What happens after activation of ascaridole? Reactive compounds and their implications for skin sensitization.

Transcription

1 What happens after activation of ascaridole? Reactive compounds and their implications for skin sensitization. Amar G. Chittiboyina,, * Cristina Avonto, and Ikhlas A. Khan., National Center for Natural Products Research; Department of Pharmacognosy, Division of BioMolecular Sciences; School of Pharmacy, University of Mississippi, University, MS 38677, USA. amar@olemiss.edu SUPPORTING INFORMATION Contents Page# 1. Figure S1. 1 H NMR (500 MHz, Chloroform-d) of ascaridole Figure S2. 13 C NMR (125 MHz, Chloroform-d) of ascaridole 3 3. Figure S3. Arrayed 1 HNMR spectra for Ascaridole and DCYA, before addition of DBN 4 4. Table S1. Select normalized integral values of DCYA and Ascaridole before addition of DBN 5 5. Figure S4. Arrayed 1 HNMR spectra for Ascaridole and DCYA, after addition of DBN 6 6. Table S2. Select normalized integral values of DCYA and Ascaridole after addition of DBN) 7 7. Figure S5. Structure of isolated compounds 2e, 2b, 2d, 2c 8 8. Table S3. 1 H and 13 C assignments of isolated ascaridole by-products 2e, 2b, 2d, 2c 8 8. Figure S6. 1 H NMR in CDCl 3 (500 MHz) of compound 2e 9 9. Figure S7. 13 C NMR in CDCl 3 (125 MHz) of compound 2e Figure S8. TLC of activated ascaridole mixture after trapping experiment with DCYA Figure S9. LC-MS of activated ascaridole mixture after treating with DCYA in ph10 buffer Table S9. 1 H and 13 C assignments of isolated diastereomeric DCYA adducts Figure S10. 1 H NMR in CDOD (500 MHz) of adduct Figure S C NMR in CDOD (125 MHz) of adduct Figure S12. 1 H NMR in CDOD (500 MHz) of adduct Figure S C NMR in CDOD (125 MHz) of adduct 2. 17

2 Figure S1. 1 H NMR (500 MHz, Chloroform-d) of ascaridole. δ 6.48 (d, J = 8.5 Hz, 1H), 6.40 (d, J = 8.5 Hz, 1H), (m, 2H), 1.91 (hept, J = 7.4, 6.9 Hz, 1H), (m, 2H), 1.36 (s, 3H), 0.98 (d, J = 7.0 Hz, 6H). 2

3 Figure S2. 13 C NMR (125 MHz, Chloroform-d) of ascaridole. δ 136.5, 133.1, 79.9, 74.5, 32.2, 29.6, 25.7, 21.5, 17.3,

4 Figure S3. Arrayed 1 HNMR spectra for Ascaridole and DCYA, before addition of DBN 4

5 Table S1. Select normalized integral values of DCYA and Ascaridole before addition of DBN (2,5-dimethylfuran was used as internal standard). Time (min) DCYA-Peak1 DCYA-Peak2 Ascaridole-Peak1 Ascaridole-Peak2 X(I) Y(X) Y2(X) Y3(X) Y4(X) ARR_DATA(I) Integral(8.620,8.520) Integral(7.230,7.180) Integral(6.520,6.460) Integral(6.440,6.380)

6 Figure S4. Arrayed 1HNMR spectra for Ascaridole and DCYA, after addition of DBN. 6

7 Table S2. Select normalized integral values of DCYA and Ascaridole after addition of DBN (2,5- dimethylfuran was used as internal standard). Time (min) DCYA-Peak1 DCYA-Peak2 Ascaridole-Peak1 Ascaridole-Peak2 X(I) Y(X) Y1(X) Y3(X) Y4(X) ARR_DATA(I) Integral(8.620,8.520) Integral(7.230,7.180) Integral(6.520,6.460) Integral(6.440,6.380)

8 Figure S5. Structure of isolated compounds 2e, 2b, 2d, 2c Table S3. 1 H and 13 C assignments of isolated ascaridole by-products 2c-2e Pos. δ H (mult,j in Hz) 2e 2b 2d 2c δ C δ H (mult, J in Hz) δ C δ H (mult, J in Hz) δ C δ H (mult, J in Hz) δ C (dd, 10.1, (bs) (m) (dt, 9.0, 1.8) ) (d, 10.1) (d, 2.9) (dd, 4.8, 1.0) (dt, 9.1, 1.8) a 2.10 a (m) (m) (m) a (m) b 2.10 a (m) (m) (dddd, 14.9, (m) , 3.2, 1.0) 6a 2.60 (m) (ddt, 4.2, (ddd, 14.1, a (m) , 1.4) 11.2, 5.5) 6b 2.39 (dddd, (dddd, 14.0, (m) , 9.3, 5.6, 2.5) 5.5, 3.4, 1.7) (d, 2.4) (hept, (hept, 7.0) a (m) ) (d, 6.9) a (dd, 6.9) * (d, 6.9) (d, 6.9) a (dd, 6.9) * (d, 6.9) (s) (s) (s) 20.2 * Inter-changeable a Overlapped with other signals 8

9 Figure S6. 1 H NMR in CDCl 3 (500 MHz) of compound 2e. 9

10 Figure S7. 13 C NMR in CDCl 3 (500 MHz) of compound 2e. 10

11 AU Figure S8. TLC of activated ascaridole mixture after trapping experiment with DCYA Figure S9. LC-MS of activated ascaridole mixture after treating with DCYA in ph10 buffer Minutes Conditions: Column: BEH Shield RP um, 100 x 2.1 mm. Column Temp: 35 o C; Sample Temp: 15 o C; UV= nm; MS Parameters: Capillary Voltage (kv): 3.0; Cone (V): 30 (pos) and 30 (neg); Extractor 11

12 Intensity AU Intensity AU Intensity Intensity AU Intensity Intensity AU Intensity (V): 3.0; Rf Lens (V): 0.5; Source Temp. 150 o C); Desolvation Temp. 350 o C; Desolvation (L/hr): 650 ; Cone (L/hr): 25 and MS Range: m/z= Peak x Peak 1 - SQ 1: MS Scan 1: ES+, Continuum, CV= Peak Peak Peak nm 2x10 6 2x Peak 2 - SQ 1: MS Scan 1: ES+, Continuum, CV= 0 4x10 6 2x x10 7 1x Peak 3 - SQ 1: MS Scan 1: ES+, Continuum, CV= Peak 4 - SQ 1: MS Scan 1: ES+, Continuum, CV= Peak 5 - SQ 1: MS Scan 1: ES+, Continuum, CV= Peak 6 - SQ 1: MS Scan 1: ES+, Continuum, CV= 4x10 6 2x x Peak 7 - SQ 1: MS Scan 1: ES+, Continuum, CV= m/z 12

13 Table S9. 1 H and 13 C assignments of isolated DCYA adducts. Pos. Adduct 1 Adduct 2 δ H (mult,j in Hz) 13 C δ H (mult,j in Hz) 13 C (dd, 7.5, 0.9) (dd, 7.5, 1.0) a (dd, 8.7, 7.4) a (dd, 8.7, 7.5) (dt, 8.7, 0.9) (dt, 8.7, 1.0) (dd, 7.3, 1.1) (dd, 7.3, 1.3) a (dd, 8.6, 7.3) a (dd, 8.6, 7.3) (dt, 8.6, 1.1) (dt, 8.6, 1.2) * * * * * (s) * (s) * (s) * (s) a (dt, 7.1, 3.16) (t, 7.1) (t, 7.1) a 2.92 a (14.5, 9.9, 6.3, 1.1) a (m) b 2.23 a (ddt, 14.5, 5.6, 1.8) (dd, 10.1, 2.2) a a (m) b 1.75 (dtd, 13.9, 6.3, 1.8) (ddd, 14.0, 6.4, 3.2) a 1.92 (ddd, 13.9, 9.9, 5.6 Hz) (td, 13.6, 5.0) a 2.50 a (m) a (m) b 2.17 a (m) (m) (s) (s) 27.9 * Inter-changeable a Overlapped with other signals 13

14 Figure S10. 1 H NMR in CDOD (500 MHz) of adduct 1. 14

15 Figure S C NMR in CDOD (125 MHz) of adduct 1. 15

16 Figure S12. 1 H NMR in CDOD (500 MHz) of adduct 2. 16

17 Figure S C NMR in CDOD (500 MHz) of adduct 2. 17