Castells E, Mulder PPJ, Pérez-Trujillo M (2014) Diversity of pyrrolizidine alkaloids in native and invasive

Castells E, Mulder PPJ, Pérez-Trujillo M (2014) Diversity of pyrrolizidine alkaloids in native and invasive Senecio pterophorus (Asteraceae): implications for toxicity. Phytochemistry 108: 137-146 DOI 10.1016/j.phytochem.2014.06.006

Supplementary information Table S1

1 H (600 MHz) and ¹³ C NMR (150 MHz) data (δ in ppm, J in Hz) of rosmarinine in CDCl 3

Id

1

H

13

C

δ

multiplicity

δ

1 2.59 m, br 48.60

2 4.29 dd, J

= 8.21, J

= 8.20 68.93

3a 3.26 m, br 60.36

3b 3.01 dd, J

= 11.24, J

= 8,21 60.36

4 - -

5a 2.69 m, br 53.28

5b 3.49 m, br 53.28

6a 2.16 m, br 34.12

6b 2.32 dd, J

=13.68, J

= 5.80 34.12

7 5.10 br 74.73

8 3.81 m, br 69.39

9a 4.92 dd, J

= 12.67, J

= 5.00 61.40

9b 4.13 dbr, J

= 12.67 61.40

10 - 11 -

179.96 12 -

76.86

13 1.79 m 37.72

14a 2.26 dbr, J

= 13. 94 39.27 14b 1.95 dd, J

= 13.94, J

= 9.62 39.27 15 -

131.27

16 - 166.33

17 -

18 1.34 d, J

= 6.5 25.69

19 0.97 d, J

= 6.78 11.43

20 5.81 q, J

= 7.04 134.84

21 1.85 d, J

= 7.04 14.81

1

Multiplicity

J

: s (singlet), d (doublet), dd (double doublet), q (quartet), m (multiplet), br (broad signal)

2 Castells E, Mulder PPJ, Pérez-Trujillo M (2014) Phytochemistry 108: 137-146

DOI 10.1016/j.phytochem.2014.06.006

Fig. S1 Rosmarinine chemical structure and stereochemistry determined by NMR spectroscopy: A)

1 H NMR spectrum with the assignment of signals corresponding to rosmarinine, B) 1D selective ¹ H

NOE spectrum when proton 7 signal is saturated, and C) 1D selective ¹ H NOE spectrum when proton

1 signal is saturated. Spectra acquired at 298.0 K and at a magnetic field of 600 MHz.

O

N H + H

O CH₃

O H CH₃

O O C

H₃

Senecionine

M+H

O

N⁺ H

O CH3

O H CH3

O O C

H3

OH Senecionine N-oxide

M+H

O

N H + H O

CH₃ O H CH₃

O O C

H₃

Platyphylline

M+H

O

N⁺ H

O CH3

O H CH₃

O O C

H3

OH Platyphylline N-oxide

M+H

Fig. S2 Typical mass fragmentation of A) senecionine and B) senecionine N-oxide as representatives of retronecine PAs; C) platyphilline and D) platyphilline N-oxide as representatives of platynecine PAs; E) rosmarinine and F) rosmarinine N-oxide as representatives of rosmarinecine PAs; G) senkirkine as representative of otonecine PAs; H) 1,2-dihydrosenkirkine as representative of

dihydrootonecine PAs; I) 2-hydroxy-1,2-senkirkine and J) 1,2-dihydro senkirkine, 2-C ₅ H ₇ O-ester as representatives of hydroxydihydrootonecine PAs.

A) B)

C) D)

4 Castells E, Mulder PPJ, Pérez-Trujillo M (2014) Phytochemistry 108: 137-146

DOI 10.1016/j.phytochem.2014.06.006

O

N H + H

O CH₃

O H CH₃

O O C

H₃

OH

Rosmarinine

M+H

OH O

N⁺ H

O CH₃

O H CH₃

O O C

H₃

OH

Rosmarinine N-oxide

M+H

O

NH⁺ O O

CH₃ O H CH₃

O O C

H₃

CH₃ Senkirkine

M+H

O

NH⁺ O

O CH3

O H CH3

O O C

H3

CH3 Dihydrosenkirkine

M+H

M+H

O

NH⁺ O

O CH3

O H CH₃

O O C

H3

CH3 OH

2-Hydroxy-1,2-dihydrosenkirkine

M+H

O

NH⁺ O

O CH₃

O H CH₃

O O C

H₃

CH₃ O

O CH₃

CH₃

1,2-Dihydrosenkirkine, 2-C₅H₇O-ester

F) E)

G) H)

I) J)

5 Castells E, Mulder PPJ, Pérez-Trujillo M (2014) Phytochemistry 108: 137-146

DOI 10.1016/j.phytochem.2014.06.006

Fig. S3 Putative fragmentation pathways for A) senecionine and B) senecionine N-oxide as

representatives of retronecine PAs; C) platyphilline and D) platyphilline N-oxide as representatives of platynecine PAs; E) rosmarinine and F) rosmarinine N-oxide as representatives of rosmarinecine PAs;

G) senkirkine as representative of otonecine PAs; H) 1,2-dihydrosenkirkine as representative of dihydrootonecine PAs; I) 2-hydroxy-1,2-senkirkine and J) 1,2-dihydro senkirkine, 2-C ₅ H ₇ O-ester as representatives of hydroxydihydrootonecine PAs.

O

N H + H O

CH₃ O H CH₃

O O C

H₃

O H CH₂

N⁺ N

H +

OH H

C₈H₁₂NO m/z: 138.091 Da

CH₂

N⁺ C₈H₁₀N m/z: 120.081 Da Senecionine

C₁₈H₂₆NO₅ M+H⁺: 336.181 Da

CH₂

N⁺ C H₂ C₆H₈N m/z: 94.065 Da

+

- H

O - HCCH

A)

O

N⁺ H O

CH₃ O H CH₃

O O C

H₃

OH Senecionine N-oxide

C₁₈H₂₆NO₆ M+H⁺: 352.175 Da

N⁺ O OH

H CH₂

N⁺

C₈H₁₀NO m/z: 136.076 Da

CH₂

N⁺ C₈H₈N m/z: 118.066 Da

C₈H₉N m/z: 119.073 Da

+

CH2

N⁺ OH H

C₈H₁₀NO m/z: 136.076 Da

- . OH

CH₂

N⁺ H

O H CH₂

N⁺ N

H + H

OH

C₈H₁₂NO m/z: 138.091 Da

CH₂

N⁺

C₈H₁₀N m/z: 120.081 Da

CH₂

N⁺ C H₂

C₆H₈N m/z: 94.065 Da

+

- H

O - HCCH

- H

O

B)

O

N H + H O

CH₃ O H CH₃

O O C

H₃

O H CH₂

N H + H

N H + H

OH

CH₂

N H + H

C H NO

C H N

CH₂

N H +

C H N

+ ^{- C}

^H

- H

O

C)

6 Castells E, Mulder PPJ, Pérez-Trujillo M (2014) Phytochemistry 108: 137-146

DOI 10.1016/j.phytochem.2014.06.006

O

N⁺ H O

CH₃ O H CH₃

O O C

H₃

OH Platyphylline N-oxide

C₁₈H₂₈NO₆ M+H⁺: 354.191 Da

O H CH₂

N⁺ N⁺

OH

C₈H₁₂NO m/z: 138.091 Da

CH₂

N⁺ C₈H₁₀N m/z: 120.081 Da

+

- H

O

H CH₂

N H + H

N H + H

OH

CH₂

N H + H

C₈H₁₄NO m/z: 140.107 Da

C₈H₁₂N m/z: 122.096 Da

+ ^{- H}

^O

CH₂

N⁺ H

OH

+

D)

E)

O

N H + H O

CH₃ O H CH₃

O O C

H₃

OH

Rosmarinine C₁₈H₂₈NO₆ M+H⁺: 354.191 Da

N H + H

OH

OH

C₈H₁₄NO₂ m/z: 156.102 Da

CH₂

N H + H

OH O

H

+

O H CH2

N H + H

N H + H

OH

C₈H₁₂NO m/z: 138.091 Da

+

CH₂

N H + H

+

CH₂

N⁺ C8H10N m/z: 120.081 Da CH2

N⁺ C H2

- HCCH

C6H8N m/z: 94.065 Da N

H + O H

H OH

+

- H

O

- H

O

F)

OH O

N⁺ H O CH₃

O H CH3

O O C

H₃

OH

Rosmarinine N-oxide C₁₈H₂₈NO₇ M+H⁺: 370.186 Da

N⁺ H

OH

OH

CH₂

N⁺ H

OH OH

N H + H

OH

OH

C₈H₁₄NO₂ m/z: 156.102 Da

CH₂

N H + H

OH O

H

+

O H CH₂

N H + H

N H + H

OH

C₈H₁₂NO m/z: 138.091 Da

+

- H

O

CH2

N H + H

+

- H

O

CH₂

N⁺ C₈H₁₀N m/z: 120.081 Da CH2

N⁺ C H2

- HCCH

C₆H₈N m/z: 94.065 Da N

H + O H

H OH

+

OH O H CH₂

N⁺ H

+ + +

- H

O

CH₂

N⁺ H

OH

O H CH2

N⁺

+ +

- H

O

CH₂

8 Castells E, Mulder PPJ, Pérez-Trujillo M (2014) Phytochemistry 108: 137-146

DOI 10.1016/j.phytochem.2014.06.006

9 Castells E, Mulder PPJ, Pérez-Trujillo M (2014) Phytochemistry 108: 137-146

O

NH⁺ O O CH3

O H CH₃

O O C

H₃

CH₃ Senkirkine C₁₉H₂₈NO₆ M+H⁺: 366.191 Da

NH⁺ O

OH

CH₃

O H CH₂

NH⁺ O

CH3 C₉H₁₄NO₂ m/z: 168.102 Da

C8H12N m/z: 122.096 Da CH₂

NH⁺ O

CH₃ C₉H₁₂NO m/z: 150.091 Da

C₇H₉N m/z: 107.073 Da

+

- CO

CH₂

N H ⁺

CH3

CH₂

N H ⁺

- .

CH

- H

O

O

NH⁺ O O CH₃

O H CH₃

O O C

H₃

CH₃ OH

2-hydroxy-1,2-dihydrosenkirkine C₁₉H₃₀NO₇ M+H⁺: 384.202 Da

NH⁺ O

OH

CH₃ O

H CH2

NH⁺ O

CH₃ NH⁺

O OH

CH₃ OH

O H CH2

NH⁺ O

CH₃ OH

C₉H₁₆NO₃ m/z: 186.112 Da

O H

NH⁺ O

OH

CH3

+ +

C9H14NO2

m/z: 168.102 Da CH₂

NH⁺ O

CH₃

OH

+ +

- H

O

CH₂

NH⁺ O

CH₃ C₉H₁₂NO m/z: 150.091 Da C₈H₁₂N

m/z: 122.096 Da CH₂

N H ⁺

CH3

- CO - H

O

G)

H)

I)

O

NH⁺ O O CH₃

O H CH₃

O O C

H₃

CH₃ Dihydrosenkirkine

C₁₉H₃₀NO₆ M+H⁺: 368.207 Da

O

NH⁺ O CH₃

O O

C H₃

CH₃

NH⁺ O

O

CH₃

C₁₄H₂₂NO₄ m/z: 268.154 Da

N⁺ OH

C7H11NO m/z: 125.084 Da C9H12NO

m/z: 150.091 Da

C₈H₁₂N m/z: 122.096 Da

- CO C₉H₁₄NO₂ m/z: 168.102 Da

C₈H₁₄NO m/z: 140.107 Da N⁺

OH O

CH3

N⁺ O

CH₃

N⁺ CH₃ - CO

+

- H₂O

-

.

OH

CH₃

10 Castells E, Mulder PPJ, Pérez-Trujillo M (2014) Phytochemistry 108: 137-146

DOI 10.1016/j.phytochem.2014.06.006

O

NH⁺ O O CH3

O H CH₃

O O C

H₃

CH₃ O

O CH₃ CH3

1,2-dihydrosenkirkine, 2-C₅H₇O ester C₂₄H₃₆NO₈ m/z: 466.244 Da

O

NH⁺ O O CH3

O H CH₃

O O C

H₃

CH₃ C₁₉H₂₈NO₆ m/z: 366.191 Da

O⁺ CH₃ CH3

C₅H₇O m/z: 83.049 Da

CH2

NH⁺ O

CH₃ O

O CH₃ CH₃

C₁₄H₂₀NO₃ m/z: 250.144 Da

C₈H₁₂N m/z: 122.096 Da

- CO

CH₂

N H ⁺

CH₃

- C

H

O

NH⁺ O

OH

CH₃

O H CH₂

NH⁺ O

CH₃ C₉H₁₄NO₂ m/z: 168.102 Da

+

CH₂

NH⁺ O

CH₃ C₉H₁₂NO m/z: 150.091 Da

- H

O

J)