SUPPORTING INFORMATION Bioassay-guided isolation and structure elucidation of fungicidal and herbicidal compounds from Ambrosia salsola (Asteraceae). Wilmer H. Perera,1* Kumudini M. Meepagala,2* Frank R. Fronczek,3 Daniel D. Cook,4 David E. Wedge4 and Stephen O. Duke4 1ORISE Fellow-Agricultural Research Service, U.S. Department of Agriculture, Natural Products Utilization Research Unit, P.O. Box 1848, University, MS, 38677, USA; [email protected] (W.H.P.) 2Agricultural Research Service, U.S. Department of Agriculture, Natural Products Utilization Research Unit, P.O. Box 1848, University, MS, 38677, USA; [email protected] (K.M.M); [email protected] (D.E.W); [email protected] (S.O.D) 3Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA; [email protected] (F.R.F) 4Agricultural Research Service, U.S. Department of Agriculture, Poisonous Plant Research Lab. 1150 E 1400 N. N Logan, UT 84341, USA; [email protected] (D.D.C) *Correspondence: [email protected]; Tel: (662)915-1046 (W.H.P); [email protected]; Tel: (662)915-1030 (K.M.M) Abstract: The discovery of potent natural and ecofriendly pesticides is one of the focuses of the agrochemical industry, and plant species are a source of many potentially active compounds. We describe the bioassay-guided isolation of antifungal and phytotoxic compounds from the ethyl acetate extract of Ambrosia salsola twigs and leaves. With this methodology, we isolated and identified twelve compounds (four chalcones, six flavonols and two pseudoguaianolide sesquiterpene lactones). Three new chalcones were elucidated as (S)-β-Hydroxy-2',3,4',6' tetrahydroxy, 5 methoxydihydrochalcone (salsolol A), (S)-β-Hydroxy- 2',4,4',6' tetrahydroxy, 3 methoxydihydrochalcone (salsolol B) and (R)-α, (R)-β-Dihydroxy-2',3,4,4',6' pentahydroxydihydrochalcone (salsolol C) together with nine known compounds: balanochalcone, six quercetin derivatives, confertin and neoambrosin. Chemical structures were determined based on comprehensive HR-DART-MS, as well as 1D and 2D NMR experiments (DQFCOSY, HMQC and HMBC), and the absolute configurations of the chalcones were confirmed by CD spectra analysis. Crystal structure of confertin was determined by X-ray diffraction. The phytotoxicity of purified compounds was evaluated, and neoambrosim was active against Agrostis stolonifera at 1 mM, while confertin was active against both, Lactuca sativa and A. stolonifera at 1 mM and 100 µM, respectively. Confertin and salsolol A and B had IC50 values of 261, 275 and 251 µM, respectively, against Lemna pausicotata (duckweed). The antifungal activity was also tested against Colletotrichum fragariae Brooks using a thin layer chromatography bioautography assay. Both, confertin and neoambrosin were antifungal at 100 µM, with confertin’s activity beings higher than that of neoambrosin at this concentration. Keywords: Ambrosia salsola, Ambrosia dumosa, β-Hydroxydihydrochalcones, fungitoxic and phytotoxic compounds, confertin, X-Ray diffraction Figures Pages Figure S1: Isolation procedure flow chart 1 Figure S2: UV spectrum of compound 1 2 Figure S3: CD spectrum of compound 1 2 Figure S4: Chemical structure of compound 1 2 Figure S5: HR-DART-MS positive mode ion of compound 1 3 Figure S6: 1H-NMR spectrum of compound 1 4 Figure S7: 13C-NMR spectrum of compound 1 5 Figure S8: DEPT 135 spectrum of compound 1 6 Figure S9: COSY spectrum of compound 1 7 Figure S10: HMQC spectrum of compound 1 8 Figure S11: HMBC spectrum of compound 1 9 Figure S12: UV spectrum of compound 2 10 Figure S13: CD spectrum of compound 2 10 Figure S14: Chemical structure of compound 2 10 Figure S15: HR-DART-MS positive mode ion of compound 2 11 Figure S16: 1H-NMR spectrum of compound 2 12 Figure S17: 13C-NMR spectrum of compound 2 13 Figure S18: DEPT 135 spectrum of compound 2 14 Figure S19: COSY spectrum of compound 2 15 Figures Pages Figure S20: HMQC spectrum of compound 2 16 Figure S21: HMBC spectrum of compound 2 17 Figure S22: UV spectrum of compound 3 18 Figure S23: CD spectrum of compound 3 18 Figure S24: Chemical structure of compound 3 18 Figure S25: HR-DART-MS positive mode ion of compound 3 19 Figure S26: 1H-NMR spectrum of compound 3 20 Figure S27: 13C-NMR spectrum of compound 3 21 Figure S28: DEPT 135 spectrum of compound 3 22 Figure S29: COSY spectrum of compound 3 23 Figure S30: HMQC spectrum of compound 3 24 Figure S31: HMBC spectrum of compound 3 25 Figure S32: UV spectrum of compound 4 26 Figure S33: CD spectrum of compound 4 26 Figure S34: Chemical structure of compound 4 26 Figure S35: HR-DART-MS positive mode ion of compound 4 27 Figure S36: 1H-NMR spectrum of compound 4 28 Figure S37: 13C-NMR spectrum of compound 4 29 Figure S38: DEPT 135 spectrum of compound 4 30 Figure S39: HPLC chromatograms of ethyl acetate extracts (10 mg/mL) recorded at 254 nm 30 for A. salsola collected in Texas (A), A. salsola from Arizona (B) and A. dumosa (C). EtOAc extract (24 g) Ambrosia salsola SiO2 Flash chromatography 1 2 3 4 5 6 7 8 9 10 11 12 13 14 32-35 36-38 39-41 42-46 47-55 56-76 77-83 84-88 89-93 94-107 108-121 122-133 122-133 Wash 212 mg 204 mg 113 mg 178 mg 292 mg 850 mg 383 mg 1.16 g 5.5 g 6.5 g 2.4 g 1.24 g column 2.74 g Solid Sup 39 mg 1.5 g Compound 8 SiO2 Flash chromatography 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 1.10 1.11 1-9 10-14 15-27 28-32 33-35 36-39 40-44 45-48 49-52 53 54-57 18 mg 17 mg 272 mg 151 mg 150 mg 247 mg 470 mg 604 mg 1.72 g 637 mg 891 mg Prep RP C-18 HPLC Precipitated Compound 1 Compound 9 SiO2 Flash chromatography 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12 2.13 28-36 37-55 56-63 64-68 69-77 78-85 86-93 94-96 97-103 104-108 109-113 114-125 Wash column 672 mg 504 mg 122 mg 33 mg 47 mg 30 mg 30 mg 25 mg 25 mg 120 mg 86 mg 80 mg 80 mg Prep RP C-18 HPLC SiO2 Flash chromatography Compounds Compound 2 11 12 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 3.10 3.11 3.12 1-69 70-75 76-97 98-100 101-102 103 104-105 106-108 109-111 112-115 116-120 Wash column 101 mg 188 mg 1.39 g 302 mg 354 mg 303 mg 519 mg 758 mg 852 mg 459 mg 119 mg 140 mg Compound 10 Compound 8 RP C-18 SiO2 Flash chromatography Compound 4 Compound 6 Compound 7 Compound 3 Compound 5 Figure S1: Isolation procedure flow chart. 1 Compound 1 (Salsolol A) *DAD1, 12.834 (422 mAU, - ) Ref=12.607 & 13.020 of 082-1301.D m A U 8000 400 350 6000 300 4000 250 [θ] 200 2000 150 0 100 280 290 300 310 320 330 340 350 50 -2000 0 220 240 260 280 300 320 340 360 380 nm -4000 Figure S2: UV spectrum of compound 1. Figure S3: CD spectrum of compound 1. Figure S4: Chemical structure of compound 1. 2 Elemental Compositions Element Limits: C 0/50 H 0/100 O 0/10 Tolerance: 7 mmu Even or odd electron ion or both: Both Electron correction: None. Charges: 1 Minimum unsaturation: -1 Maximum unsaturation: 100 Calc. m/z Abund % mmu DBE Composition 303.086863 100.000 -5.09 9.5 C16H15O6 Figure S5: HR-DART-MS positive mode ion of compound 1. 3 Figure S6: 1H-NMR spectrum of compound 1. 4 Figure S7: 13C-NMR spectrum of compound 1. 5 Figure S8: DEPT 135 spectrum of compound 1. 6 Figure S9: COSY spectrum of compound 1. 7 Figure S10: HMQC spectrum of compound 1. 8 Figure S11: HMBC spectrum of compound 1. 9 Compound 2 (Salsolol B) *DAD1, 13.035 (449 mAU, - ) Ref=11.808 & 13.615 of 091-0101.D m A U 600 300 250 100 200 [θ] 280 290 300 310 320 330 340 350 150 -400 100 50 -900 0 -1400 220 240 260 280 300 320 nm Figure S13: CD spectrum of compound 2. Figure S12: UV spectrum of compound 2. Figure S14: Chemical structure of compound 2. 10 Elemental Compositions Element Limits: C 0/50 H 0/100 O 0/10 Tolerance: 7 mmu Even or odd electron ion or both: Both Electron correction: None. Charges: 1 Minimum unsaturation: -1 Maximum unsaturation: 100 Calc. m/z Abund % mmu DBE Composition 303.086863 100.000 -4.60 9.5 C16H15O6 Figure S15: HR-DART-MS positive mode ion of compound 2. 11 Figure S16: 1H-NMR spectrum of compound 2. 12 Figure S17: 13C-NMR spectrum of compound 2. 13 Figure S18: DEPT 135 spectrum of compound 2. 14 Figure S19: COSY spectrum of compound 2. 15 Figure S20: HMQC spectrum of compound 2. 16 Figure S21: HMBC spectrum of compound 2. 17 Compound 3 (Salsolol C) *DAD1, 10.394 (1563 mAU, - ) Ref=9.887 & 12.460 of 042-0201.D m A U 5800 1400 3800 1200 1800 1000 ] θ -200 800 [ 283 293 303 313 323 333 343 600 -2200 400 -4200 200 -6200 0 220 240 260 280 300 320 340 360 380 nm -8200 Figure S22: UV spectrum of compound 3.