 [CH₃CHN(H)C²H₃]⁺; and isotopic distribution of ²H-labeled oripavine, 0.1% [²H₀], 0.2% [²H₁], 0.3% [²H₂], 30.3% [²H₃], 69% [²H₄], and 0.1% [²H₅]. Synthesis of [6-²H]Codeine.
[6-²H]Codeine was synthesized by the reduction of unlabeled codeinone with NaB²H₄ by using the standard method as described above (synthesis of [7-²H]salutaridinol). Pure [6-²H]codeine was obtained in 80% yield: Mass spectra m/z 301 [M + H]⁺, 58 [CH₃CHN(H)C²H₃]⁺; isotopic distribution of ²H-labeled codeine, 0.1% [²H₀], 99.0% [²H₁], and 0.9% [²H₂].

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