Supplemental Dataset 1. Identification of C. Roseus Alkaloids. Metabolites

Home , Catharanthine, Strictosidine, Tabersonine

Supplemental Dataset 1. Identification of C. roseus alkaloids.

Metabolites identified in C. roseus flowers by LC-FT-ICR-MS. For each metabolite it is indicated whether the metabolite was identified based on an authentic standard or whether it was tentatively identified based on its accurate mass and MSn fragmentation.

Compound Identification Fragmentation spectra Catharanthine Standard Schweizer et al., 2018 Vindoline Standard Schweizer et al., 2018 Strictosidine Standard Schweizer et al., 2018 Secologanin Standard Schweizer et al., 2018 16-Hydroxytabersonine Standard Schweizer et al., 2018 19-Hydroxytabersonine Standard Schweizer et al., 2018 Hörhammericine Standard Schweizer et al., 2018 Vinblastine Standard Schweizer et al., 2018 Vincristine Standard Schweizer et al., 2018 Akuammicine Standard Schweizer et al., 2018 Vincadifformine Standard Schweizer et al., 2018 Loganin Standard Schweizer et al., 2018 Isositsirikine Standard Schweizer et al., 2018 16-Methoxyhörhammericine Predicted Schweizer et al., 2018 16-Hydroxyhörhammericine Predicted Schweizer et al., 2018 Anhydrovinblastine Predicted Schweizer et al., 2018 Geissoschizine Predicted Schweizer et al., 2018 Serpentine Predicted Schweizer et al., 2018 16‑Hydroxyvincadifformine Predicted Schweizer et al., 2018 Perivine Predicted Schweizer et al., 2018 O-acetylstemmadenine Predicted Schweizer et al., 2018 Minovincinine Predicted Schweizer et al., 2018 Unknown 1 (isositsirikine isomer) Predicted Schweizer et al., 2018 Unknown 2 (strictosidine aglycone isomer) Predicted Schweizer et al., 2018 16-hydroxy-19-O-acetyltabersonine Predicted Schweizer et al., 2018 Strictosidine secologanoside Predicted Schweizer et al., 2018 Deacetylvindoline Predicted This study Desacetoxyvindoline Predicted This study Demethoxyvindoline = vindorosine Predicted This study Desacetoxyvindorosine Predicted This study Deacetylvindorosine = catharosine Predicted This study Strictosidinic Acid Predicted This study 19-O-acetyltabersonine Predicted This study Supplemental Dataset 1. Identification of C. roseus alkaloids.

Compound Identification Fragmentation spectra 16-Hydroxylochnericine Predicted This study 16-Hydroxylochnericine glucoside Predicted This study Unknown MIA (geissoschizine isomer 1) Predicted This study Vandrikidine Predicted This study Unknown MIA (geissoschizine isomer 2) Predicted This study 16-Hydroxytabersonine glucoside Predicted This study Unknown MIA (catharanthine isomer 1) Predicted This study Unknown MIA (catharanthine isomer 2) Predicted This study Unknown MIA (catharanthine isomer 3) Predicted This study Unknown MIA (serpentine isomer) Predicted This study

Below, for each metabolite identified in this study a detailed description of its identification is given. (a) (b) 15.53 415.22264 100 100

50 50 416.22597 Relative Abundance Relative Abundance Relative 0 0 0 5 10 15 20 25 time (min) 405 410 415 420 m/z

50 50 365.1

162.1 188.1 295.1 397.1

Relative Abundance Relative Abundance Relative 326.1 258.2 337.2 0 0 100 150 200 250 300 350 m/z 100 150 200 250 300 m/z

Figure 1. Identification of deacetylvindoline. (a) Extracted Ion Current (EIC = 415.22 ± 0.01 Da) chromatogram of a C. roseus flower sample. Only one major peak with a mass corresponding to the mass of deacetylvindoline was observed. (b) FT-MS scan of the compound eluting at 15.53 min revealed an accurate mass of the [M+H]+ ion at m/z

415.22264, corresponding to the chemical formula of deacetylvindoline, C23H30N2O5 (δ ppm = -0.261). (c) MS2 fragmentation spectrum of the [M+H]+ ion at m/z 415.22. Major

daughter ions are observed at m/z 397 (loss of H2O), m/z 365 (loss of H2O + MeOH), m/z 355 (loss of methyl formate), and m/z 188. (d) MS3 fragmentation spectrum of the daughter ion at m/z 355. N H OH OH O O N H O Deacetylvindoline [M+H]+ = 415.222749

N N H H OH OH

O O O N H O N H O [M+H]+ = 397.212184 [M+H]+ = 355.201619

Figure 2. Chemical structure and proposed fragmentation of deacetylvindoline. (a) (b) 19.84 399.22764 100 100

50 50 10.28 400.23101 Relative Abundance Relative Abundance Relative 0 0 0 5 10 15 20 25 time (min) 390 395 400 405 m/z

50 50 274.1 349.1 321.2 293.2 339.2 246.1 Relative Abundance Relative 274.1 Abundance Relative 188.1 0 0 100 150 200 250 300 350 m/z 100 150 200 250 300 350 m/z

Figure 3. Identification of desacetoxyvindoline. (a) Extracted Ion Current (EIC = 399.22 ± 0.01 Da) chromatogram of a C. roseus flower sample. Two major peaks with this mass were observed in the chromatogram; the first peak eluting at 10.28 min was previously identified as 16-methoxyhörhammericine (Schweizer et al., 2018). (b) FT-MS scan of the compound eluting at 19.84 min revealed an accurate mass of the [M+H]+ ion at m/z

399.22764, corresponding to the chemical formula of desacetoxyvindoline, C23H30N2O4 (δ ppm = -0.486). (c) MS2 fragmentation spectrum of the [M+H]+ ion at m/z 399.23. Major

daughter ions are observed at m/z 381 (loss of H2O), and m/z 321 (loss of H2O + methyl formate). (d) MS3 fragmentation spectrum of the daughter ion at m/z 381. N H

OH O O N H O Desacetoxyvindoline [M+H]+ = 399.227834

N N H H

O O N H O N O [M+H]+ = 381.217269 [M+H]+ = 321.196140

Figure 4. Chemical structure and proposed fragmentation of desacetoxyvindoline. (a) (b) 19.43 427.22256 100 100

50 50 428.22589 Relative Abundance Relative Abundance Relative 0 0 0 5 10 15 20 25 time (min) 420 425 430 435 m/z

50 50

158.1 409.2 307.2 325.2 Relative Abundance Relative 335.1 Abundance Relative 132.1 222.1 0 0 150 200 250 300 350 m/z 100 150 200 250 300 m/z

Figure 5. Identification of vindorosine. (a) Extracted Ion Current (EIC = 427.22 ± 0.01 Da) chromatogram of a C. roseus flower sample. Only one major peak with a mass corresponding to the mass of vindorosine was observed. (b) FT-MS scan of the compound eluting at 19.43 min revealed an accurate mass of the [M+H]+ ion at m/z 427.22256,

corresponding to the chemical formula of vindorosine, C24H30N2O5 (δ ppm = -0.441). (c) MS2 fragmentation spectrum of the [M+H]+ ion at m/z 427.22. Major daughter ions are observed at m/z 409 (loss of MeOH), m/z 367 (loss of acetate), and m/z 158. These fragments ions are similar to those of vindoline (Schweizer et al., 2018), which has an additional O-methyl group compared to vindorosine, leading to a difference of 30 amu for all fragment ions. (d) MS3 fragmentation spectrum of the daughter ion at m/z 367. 367 N H O H O N HO O O Vindorosine [M+H]+ = 427.222749

N N H H O H O N HO O N O O O [M+H]+ = 367.201619 [M+H]+ = 409.212184

Figure 6. Chemical structure and proposed fragmentation of vindorosine. (a) (b) 12.80 369.21713 100 100

20.06

50 50 378.52419 367.16487

370.22093 Relative Abundance Relative Abundance Relative 0 0 0 5 10 15 20 25 time (min) 360 365 370 375 m/z

50 50 244.2 291.3 319.2 309.2 263.2 Relative Abundance Relative 244.2 Abundance Relative 158.1 0 0 100 150 200 250 300 m/z 100 150 200 250 300 m/z

Figure 7. Identification of desacetoxyvindorosine. (a) Extracted Ion Current (EIC = 369.22 ± 0.01 Da) chromatogram of a C. roseus flower sample. Two low abundant peaks with this mass were observed in the chromatogram. As the very similar desacetoxyvindoline elutes at 19.84 min, the compound eluting at 20.06 minutes is our prime candidate. (b) FT-MS scan of the compound eluting at 20.06 min revealed an accurate mass of the [M+H]+ ion at m/z 369.21713, corresponding to the chemical formula 2 of desacetoxyvindorosine, C22H28N2O3 (δ ppm = -0.377). (c) MS fragmentation spectrum of the [M+H]+ ion at m/z 369.22. Major daughter ions are observed at m/z 351 (loss of 3 H2O) and m/z 291 (loss of H2O + methyl formate). (d) MS fragmentation spectrum of the daughter ion at m/z 351. N H

OH O N H O Desacetoxyvindorosine [M+H]+ = 369.217269

N N H H

O N H N H O [M+H]+ = 351.206705 [M+H]+ = 291.185575

Figure 8. Chemical structure and proposed fragmentation of desacetoxyvindorosine. (a) (b) 15.65 391.28415 100 100

16.46 385.21203

50 17.16 50

371.19637 Relative Abundance Relative Abundance Relative 0 0 0 5 10 15 20 25 time (min) 375 380 385 390 m/z

50 50 355.3

158.2 367.1 Relative Abundance Relative 265.1 Abundance Relative 132.2 180.2 307.1 0 0 100 150 200 250 300 350 m/z 100 150 200 250 300 m/z

Figure 9. Identification of deacetylvindorosine. (a) Extracted Ion Current (EIC = 385.21 ± 0.01 Da) chromatogram of a C. roseus flower sample. A few low abundant peaks with this mass were observed in the chromatogram. As the very similar deacetylvindoline elutes at 15.53 min, the compound eluting at 15.65 minutes is our prime candidate. (b) FT-MS scan of the compound eluting at 15.65 min revealed an accurate mass of the [M+H]+ ion at m/z 385.21201, corresponding to the chemical formula of 2 deacetylvindorosine, C22H28N2O4 (δ ppm = -0.451). (c) MS fragmentation spectrum of the [M+H]+ ion at m/z 385.21. Major daughter ions are observed at m/z 355 and m/z 325 (loss of methyl formate). (d) MS3 fragmentation spectrum of the daughter ion at m/z 325. N H OH OH O N H O Deacetylvindorosine [M+H]+ = 385.212184

N N H H OH OH

O N H N H O O [M+H]+ = 367.201619 [M+H]+ = 325.191054

Figure 10. Chemical structure and proposed fragmentation of deacetylvindorosine. (a) (b) 517.21793 500.1 100 100

50 50

337.1 355.1 Relative Abundance Relative Abundance Relative 0 0 510 515 520 525 m/z 150 200 250 300 350 400 450 m/z

320.2 337.1 50 50

144.1 285.0 251.1 Relative Abundance Relative 482.0 Abundance Relative 0 0 150 200 250 300 350 400 450 m/z 100 150 200 250 300 m/z

Figure 11. Identification of strictosidinic acid. (a) FT-MS scan of the unknown peak revealed an accurate mass of the [M+H]+ ion at m/z 517.21793, corresponding to the 2 chemical formula C26H32N2O9 (δ ppm = -0.246). (b) MS fragmentation spectrum of the ion at m/z 517.22. The major daughter ion observed at m/z 500 arises from the loss of an

NH3 molecule (17 Da), a neutral loss previously observed for strictosidine (Schweizer et al., 2018). Additional fragment ions were observed at m/z 355 and m/z 337, resulting from

the loss of a hexose moiety (162 Da), or a hexose and a H2O molecule (162 + 18 Da), respectively. (c) MS3 fragmentation spectrum of the daughter ion at m/z 500. In analogy with the fragmentation of strictosidine, major granddaughter ions were observed at m/z 338 and m/z 320, resulting from the loss of a hexose moiety (162 Da), or a hexose and a 3 H2O molecule (162 + 18 Da), respectively. (d) MS fragmentation spectrum of the daughter ion at m/z 355. Major granddaughter ions were observed at m/z 338 (loss of

NH3), m/z 337 (loss of H2O), and m/z 144, the latter corresponding to an indole molecule. 500 355 OH NH O O N H H OH O HO OH HO

O Strictosidinic acid [M+H]+ = 517.218057

144 338 320 338 OH O NH O OH N N H H OH H O HO O OH 337 HO HO O O [M+H]+ = 500.191508 [M+H]+ = 355.165234

N OH N N N H N H H H H O O O [M+H]+ = 144.080776 HO HO HO

O O O [M+H]+ = 320.128125 [M+H]+ = 338.138685 [M+H]+ = 337.154669

Figure 12. Chemical structure and proposed fragmentation of strictosidinic acid. (a) (b) 395.19642 335.1 100 100

228.1 50 396.22799 50 222.0 275.1 303.1 366.1 391.28415

Relative Abundance Relative Abundance Relative 168.1 0 0 385 390 395 400 m/z 100 150 200 250 300 350 m/z

50 228.2 50 247.1 200.1

Relative Abundance Relative 168.1 Abundance Relative 0 0 100 150 200 250 300 m/z 50 100 150 200 m/z

Figure 13. Identification of 19-O-acetyltabersonine. (a) FT-MS scan of the unknown peak revealed an accurate mass of the [M+H]+ ion at m/z 395.19642, corresponding to the 2 chemical formula C23H26N2O4 (δ ppm = -0.288). (b) MS fragmentation spectrum of the ion at m/z 395.20. The major daughter ion observed at m/z 335 arises from the loss of either an acetate moiety, which would point to an acetylated molecule, or the loss of methyl formate. Additional daughter ions are observed at m/z 366, m/z 303, m/z 275, m/z 228, and m/z 222. (c) MS3 fragmentation spectrum of the daughter ion at m/z 335. Major granddaughter ions are observed at m/z 303 (loss of MeOH, and indicating an O-methyl group), m/z 275 (loss of methyl formate), m/z 247, and m/z 228. (d) MS3 fragmentation spectrum of the daughter ion at m/z 228. Two major granddaughter ions were observed at m/z 168, and m/z 196. The occurrence of a daughter ion at m/z 228 and its MS3 fragmentation into these two major ions was also observed for tabersonine (Schweizer et al., 2018). Combined with the observed loss of an acetate moiety, this fragmentation indicates the unknown metabolite corresponds to 19-O-acetyltabersonine. 228

335 N H O O

N O H O 19-O-acetyltabersonine [M+H]+ = 395.196534

228

N H

N O N O H O H O [M+H]+ = 228.101905 [M+H]+ = 335.175404

N N H H

N N N H H O N O [M+H]+ = 168.080776 [M+H]+ = 196.07569 [M+H]+ = 275.154275 [M+H]+ = 303.149190

Figure 14. Chemical structure and proposed fragmentation of 19-O-acetyltabersonine. (a) (b) 369.18078 244.1 100 100 337.1

50 50 309.1 370.18412

351.1 Relative Abundance Relative Abundance Relative 138.2 222.2 0 0 360 365 370 375 m/z 100 150 200 250 300 m/z

184.1

50 50

186.2 Relative Abundance Relative Relative Abundance Relative 230.1 214.1 253.2 0 0 100 150 200 m/z 100 150 200 250 300 m/z

Figure 15. Identification of 16-hydroxylochnericine. (a) FT-MS scan of the unknown peak revealed an accurate mass of the [M+H]+ ion at m/z 369.18078, corresponding to the 2 chemical formula C21H24N2O4 (δ ppm = -0.281). (b) MS fragmentation spectrum of the [M+H]+ ion at m/z 369.18. The fragmentation spectrum is similar to that of lochnericine, which has major daughter ions at m/z 228, m/z 293, and m/z 321 (Schweizer et al., 2018). The ions observed for this unknown metabolite are 16 Da heavier, pointing to a lochnericine-like molecule with an additional hydroxy group. (c) MS3 fragmentation spectrum of the daughter ion at m/z 244. Two major granddaughter ions were observed at m/z 184 and m/z 212. The occurrence of a daughter ion at m/z 244 and its MS3 fragmentation into these two major granddaughter ions was also observed for 16- hydroxytabersonine (Schweizer et al., 2018). As such, the unknown molecule is likely lochnericine, with an additional hydroxy group at the C-16 position: 16- hydroxylochnericine (d) MS3 fragmentation spectrum of the daughter ion at m/z 337. 244 O

N H

O HO N H O 16-hydroxylochnericine [M+H]+ = 369.180884

N H

HO N N O HO O O [M+H]+ = 337.154669 [M+H]+ = 244.096820

N H

HO N HO N H H O HO N [M+H]+ = 184.075690 [M+H]+ = 212.070605 [M+H]+ = 309.159754

Figure 16. Chemical structure and proposed fragmentation of 16-hydroxylochnericine. (a) (b) 531.23351 369.1 100 100

50 50 406.1 532.23681 499.2 244.1

Relative Abundance Relative Abundance Relative 337.2 0 0 520 525 530 535 m/z 150 200 250 300 350 400 450 m/z

309.2 50 50

184.1 Relative Abundance Relative 244.2 Abundance Relative 212.0 0 0 100 150 200 250 300 m/z 100 150 200 250 300 350 m/z

Figure 17. Identification of 16-hydroxylochnericine glucoside. (a) FT-MS scan of the unknown peak revealed an accurate mass of the [M+H]+ ion at m/z 531.23351, 2 corresponding to the chemical formula C27H34N2O9 (δ ppm = -0.371). (b) MS fragmentation spectrum of the [M+H]+ ion at m/z 531.23. The major daughter ion at m/z 369 points to the loss of a hexose residue. Other major daughter ions are observed at m/z 499 (loss of MeOH), m/z 406, m/z 337, and m/z 244. This fragmentation pattern indicates the unknown metabolite corresponds to a MIA glucoside, with the MIA having a [M+H]+ of 369.18 Da. (c) MS3 fragmentation spectrum of the daughter ion at m/z 369. (d) MS3 fragmentation spectrum of the daughter ion at m/z 406. the presence of a granddaughter ion at m/z 244 and two minor granddaughter ions at m/z 184 and m/z 212 indicates a 16- hydroxylochnericine backbone. As such, the unknown glucoside likely corresponds to 16- hydroxylochnericine glucoside. 406 O

OH N H 369 HO O

O HO O N H O HO O 16-hydroxylochnericine glucoside OH N [M+H]+ = 531.233707 H HO O

HO O N OH O HO [M+H]+ = 499.207492 HO 244 O 244 O O HO O N N H O HO H [M+H]+ = 406.1496430

O HO N H O + [M+H] = 369.180884 O

N H

HO N N O HO O O [M+H]+ = 337.154669 [M+H]+ = 244.096820

N H

HO N HO N H H O HO N [M+H]+ = 184.075690 [M+H]+ = 212.070605 [M+H]+ = 309.159754

Figure 18. Chemical structure and proposed fragmentation of 16-hydroxylochnericine glucoside. (c) (c) (a) (a) illustrative forthe fragmentation ofthe metabolite. unknown 19 20, Figure in proposed isomer The group. hydroxy free a of an H a be of loss to metabolite unknown the propose of isomer we spectrum, fragmentation this on Based at ( ( a to corresponds likely metabolite unknown for observed also at observed [M+H] C the formula of spectrum chemical fragmentation the to [M+H] the of corresponding mass accurate an revealed peak unknown the of 19. Figure Schweizer d

Relative Abundance 100 Relative Abundance 100

MS ) m 50 50 0 0 /

345 z 108and

3 75 rgetto setu o te agtr o at ion daughter the of spectrum fragmentation

350 geissoschizine Identification of an unknown MIA ( MIA unknown an of Identification 2 et al., 2018), fragmentation of this ion leads to two major granddaughter ions granddaughter major two to leads ion this of fragmentation 2018), al., et 353.18581 O molecule in the MS the in molecule O 100 m 108.1

z m 321 (loss of (loss 321 354.18920 355 125 / z isositsirikine 144.( 144.1

150 360

171.1 175

with an open E-ring. This is further supported by the observed the by supported further is This E-ring. open an with d ) MS m/z

200

MeOH and

3 222.1 fragmentation spectrum ofthe daughter ion at 225 2 fragmentation spectrum, which indicates the presence the indicates which spectrum, fragmentation

geissoschizine ) and ) m/z

+ o at ion m / z corynanthe 251. A major daughter ion at ion daughter major A251. 21 H (d) (b) geissoschizine 24 ( m

Relative Abundance 100 Relative Abundance 100

N Schweizer 50 50 / 0 0 z 2

5.9 Mjr agtr os are ions daughter Major 353.19. O 100 3 108.2 ( -type MIA with an open E-ring. open an with MIA -type 100 m

p = 041. ( -0.451). = ppm 144.1 z 5. ie for Like 251. 158.2 150 et al., 2018), indicating the indicating 2018), al., et isomer 1). ( 1). isomer 150 170.2

Z + 170.2 o at ion -geissoschizine, is only is -geissoschizine,

200 210.1 172.2 200

251.2 250 m a ) FT-MSscan ) / 251.2 z

250 m isositsirikine 353.18581, m / z

303.1 300 / 251 was was 251 z b 293.1 321. 321.1 MS )

300 335.2

m/z m/z 2

N H H 251 O OH O Geissoschizine isomer [M+H]+ = 353.185969 170

N H H 144 251 108 N O O N [M+H]+ = 321.1597545 H H

[M+H]+ = 251.154275

N N N H H [M+H]+ = 108.080776 [M+H]+ = 144.080776 [M+H]+ = 170.096426

Figure 20. Proposed fragmentation of the unknown MIA (geissochizine isomer 1). The isomer depicted here, 19Z-geissoschizine, is only illustrative for the possible fragmentation of the unknown metabolite. (a) (b) 383.19642 351.1 100 100

50 50 365.1 384.19983 339.1

Relative Abundance Relative Abundance Relative 258.2 307.2 200.1 0 0 375 380 385 390 m/z 100 150 200 250 300 350 m/z

258.2 50 50 228.2 333.1 305.1 200.1 276.0 Relative Abundance Relative Abundance Relative 0 0 100 150 200 250 300 m/z 100 150 200 250 300 m/z

Figure 21. Identification of vandrikidine. (a) FT-MS scan of the unknown peak revealed an accurate mass of the [M+H]+ ion at m/z 383.19642, corresponding to the chemical 2 + formula C22H26N2O4 (δ ppm = -0.297). (b) MS fragmentation spectrum of the [M+H] ion at m/z 383.20. Major daughter ions are observed at m/z 365 (loss of H2O) and m/z 351 (loss of MeOH). A less abundant daughter ion was observed at m/z 258, a daughter ion previously observed for 16-methoxyhörhammericine (Schweizer et al., 2018), suggesting an aspidosperma-type MIA backbone with a methoxy group at the C-16 position as is the case for vandrikidine. (c) MS3 fragmentation spectrum of the daughter ion at m/z 351. (d) MS3 fragmentation spectrum of the daughter ion at m/z 365. Both MS3 fragmentation spectra support that the unknown metabolite corresponds to vandrikidine. 258

365 N H OH 351

O O N H O Vandrikidine [M+H]+ = 383.196534

258 307

N N 333 H H OH 333

O O O N O N O N H H O O O [M+H]+ = 258.112470 [M+H]+ = 365.185969 [M+H]+ = 351.170319

N N N H H H

O N O N O N O O [M+H]+ = 305.164840 [M+H]+ = 333.159754 [M+H]+ = 307.144104

Figure 22. Chemical structure and proposed fragmentation of vandrikidine. (a) (b) 353.18582 321.1 100 100

50 50 354.18917

Relative Abundance Relative Abundance Relative 251.1 0 0 345 350 355 360 m/z 100 150 200 250 300 m/z

108.1 50 172.1 251.1 50 293.1

158.1 Relative Abundance Relative Relative Abundance Relative 184.1 222.2 0 0 100 150 200 250 300 m/z 75 100 125 150 175 200 225 m/z

Figure 23. Identification of an unknown MIA (geissoschizine isomer 2). (a) FT-MS scan of the unknown peak revealed an accurate mass of the [M+H]+ ion at m/z 353.18582, 2 corresponding to the chemical formula C21H24N2O3 (δ ppm = -0.422). (b) MS fragmentation spectrum of the [M+H]+ ion at m/z 353.19. A single major daughter ion was observed at m/z 321 (loss of MeOH). A minor ion was observed at m/z 251. (c) MS3 fragmentation spectrum of the daughter ion at m/z 321. (d) MS3 fragmentation spectrum of the daughter ion at m/z 251 yields two major granddaughter ions at m/z 108 and m/z 144.

Fragmentation of a daughter ion at m/z 251 into two major granddaughter ions at m/z 108 and m/z 144 was observed before for isositsirikine. Hence, it is likely that the unknown metabolite also corresponds to a corynanthe-type MIA with an open E-ring. Since only one major fragment ion, resulting from the loss of a MeOH moiety was observed in the MS2 fragmentation spectrum, only one functional group that can be easily lost upon collision induced dissociation is present on the molecule. To illustrate the fragmentation of the unknown geissoschizine isomer, a methoxylated dioxolene was used (Figure 24). N

N H H 251 O O O Geissoschizine isomer [M+H]+ = 353.185969 170

N H H 293 144 251 108 N O O N [M+H]+ = 321.1597545 H H

[M+H]+ = 251.154275

N N H H [M+H]+ = 108.080776 [M+H]+ = 144.080776 [M+H]+ = 170.096426

Figure 24. Proposed fragmentation of the unknown MIA (geissochizine isomer 2). The isomer depicted here is only illustrative for the possible fragmentation of the unknown metabolite. (a) (b) 515.23862 353.2 100 100

50 50 516.24191 483.1 Relative Abundance Relative Relative Abundance Relative 0 0 505 510 515 520 m/z 150 200 250 300 350 400 450 m/z

50 50 244.2 265.1 293.1

Relative Abundance Relative 122.2 186.2 Abundance Relative 264.1 0 0 100 150 200 250 300 m/z 150 200 250 300 350 400 m/z

(e) 293.1 100

186.1 278.1 214.1 Relative Abundance Relative 160.2 0 100 150 200 250 300 m/z

Figure 25. Identification of 16-hydroxytabersonine glucoside. (a) FT-MS scan of the unknown peak revealed an accurate mass of the [M+H]+ ion at m/z 515.23862, 2 corresponding to the chemical formula C27H34N2O8 (δ ppm = -0.335). (b) MS fragmentation spectrum of the ion at m/z 515.24. The major daughter ion observed at m/z 353 arises from the loss of a hexose moiety, whereas the ion observed at m/z 483 is derived from the neutral loss of MeOH. Together this indicates the unknown compound is a glycoside of a MIA with an O-methyl group. (c) MS3 fragmentation spectrum of the daughter ion at m/z 353. Major granddaughter ions are observed at m/z 321 (loss of MeOH, and again indicating an O-methyl group), m/z 293 (loss of methyl formate), m/z 265, and m/z 244. (d) MS3 fragmentation spectrum of the daughter ion at m/z 483. The major granddaughter ion observed at m/z 321 results from the loss of a hexose moiety. (e) MS4 fragmentation spectrum of the granddaughter ion at m/z 321. This MS4 fragmentation spectrum is identical to that of the MS3 fragmentation spectrum of the daughter ion at m/z 321 of 16-hydroxytabersonine (Schweizer et al., 2018). This indicates the unknown metabolite is likely a glucoside of 16-hydroxytabersonine. OH N H 353 HO O

O HO O N H O HO 16-hydroxytabersonine glucoside [M+H]+ = 515.238792

244

OH N N H H HO 321 O

N N O HO O HO O H O HO [M+H]+ = 353.185969 [M+H]+ = 483.212578

N N H H

O HO N HO N O HO N O [M+H]+ = 244.096820 [M+H]+ = 293.164840 [M+H]+ = 321.159754

Figure 26. Chemical structure and proposed fragmentation of 16-hydroxytabersonine glucoside. (a) (b) 337.19092 320.1 100 100

50 50 338.19429 308.1 Relative Abundance Relative Abundance Relative 0 0 330 335 340 345 m/z 100 150 200 250 300 m/z

144.1

50 288.2 50 117.2 260.1 248.1 280.1 Relative Abundance Relative Relative Abundance Relative 0 0 100 150 200 250 300 m/z 100 150 200 250 m/z

Figure 27. Identification of an unknown MIA (catharanthine isomer 1). (a) FT-MS scan of the unknown peak revealed an accurate mass of the [M+H]+ ion at m/z 337.19092, 2 corresponding to the chemical formula C21H24N2O2 (δ ppm = -0.399). (b) MS fragmentation spectrum of the [M+H]+ ion at m/z 337.19. A single major daughter ion was 3 observed at m/z 320 (loss of NH3). A minor ion was observed at m/z 308. (c) MS fragmentation spectrum of the daughter ion at m/z 320. (d) MS3 fragmentation spectrum of the daughter ion at m/z 308.

The unknown metabolite has a calculated chemical formula that is identical to

catharanthine. The observed loss of ammonia (NH3) was also observed for strictosidine and may point to a similar A-B-C-ring structure in the unknown catharanthine isomer. (c) (c) (a) (a) is nearly identical to that of metabolite unknown this of spectrum fragmentation and formula chemical calculated The of the daughter ion at at ion daughter the of spectrum fragmentation at observed C formula chemical [M+H] the of spectrum fragmentation the to [M+H] the of corresponding mass accurate an revealed peak unknown the of 28. Figure

Relative Abundance 100 Relative Abundance 100 50 50 0 0

330

100 117.1 117.1 337.19092 335

Identification of an unknown MIA ( MIA unknown an of Identification

144.1 m 150 338.19429 / z 340 2 (os f NH of (loss 320

177.1

200 m 345 /

308. m/z catharanthine 250 260.1

288.2 305.1 300 3 . mnr o ws bevd at observed was ion minor A ).

+ m/z ion at ion

isomer 1. m 21 / m H (d) (b) z catharanthine 337.19. A was ion daughter major single / 24 z 100 Relative Abundance Relative Abundance 100 320. ( 320.

N 50 50 0 0 2

100 O 2

p = 039. ( -0.399). = ppm (δ d 100 ) MS )

150 isomer 2). ( 2). isomer

3 150 fragmentation spectrum fragmentation + o at ion

200

m 200 / z m

250 a 0. ( 308. ) FT-MSscan ) / z

337.19092, 248.2 250 308.1

300 b 276.1 c MS ) MS ) 320.1

280.1 m/z m/z 3 2

(c) (c) (a) (a) 1 and 2. as annotated were that metabolites unknown the of that to similar is metabolite unknown this of spectrum fragmentation and formula chemical calculated The of the daughter ion at at ion daughter the of spectrum fragmentation at observed C formula chemical [M+H] the of spectrum fragmentation the to [M+H] the of corresponding mass accurate an revealed peak unknown the of 29. Figure 100 Relative Abundance Relative Abundance 100 50 50 0 0

330

100 117.1 117.1 337.19092 335

Identification of an unknown MIA ( MIA unknown an of Identification

144.1 m 150 338.19422 / z 340 2 (os f NH of (loss 320

345.26816 200 m 345 /

305. m/z 250 260.1

288.1 300 3 . mnr o ws bevd at observed was ion minor A ).

+ m/z ion at ion

m 21 / m H (d) (b) z catharanthine 337.19. A was ion daughter major single / 24 z

Relative Abundance 100 Relative Abundance 100 320. ( 320.

N 50 50 0 0 2

100 O 2

p = 039. ( -0.399). = ppm (δ d 100 ) MS )

150 isomer 3). ( 3). isomer

3 150 156.2 fragmentation spectrum fragmentation + o at ion

200 catharanthine 194.2

m 200 / z 222.2 m

250 a 0. ( 305. ) FT-MSscan ) / z

337.19092, 249.2 276.1 305.2 250

isomers 300 b c MS ) 287.3 MS ) 320.1

m/z m/z 3 2

(a) (b) 349.15450 317.1 100 100

263.1 50 50 350.15784 321.1 353.18586 Relative Abundance Relative Abundance Relative 0 0 340 345 350 355 m/z 100 150 200 250 300 m/z

261.0 50 50 289.2 247.0 205.1 Relative Abundance Relative Abundance Relative 0 0 100 150 200 250 300 m/z 75 100 125 150 175 200 225 m/z

Figure 30. Identification of an unknown MIA (serpentine isomer). (a) FT-MS scan of the unknown peak revealed an accurate mass of the [M+H]+ ion at m/z 349.15450,

corresponding to the chemical formula of serpentine, C21H20N2O3 (δ ppm = -0.484). (b) MS2 fragmentation spectrum of the [M+H]+ ion at m/z 349.15. Major daughter ions are observed at m/z 317 (loss of MeOH) and m/z 263. (c) MS3 fragmentation spectrum of the daughter ion at m/z 317. (d) MS3 fragmentation spectrum of the daughter ion at m/z 263.

The observed chemical formula and fragmentation spectrum is nearly identical to that of serpentine (Schweizer et al., 2018), hence, this unknown metabolite was annotated as a serpentine isomer.