SUPPORTING INFORMATION

SUPPORTING INFORMATION Trichodermides A E: New Peptaibols isolated from Australian Termite Nestderived Fungus Trichoderma virens CMB-TN16 Wei-Hua Jiao,, Zeinab Khalil, Pradeep Dewapriya, Angela A. Salim, Hou-Wen Lin, and Robert J. Capon* Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD, 4072, Australia Research Center for Marine Drugs, State Key Laboratory of Oncogenes and Related Genes, Department of Pharmacy, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, China 1

Table of contents 1. DNA taxonomy of the fungus CMB-TN16... 4 1.1. ITS gene sequence of the fungus CMB-TN16... 4 1.2. BLAST search (closest match)... 5 2. Spectroscopic characterization for trichodermides A-E (1-5)... 6 3. MS/MS fragmentation for tichodermides A-E (1-5)... 30 4. C3 Marfey analysis... 33 5. Cytotoxicity assay... 38 6. Antimicrobial assay... 39 List of Tables Table S1. 1D and 2D NMR (600 MHz, DMSO-d 6 ) data for 1... 6 Table S2. 1D and 2D NMR (600 MHz, DMSO-d 6 ) data for 2... 8 Table S3. 1D and 2D NMR (600 MHz, DMSO-d 6 ) data for 3... 10 Table S4. 1D and 2D NMR (600 MHz, DMSO-d 6 ) data for 4... 12 Table S5. 1D and 2D NMR (600 MHz, DMSO-d 6 ) data for 5... 14 List of Figures Figure S1. 1 H NMR (600 MHz, DMSO-d 6 ) spectrum of 1... 16 Figure S2. 13 C NMR (150 MHz, DMSO-d 6 ) spectrum of 1... 17 Figure S3. HSQC (600 MHz, DMSO-d 6 ) spectrum of 1... 17 Figure S4. COSY (600 MHz, DMSO-d6) spectrum of 1... 18 Figure S5. COSY (600 MHz, DMSO-d 6 ) spectrum of 1... 18 Figure S6. HMBC (600 MHz, DMSO-d 6 ) spectrum of 1... 19 Figure S7. HMBC (600 MHz, DMSO-d 6 ) spectrum of 1... 19 Figure S8. HMBC (600 MHz, DMSO-d 6 ) spectrum of 1... 20 Figure S9. ROESY (600 MHz, DMSO-d 6 ) spectrum of 1... 20 Figure S10. ROESY (600 MHz, DMSO-d 6 ) spectrum of 1... 21 Figure S11. HR-QTOF-MS spectrum of 1... 21 Figure S12. 1 H NMR (600 MHz, DMSO-d 6 ) of 2... 22 Figure S13. 13 C NMR (150 MHz, DMSO-d 6 ) of 2... 23 Figure S14. HR-QTOF-MS spectrum of 2... 23 Figure S15. 1 H NMR (600 MHz, DMSO-d 6 ) spectrum of 3... 24 Figure S16. 13 C NMR (150 MHz, DMSO-d 6) spectrum of 3... 25 Figure S17. HR-QTOF-MS spectrum of 3... 25 Figure S18. 1 H NMR (600 MHz, DMSO-d 6 ) spectrum of 4... 26 Figure S19. 13 C NMR (150 MHz, DMSO-d 6 ) of 4... 27 Figure S20. HR-QTOF-MS spectrum of 4... 27 Figure S21. 1 H NMR (600 MHz, DMSO-d 6 ) of 5... 28 Figure S22. 13 C NMR (150 MHz, DMSO-d 6 ) of 5... 29 Figure S23. HR-QTOF-ESI-MS spectrum of 5... 29 Figure S24. Positive and negative ion HR-QTOF-ESI-MS/MS spectra of 1... 30 Figure S25. Positive and negative ion HR-QTOF-ESI-MS/MS spectra of 2... 30 Figure S26. Positive and negative ion HR-QTOF-ESI-MS/MS spectra of 3... 31 Figure S27. Positive and negative ion HR-QTOF-ESI-MS/MS spectra of 4... 31 Figure S28. Positive and negative ion HR-QTOF-ESI-MS/MS spectra of 5... 32 Figure S29. C 3 Marfey s method analysis of 1.... 33 2

Figure S30. C 3 Marfey s method analysis of 2.... 34 Figure S31. C 3 Marfey s method analysis of 3... 35 Figure S32. C 3 Marfey s method analysis of 4.... 36 Figure S33. C 3 Marfey s method analysis of 5... 37 Figure S34. Cytotoxicity assay of the trichodermids 1 5 against (a) NCI-H460 (lung cancer cell line) and (b) SW620 (human colon cancer cell line)... 38 Figure S35. Antimicrobial assay screening graphs of trichodermides A-E (1 5)... 39 3

1. DNA taxonomy of the fungus CMB-TN16 Genomic DNA of the fungus CMB-TN16 was extracted from the mycelia using the DNeasy Plant Mini Kit (Qiagen) as per the manufacturers protocol. The 18S rrna genes were amplified from genomic DNA by PCR using the universal primers ITS 1 (5ʹ-TCCGTAGGTGAACCTGCGG-3ʹ) and ITS 4 (5ʹ- TCCTCCGCTTATTGATATGC-3ʹ). The PCR mixture (50 μl) contained 1 μl of genomic DNA (20 40 ng), 200 μm of each deoxynucleoside triphosphate (dntp), 1.5 mm MgCl2, 0.3 μm of each primer, 1 U of Taq DNA polymerase (Fisher Biotec) and 5 μl of PCR buffer. PCR was performed using the following conditions: initial denaturation at 95 C for 3 min, 30 cycles in series of 94 C for 30 s (denaturation), 55 C for 60 s (annealing) and 72 C for 60 s (extension), followed by one cycle at 72 C for 6 min. The PCR products were purified with PCR purification kit (Qiagen) and sequenced. The BLAST search showed the amplified ITS sequence (GenBank accession no. KU521856.1) has 99% homology with other members of the genus Trichoderma virens. 1.1. ITS gene sequence of the fungus CMB-TN16 TCCCAACCCATGTGACGTTACCAAACTGTTGCCTCGGCGGGATCTCTGCCCCGGGTG CGTCGCAGCCCCGGACCAAGGCGCCCGCCGGAGGACCAACCAAAACTCTTATTGTAT ACCCCCTCGCGGGTTTTTTACTATCTGAGCCATCTCGGCGCCCCTCGTGGGCGTTTC GAAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAAC GCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTT GAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTCCGAGCGTCATTTC AACCCTCGAACCCCTCCGGGGGGTCGGCGTTGGGGATCGGCCCTTTACGGGGCCGGC CCCGAAATACAGTGGCGGTCTCGCCGCAGCCTCTCCTGCGCAGTAGTTTGCACACTC GCATCGGGAGCGCGGCGCGTCCACAGCCGTTAAACACCCCAAACTTCTGAAATGTTG ACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCATAA CGGGAGG 4

1.2. BLAST search (closest match) 5

2. Spectroscopic characterization for trichodermides A-E (1-5) Table S1. 1D and 2D NMR (600 MHz, DMSO-d 6 ) data for 1 No. C H (J in Hz) COSY HMBC ROESY Ac 1 170.8, a C 2 22.9, CH 3 1.93, s 1 Iva 1 -NH D-Iva 1 1 176.0, C 2 58.6, C 3 27.3, CH 2 1.89, i m 4 NH 1.69, m 4 4 7.6, CH 3 0.76, o dd 3 2, 3 NH 5 22.4, CH 3 1.28, s 1, 2, 3 NH NH 8.48, s Ac-1, 1, 2, 3,5 Ac-2, Gln 2 -NH, 3, 4, 5 L-Gln 2 1 172.7, b C 2 55.0, CH 3.96, m NH, 3 1, 2 Ile 3 -NH 3 25.6, c CH 2 1.91, i m 2, 4 NH 1.85, j m 2, 4 2 4 31.3, CH 2 2.24, m 3, 5 2, 3 2.16, m 3, 5 2, 3 5 174.4, C NH 8.73, d (5.4) 2 Iva 1-1, 2, 3 Iva 1 -NH, Ile 3 -NH, 3 5-NH 2 7.42, s 5-NH 5 6.91, s 5-NH 5 L-Ile 3 1 170.9, a C 2 59.2, CH 3.89, m NH, 3 1, 3, 4, 5 3 34.9, d CH 1.91, i m 2, 4, 6 2, 4 4 25.3, c CH 2 1.74, k m 3, 5 1.50, m 3, 5 5 10.9, CH 3 0.83, l t 4 3 6 15.3, e CH 3 0.88, q d 3 2 NH NH 7.73, d (7.2) 2 Gln 2-1, 2, 3 Gln 2 -NH, Val 4 -NH L-Val 4 1 172.6, b C 2 58.6, CH 4.09, m NH, 3 1, 3, 4 NH 3 29.6, CH 2.09, m 2, 4, 5 1, 2 4 19.1, CH 3 0.83, l d 3 2, 3, 5 NH 5 17.9, CH 3 0.85, l d 3 NH, Aib 5 -NH NH 7.24, d (8.4) 2 Ile 3-1, 2, 3 Ile 3 -NH, Aib 5 -NH Aib 5 1 171.6, C 2 56.1, C 3 25.4, c CH 3 1.38, s 1, 2, 4 NH 4 23.0, f CH 3 1.44, s 1, 2, 3 NH NH 7.92, s Val 4-1, 1, 3, 4 Val 4 -NH, 3, 4, Pro 6-5 6

No. C H (J in Hz) COSY HMBC ROESY L-Pro 6 1 172.4, n C 2 63.3, CH 4.20, d (7.8, 7.8) 3 1, 3, 4 Ile 7 -NH 3 28.6, g CH 2 2.06, m 2, 4 1, 5 1.23, m 2, 4 1, 4 4 25.7, c CH 2 1.86, j m 3, 5 1.73, k m 3, 5 5 48.5, CH 2 3.63, m 4 Aib 5 -NH 3.39, m 4 Aib 5 -NH L-Ile 7 1 173.0, C 2 59.4, CH 3.80, t (7.8) NH, 3 1, 3, 6 3 34.9, d CH 1.91, i m 2, 4, 6 4 25.2, c CH 2 1.72, k m 3, 5 1.50, m 3, 5 5 10.8, CH 3 0.83, l t 4 3 6 15.3, e CH 3 0.84, l d 3 2 NH 7.38, d (7.8) 2 1, 2, 3 Pro 6-2 L-Leu 8 1 172.3, n C 2 51.1, CH 4.24, m NH, 3 1, 3 Aib 9 -NH 3 39.6, CH 2 1.53, m 2, 4 4 24.0, CH 1.37, m 3, 5 5 21.5, CH 3 0.80, p d 4 4 6 20.1, CH 3 0.75, o d 4 Leu 8-3 NH 7.08, m d 2 2, 3 Aib 9 1 171.4, h C 2 55.9, C 3 25.5, c CH 3 1.32, s 1, 2, 4 NH 4 23.7, CH 3 1.36, s 1, 2, 3 NH 7.60, s Leu 8-1, 1, 2, 4 3, Pro 10-5, Leu 8-2 L-Pro 10 1 171.4, h C 2 62.0, CH 4.25, m NH, 3 1, 4, 5 3 28.7, g CH 2 2.22, m 2, 4 1, 4 1.61, m 2, 4 4 25.5, c CH 2 1.86, j m 3, 5 1.73, k m 5 47.9, CH 2 3.36, m 4 Aib 9 -NH 3.25, m 4 Aib 9 -NH L-Leuol 11 1 48.7, CH 3.72, m NH, 2, 3, 6 6 2 39.2, CH 2 1.41, m 1, 3 1.36, m 1, 3 3 24.3, CH 1.59, m 2, 4, 5 4 24.0, CH 3 0.88, q d 3 5 23.0, f CH 3 0.80, p d 3 6 64.0, CH 2 3.31, m 1 NH 3.12, m 1 NH NH 7.08, m d 1 1 a-q Assignments for overlapping 1 H and 13 C NMR resonances with the same superscript may be interchanged. 7

Table S2. 1D and 2D NMR (600 MHz, DMSO-d 6 ) data for 2 No. C H (J in Hz) COSY 1 H 13 C HMBC ROESY Ac 1 170.9, a C 2 23.0, CH 3 1.92, s 1 Aib 1 -NH Aib 1 1 175.9, C 2 55.7, C 3 26.4, CH 3 1.34, s 1, 4 4 23.7, CH 3 1.36, s 1, 2, 3 NH 8.74, s Ac-1, 1, 2, 3 Gln 2 -NH, Ac-2 L-Gln 2 1 172.5, b C 2 55.1, CH 3.94, t (5.4) NH, 3 1, 3, 4 Ile 3 -NH, NH 3 25.8, c CH 2 1.96, f m 2, 4 1.86, g m 2, 4 2 4 31.4, CH 2 2.25, m 3 2, 3 2.16, m 3 2, 3 5 174.4, C NH 8.79, d (5.4) 2 Aib 1-1 Ile 3 -NH, 2 5-NH 2 7.45, s 5 6.90, s 4, 5 L-Ile 3 1 171.5, d C 2 59.2, CH 3.89, m NH, 3 1, 3, 4, 6 3 35.0, n CH 1.93, o m 2, 4, 6 2, 4, 6 4 25.3, c CH 2 1.51, m 3, 5 1.23, m 3, 5 5 10.9, CH 3 0.84, h t 4 3 6 15.4, CH 3 0.86, h d 3 2 NH 7.68, d (7.2) 2 Gln 2-1, 2, 3 Gln 2 -NH, Val 4 -NH L-Val 4 1 172.4, b C 2 58.8, CH 4.07, m NH, 3 1, 3, 4 Aib 5 -NH 3 29.6, CH 2.08, m 2, 4, 5 1, 3 4 19.1, CH 3 0.84, h d 3 2, 3, 5 5 18.2, CH 3 0.89, q d 3 NH 7.31, d (8.4) 2 Ile 3-1 Ile 3 -NH, Aib 5 -NH Aib 5 1 172.8, e C 2 56.1, C 3 25.6, c CH 3 1.39, s 1, 2, 4 4 23.0, CH 3 1.44, s 1, 2, 3 NH NH 7.96, s Val 4-1, 2, 3, 4 Val 4 -NH, Pro 6-2 8

No. C H (J in Hz) COSY 1 H 13 C HMBC ROESY L-Pro 6 1 173.0, C 2 63.3, CH 4.20, dd (7.8, 7.8) 3 1, 3, 4 Ile 7 -NH 3 28.7, i CH 2 2.22, m 2, 4 1, 5 1.23, m 2, 4 1, 4 4 25.8, c CH 2 1.96, f m 3, 5 1.86, g m 3, 5 5 48.5, CH 2 3.64, m 4 3 3.40, m 4 L-Ile 7 1 171.6, d C 2 59.5, CH 3.80, t (7.8) NH, 3 1, 3, 4, 6 3 34.9, n CH 1.93, o m 2, 4, 6 4 25.4, c CH 2 1.73, j m 3, 5 1.50, m 3, 5 5 10.8, CH 3 0.84, h t 4 3 6 15.3, CH 3 0.86, h d 3 2 NH 7.39, d (7.8) 2 Pro 6-1, 2 Pro 6-2, Pro 6-5 L-Leu 8 1 172.6, e C 2 51.1, CH 4.24, k m NH, 3 1, 3 Aib 9 -NH 3 39.6, CH 2 1.54, m 2, 4 4 24.0, r CH 1.59, l m 3, 5 5 21.5, CH 3 0.79, p d (7.2) 4 4, 6 6 20.1, CH 3 0.73, d (6.6) 4 3, 5 NH 7.09, m d Aib 9 1 171.4, d C 2 55.9, C 3 23.7, s CH 3 1.36, s 1, 2, 4 NH 4 23.9, s CH 3 1.32, s 1, 2, 3 NH 7.61, s 1, 2, 3 Pro 10-2 L-Pro 10 1 171.0, a C 2 62.0, CH 4.25, k m 3 3, 4, 5 Aib 9 -NH 3 28.6, i CH 2 2.07, m 2, 4 1.61, m 2, 4 4 25.2, c CH 2 1.73, j m 3, 5 5 48.0, CH 2 3.61, m 4 3.24, m 4 L-Leuol 11 1 48.7, CH 3.71, m NH, 2, 6 2 39.2, CH 2 1.41, m 1, 3 1.37, m 1, 3 3 24.3, r CH 1.59, l m 2, 4, 5 4 23.7, CH 3 0.88, q d 3 5 23.0, CH 3 0.80, p d (6.6) 3 6 64.0, CH 2 3.31, m 1 NH 3.12, m 1 NH NH 7.09, m d a-s Assignments for overlapping 1H and 13C NMR resonances with the same superscript may be interchanged. 9

Table S3. 1D and 2D NMR (600 MHz, DMSO-d 6 ) data for 3 position C, mult H (J in Hz) COSY 1 H 13 C ROESY HMBC Ac 1 170.8, C 2 22.9, CH 3 1.92, s Ac-1 Iva 1 -NH D-Iva 1 1 176.0, C 2 58.6, a C 3 27.4, CH 2 1.89, i m 4 1.69, m 4 4 7.6, CH 3 0.76, t (6.6) 3 2, 3 NH 5 22.4, CH 3 1.28, s 1, 2, 3 NH, Gln 2 -NH NH 8.50, s Ac-1, 1, 2, 3, 5 Ac-2, Gln 2 -NH, 3, 5 L-Gln 2 1 172.3, b C 2 55.0, c CH 3.97, m NH, 3 1, 3 Ile 3 -NH 3 25.8, CH 2 1.95, m 2, 4 1.86, j m 2, 4 2 4 31.4, CH 2 2.22, k m 3 2, 3 2.15, m 3 2, 3 5 174.5, C NH 8.72, d (5.4) 2 Iva 1-1, 2, 3 Iva 1 -NH, Ile 3 -NH 5-NH 2 7.44, s 5 5-NH 6.91, s 5 5-NH L-Ile 3 1 171.6, d C 2 59.1, CH 3.90, m NH, 3 1, 3, 4, 5 3 34.9, e CH 1.91, i m 2, 4, 6 2, 4 4 25.2, CH 2 1.50, l m 3, 5 NH 1.21, m 3, 5 5 10.9, CH 3 0.83, m t 4 3 6 15.3, f CH 3 0.88, o d 3 2 NH NH 7.75, d (7.2) 2 Gln 2 -NH, Val 4 -NH L-Val 4 1 172.3, b C 2 58.6, a CH 4.09, m NH, 3 1, 3, 4 NH 3 29.6, CH 2.08, m 2, 4, 5 1, 2 4 19.1, CH 3 0.85, m d 3 2, 3, 5 NH 5 18.0, CH 3 0.80, n d 3 NH, Aib 5 -NH NH 7.30, d (8.4) 2 Ile 3-1 Ile 3 -NH, Aib 5 -NH Aib 5 1 172.6, C 2 56.1, C 3 25.4, g CH 3 1.38, s 1, 2, 4 NH 4 22.9, h CH 3 1.43, s 1, 2, 3 NH 10

position C, mult H (J in Hz) COSY 1 H 13 C ROESY HMBC NH 7.97, s Val 4-1, 1, 3, 4 Val 4 -NH, Pro 6-5 L-Pro 6 1 173.1, C 2 63.3, CH 4.20, dd (7.8, 7.8) 3 1, 3, 4 Ile 7 -NH 3 28.6, CH 2 2.21, k m 2, 4 1, 5 1.61, m 2, 4 1, 4 4 25.5, g CH 2 1.86, j m 3, 5 1.60, m 3, 5 5 48.5, CH 2 3.64, p m 4 Aib 5 -NH 3.40, m 4 L-Ile 7 1 171.4, d C 2 59.4, CH 3.81, t (7.8) NH, 3 1, 3, 6 Leu 8 -NH 3 35.0, e CH 1.94, m 2, 4, 6 4 25.4, g CH 2 1.51, l m 3, 5 1.25, m 3, 5 5 10.8, CH 3 0.83, m t 4 3 6 15.4, f CH 3 0.86, o d 3 3 Leu 8 -NH NH 7.42, d (7.8) 2 Pro 6-1 Pro 6-2, Leu 8 -NH L-Leu 8 1 172.7, C 2 51.0, CH 4.24, q m NH, 3 1, 3 Aib 9 -NH 3 39.8, CH 2 1.55, m 2, 4 4 24.0, CH 1.59, m 3, 5 5 22.9, h CH 3 0.79, n d 4 4 6 20.2, CH 3 0.73, d (6.6) 4 3 NH 7.12, d (8.4) 2 Ile 7-1 Ile 7 -NH, Aib 9 -NH Aib 9 1 171.6, d C 2 55.9, C 3 23.1, h CH 3 1.43, s 1, 2, 4 NH 4 23.7, CH 3 1.36, s 1, 2, 3 NH 7.62, s Leu 8-1, 1, 2, 4 Leu 8 -NH, Pro 10-5 L-Pro 10 1 171.4, d C 2 62.3, CH 4.25, q m 3 1, 4, 5 Valol 11 -NH 3 29.0, CH 2 2.08, m 2, 4 1, 4 1.63, m 2, 4 4 25.6, g CH 2 1.75, m 3, 5 1.53, l m 3, 5 5 48.0, CH 2 3.61, m 4 Aib 9 -NH 3.31, m 4 L-Valol 11 1 55.0, c CH 3.64, p m NH, 2, 6 Pro 10-1, 3, 5 2 28.1, CH 1.84, m 3, 4 3 20.0, CH 3 0.80, n d 2 4 17.2, CH 3 0.80, n d 2 5 61.4, CH 2 3.42, m 1 2 NH 3.35, m 1 2 NH NH 7.00, d (9.6) 2 Pro 10-1 Pro 10-2, 5 a-q Assignments for overlapping 1H and 13C NMR resonances with the same superscript may be interchanged. 11

Table S4. 1D and 2D NMR (600 MHz, DMSO-d 6 ) data for 4 position C H (J in Hz) COSY 1 H 13 C HMBC ROESY Ac 1 171.0, C 2 23.0, i CH 3 1.92, s 1 Aib 1 -NH Aib 1 1 176.0, C 2 55.9, C 3 26.4, CH 3 1.34, k s 2, 4 NH 4 24.0, CH 3 1.32, k s 1, 2, 3 NH 8.72, s Ac-1, 1, 2, 3 Gln 2 -NH, 3 L-Gln 2 1 172.6, a C 2 55.1, CH 3.94, m NH, 3 1, 3 Ile 3-3, Ile 3-3, NH 3 25.7, b CH 2 1.96, m 2, 4 1.52, l m 2, 4 2 4 31.4, CH 2 2.22, m 3 2, 3 2.16, m 3 2, 3 5 174.4, C NH 8.78, d (5.4) 2 Aib 1-1 Aib 1-4, Ile 3 -NH, 2 5-NH 2 7.45, s 5 6.91, s 5 L-Ile 3 1 171.6, c C 2 59.2, d CH 3.89, m NH, 3 1, 3, 4, 6 3 35.0, e CH 1.93, m 2, 4, 6 2, 4 Gln 2-2 4 25.2, b CH 2 1.51, l m 3, 5 1.22, m 3, 5 5 10.9, f CH 3 0.84, m t (6.6) 4 3 6 15.4, g CH 3 0.89, n d (7.2) 3 2 NH 7.66, d (7.2) 2 Gln 2-2, 2, 3 Gln 2 -NH, Val 4 -NH L-Val 4 1 172.3, h C 2 58.8, CH 4.07, m NH, 3 1, 3, 4 NH 3 29.6, CH 2.07, o m 2, 4, 5 1, 2 4 19.1, CH 3 0.89, n d (6.6) 3 2, 3, 5 5 18.2, CH 3 0.87, n d (6.0) 3 NH 7.30, d (8.4) 2 Ile 3-1 Ile 3 -NH, Aib 5 -NH Aib 5 1 172.7, a C 12

position C H (J in Hz) COSY 1 H 13 C HMBC ROESY 2 56.1, C 3 25.5, b CH 3 1.38, s 1, 2, 4 4 23.0, i CH 3 1.44, s 1, 2, 3 NH NH 7.95, s Val 4-1, 3, 4 Val 4 -NH, Val 4-2, Pro 6-5 L-Pro 6 1 173.0, C 2 63.3, CH 4.20, dd (7.8, 7.8) 3 1, 3, 4 Ile 7 -NH 3 28.6, CH 2 2.09, o m 2, 4 1, 5 1.61, p m 2, 4 1, 4 4 25.7, b CH 2 1.96, m 3, 5 1.52, l m 3, 5 5 48.5, j CH 2 3.63, m Ile 7-1, Aib 5 -NH 3.40, m 4 Aib 5-1, 2 L-Ile 7 1 171.4, c C 2 59.4, d CH 3.81, t (7.8) NH, 3 1, 3, 6 3 34.9, e CH 1.93, m 2, 4, 6 4 25.4, b CH 2 1.76, m 3, 5 1.23, m 3, 5 5 10.8, f CH 3 0.84, m t (6.6) 4 3 6 15.3, g CH 3 0.84, m d (7.2) 3 2 NH 7.41, d (7.8) 2 Pro 6-1 Pro 6-2, Leu 8 -NH L-Leu 8 1 172.3, h C 2 50.9, CH 4.26, q m NH, 3 1, 3 Aib 9 -NH 3 39.9, CH 2 1.53, l m 2, 4 4 24.0, CH 1.60, m 3, 5 5 22.9, i CH 3 0.92, d (7.2) 4 4 6 19.9, CH 3 0.74, d (6.6) 4 3 NH 7.12, d (8.4) 2 Ile 7-1 Ile 7 -NH, Aib 9 -NH Aib 9 1 171.6, c C 2 55.7, C 3 25.5, b CH 3 1.32, k s 1, 2, 4 NH 4 23.8, CH 3 1.36, s 1, 2, 3 NH 7.62, s 1, 2, 4 Leu 8 -NH, Pro 10-5 L-Pro 10 1 171.3, c C 2 62.2, CH 4.25, q m 3 1, 4, 5 Valol 11 -NH 3 29.0, CH 2 2.22, m 2, 4 1, 4 1.61, p m 2, 4 4 25.6, b CH 2 1.86, m 3, 5 1.51, l m 3, 5 5 48.0, j CH 2 3.62, m 4 Aib 9 -NH 3.30, m 4 Aib 9 -NH L-Valol 11 1 55.0, CH 3.64, m NH, 2, 6 3, 5 2 28.1, CH 1.85, m 3, 4 3 20.1, CH 3 0.82, m d 2 4 17.2, CH 3 0.79, d 2 5 61.4, CH 2 3.43, m 1 2 NH 3.34, m 1 2 NH NH 7.00, d (9.6) 2 Pro 10-1 Pro 10-2, 5 a-q Assignments for overlapping 1 H and 13 C NMR resonances with the same superscript may be interchanged. 13

Table S5. 1D and 2D NMR (600 MHz, DMSO-d 6 ) data for 5 position C H (J in Hz) COSY 1 H 13 C HMBC ROESY Ac 1 170.7, C 2 22.9, CH 3 1.91, h s 1 Iva 1 -NH D-Iva 1 1 175.8, C 2 58.9, C 3 27.5, CH 2 1.90, h m 4 1.70, m 4 4 7.6, CH 3 0.76, i dd 3 2 5 22.4, CH 3 1.29, s 1, 2, 3 NH 8.47, s Ac-1, 1, 2, 5 L-Gln 2 1 172.7, a C 2 54.9, b CH 3.99, m NH, 3 1, 3 3 25.8, c CH 2 1.96, m 2, 4 1.86, p m 2, 4 2 4 31.3, CH 2 2.21, j m 3 2, 3 2.16, m 3 2, 3 5 174.4, C NH 8.71, d (4.8) 2 Iva 1-1, 1, 2 Val 3 -NH 5-NH 2 7.43, k s 6.89, s L-Val 3 1 171.6, d C 2 60.3, CH 3.91, m NH, 3 1, 3, 4, 5 3 29.0, e CH 2.13 m 2, 4, 5 2, 3 4 19.1, f CH 3 0.93, l d 3 2 5 19.1, f CH 3 0.89, m d 3 2 NH 7.75, d (7.2) 2 Gln 2-1 Gln 2 -NH, Val 4 -NH L-Val 4 1 172.3, g C 2 58.5, CH 4.11, m NH, 3 1, 3, 4 3 29.6, CH 2.09, m 2, 4, 5 1, 2 4 17.9, CH 3 0.88, m d 3 2, 3, 5 5 18.8, CH 3 0.95, l d 3 NH 7.38, d (7.2) 2 Val 3-1, 1 Val 3 -NH, Aib 5 -NH 14 5

position C H (J in Hz) COSY 1 H 13 C HMBC ROESY Aib 5 1 172.7, a C 2 56.0, C 3 25.4, c CH 3 1.38, s 1,2, 4 4 23.1, CH 3 1.43, s 1, 2, 3 NH 8.02, s Val 4-1, 1, 3, 4 Val 4 -NH L-Pro 6 1 172.2, g C 2 62.2, CH 4.23, q m 3 1, 3, 4 Ile 7 -NH 3 28.6, CH 2 2.22, j m 2, 4 1, 5 2.08, m 2, 4 1, 4 4 25.3, c CH 2 1.76, m 3, 5 1.50, n m 3, 5 5 48.4, CH 2 3.65, o m 3.39, m 4 Aib 5-1, 2 L-Ile 7 1 171.5, d C 2 59.3, CH 3.81, t (5.4) NH, 3 1, 3, 6 3 34.9, CH 1.93, m 2, 4, 6 4 25.3, c CH 2 1.76, m 3, 5 1.50, n m 3, 5 5 10.9, CH 3 0.83, dd 4 3 6 15.4, CH 3 0.87, m d 3 2 NH 7.42, k d 2 Pro 6-1, 1 Leu 8 -NH, Pro 6-2 L-Leu 8 1 171.3, d C 2 50.9, l CH 4.25, q m NH, 3 1, 3 3 39.6, m CH 2 1.53, m 2, 4 4 24.0, CH 1.59, m 3, 5 5 20.2, CH 3 0.75, i d 4 4 6 19.9, CH 3 0.79, i d 4 3 NH 7.12, d (8.4) 2 1 Ile 7 -NH, Aib 9 -NH Aib 9 1 172.7, a C 2 55.9, C 3 25.5, c CH 3 1.32, s 1, 2, 4 4 23.6, CH 3 1.36, s 1, 2, 3 NH 7.62, s Leu 8-1/ Pro 10-1, 1, 2, 4 Leu 8 -NH, Pro 10-5 L-Pro 10 1 171.4, d C 2 63.2, CH 4.20, dd (7.8, 7.8) 3 1, 4, 5 3 28.9, e CH 2 2.22, j m 2, 4 1, 4 1.62, m 2, 4 4 25.6, c CH 2 1.86, p m 3, 5 1.51, n m 3, 5 5 48.0, CH 2 3.62, o m 4 Aib 9-1, 2 Aib 9 -NH 3.30, m 4 Aib 9-1, 3 L-Valol 11 1 54.9, b CH 3.64, o m 2 28.1, CH 1.89, h m 3, 4 3 20.2, CH 3 0.75, i d 2 4 17.2, CH 3 0.79, i d 2 5 61.4, CH 2 3.43, m 3.35, m NH 6.99, d (9.6) 2 Pro 10-1 Pro 10-2, 5 a-q Assignments for overlapping 1 H and 13 C NMR resonances with the same superscript may be interchanged. 15

Figure S1. 1 H NMR (600 MHz, DMSO-d 6 ) spectrum of 1 16

Figure S2. 13 C NMR (150 MHz, DMSO-d 6 ) spectrum of 1 Figure S3. HSQC (600 MHz, DMSO-d 6 ) spectrum of 1 17

Figure S4. COSY (600 MHz, DMSO-d 6 ) spectrum of 1 Figure S5. COSY (600 MHz, DMSO-d 6 ) spectrum of 1 18

Figure S6. HMBC (600 MHz, DMSO-d 6 ) spectrum of 1 Figure S7. HMBC (600 MHz, DMSO-d 6 ) spectrum of 1 19

Figure S8. HMBC (600 MHz, DMSO-d 6 ) spectrum of 1 Figure S9. ROESY (600 MHz, DMSO-d 6 ) spectrum of 1 20

Figure S10. ROESY (600 MHz, DMSO-d 6 ) spectrum of 1 Figure S11. HR-QTOF-MS spectrum of 1 21

Figure S12. 1 H NMR (600 MHz, DMSO-d 6 ) spectrum of 2 22

Figure S13. 13 C NMR (150 MHz, DMSO-d 6 ) spectrum of 2 Figure S14. HR-QTOF-MS spectrum of 2 23

Figure S15. 1 H NMR (600 MHz, DMSO-d 6 ) spectrum of 3 24

Figure S16. 13 C NMR (150 MHz, DMSO-d 6 ) spectrum of 3 Figure S17. HR-QTOF-MS spectrum of 3 25

Figure S18. 1 H NMR (600 MHz, DMSO-d 6 ) spectrum of 4 26

Figure S19. 13 C NMR (150 MHz, DMSO-d 6 ) spectrum of 4 Figure S20. HR-QTOF-MS spectrum of 4 27

Figure S21. 1 H NMR (600 MHz, DMSO-d 6 ) spectrum of 5 28

Figure S22. 13 C NMR (150 MHz, DMSO-d 6 ) spectrum of 5 Figure S23. HR-QTOF-ESI-MS spectrum of 5 29

3. MS/MS fragmentation for trichodermides A-E (1-5) Figure S24. Positive and negative ion HR-QTOF-ESI-MS/MS spectra of 1 Figure S25. Positive and negative ion HR-QTOF-ESI-MS/MS spectra of 2 30

Figure S26. Positive and negative ion HR-QTOF-ESI-MS/MS spectra of 3 Figure S27. Positive and negative ion HR-QTOF-ESI-MS/MS spectra of 4 31

Figure S28. Positive and negative ion HR-QTOF-ESI-MS/MS spectra of 5 32

4. C3 Marfey analysis (a) * (b) L-Glu D-Glu (c) L-Pro D-Pro ion abundance (d) L-Ile L-allo-Ile L-Leu D-allo-Ile D-Leu D-Ile (e) L-Val L-Iva L-Leuol D-Iva D-Val D-Leuol (f) Aib 0 5 10 15 20 25 30 35 40 45 50 retention time (min) Figure S29. HPLC-DAD-MS spectrum extracted from a C 3 Marfey s method analysis of 50 g of 1 derivatized with L-FDAA: (a) UV (340 nm), where * = residue L-FDDA; (b) SIE (m/z 398) for L-FDAA- L-Glu; (c) SIE (m/z 366) for L-FDDA-L-Pro; (d) SIE (m/z 382) for L-FDAA-L-Ile and L-FDAA-L-Leu; (e) SIE (m/z 368) for L-FDAA-L-Val, L-FDAA-D-Iva, and L-FDAA-L-Leuol; (f) SIE (m/z 354) for L- FDAA-Aib. Note for (b)-(f) L-FDAA derivatives of authentic amino acid standards are also displayed (dotted line). 33

(a) * (b) L-Glu D-Glu (c) L-Pro D-Pro ion abandance (d) L-Leu L-Ile L-allo-Ile D-Leu D-allo-Ile D-Ile (e) L-Val L-Leuol D-Val D-Leuol (f) Aib 0 5 10 15 20 25 30 35 40 45 50 retention time (min) Figure S30. HPLC-DAD-MS spectrum extracted from a C 3 Marfey s method analysis of 50 g of 2 derivatized with L-FDAA: (a) UV (340 nm), where * = residue L-FDDA; (b) SIE (m/z 398) for L-FDAA- L-Glu; (c) SIE (m/z 366) for L-FDAA-L-Pro; (d) SIE (m/z 382) for L-FDAA-L-Ile and L-FDAA-L-Leu; (e) SIE (m/z 368) for L-FDAA-L-Val and L-FDAA-L-Leuol; (f) SIE (m/z 354) for L-FDAA-Aib. Note for (b)-(f) L-FDAA derivatives of authentic amino acid standards are also displayed (dotted line). 34

(a) * (b) L-Glu D-Glu (c) L-Pro D-Pro ion abundance (d) L-Ile L-Leu L-allo-Ile D-Leu D-allo-Ile D-Ile (e) L-Val L-Iva D-Iva D-Val (f) L-Valol Aib D-Valol 0 5 10 15 20 25 30 35 40 45 50 retention time (min) Figure S31. HPLC-DAD-MS spectrum extracted from a C 3 Marfey s method analysis of 50 g of 3 derivatized with L-FDAA: (a) UV (340 nm), where * = residue L-FDAA; (b) SIE (m/z 398) for L-FDAA- L-Glu; (c) SIE (m/z 366) for L-FDAA-L-Pro; (d) SIE (m/z 382) for L-FDAA-L-Ile and L-FDAA-L-Leu; (e) SIE (m/z 368) for L-FDAA-L-Val and L-FDAA-D-Iva; (f) SIE (m/z 354) for L-FDAA-Aib and L- FDAA-L-Valol. Note for (b)-(f) L-FDAA derivatives of authentic amino acid standards are also displayed (dotted line). 35

(a) * (b) L-Glu D-Glu (c) L-Pro D-Pro ion abundance (d) L-Ile L-Leu D-Leu D-Ile L-allo-Ile D-allo-Ile (e) L-Val D-Val (f) L-Valol Aib D-Valol 0 5 10 15 20 25 30 35 40 45 50 retention time (min) Figure S32. HPLC-DAD-MS spectrum extracted from a C 3 Marfey s method analysis of 50 g of 4 derivatized with L-FDAA: (a) UV (340 nm), where * = residue L-FDAA; (b) SIE (m/z 398) for L-FDAA- L-Glu; (c) SIE (m/z 366) for L-FDAA-L-Pro; (d) SIE (m/z 382) for L-FDAA-L-Ile and L-FDAA-L-Leu; (e) SIE (m/z 368) for L-FDAA-L-Val; (f) SIE (m/z 354) for L-FDAA-Aib and L-FDAA-L-Valol. Note for (b)-(f) L-FDAA derivatives of authentic amino acid standards are also displayed (broken line). 36

ion abundance (a) * (b) L-Glu D-Glu (c) L-Pro D-Pro (d) L-Ile L-Leu D-Leu D-Ile L-allo-Ile D-allo-Ile (e) L-Val L-Iva D-Iva D-Val (f) L-Valol Aib D-Valol 0 5 10 15 20 25 30 35 40 45 50 retention time (min) Figure S33. HPLC-DAD-MS spectrum extracted from a C 3 Marfey s method analysis of 50 g of 5 derivatized with L-FDAA: (a) UV (340 nm), where * = residue L-FDAA; (b) SIE (m/z 398) for L-FDAA- L-Glu; (c) SIE (m/z 366) for L-FDAA-L-Pro; (d) SIE (m/z 382) for L-FDAA-L-Ile and L-FDAA-L-Leu; (e) SIE (m/z 368) for L-FDAA-L-Val and L-FDAA-D-Iva; (f) SIE (m/z 354) for L-FDAA-Aib and L- FDAA-L-Valol. Note for (b)-(f) L-FDDA derivatives of authentic amino acid standards are also displayed (dotted line). 37

% cell viability % cell viability 5. Cytotoxicity assay 150 (a) 100 50 5 4 3 2 1 DMSO (1%) doxorubicin (30 mm) 150 (b) 100 50 5 4 3 2 1 DMSO (1%) doxorubicin (30 mm) 0 0.001 0.01 0.1 1 10 100 Concentration (mm) 0.001 0.01 0.1 1 10 100 Concentration (mm) Figure S34. Cytotoxicity assay of the trichodermides 1 5 against (a) NCI-H460 (lung cancer cell line) and (b) SW620 (human colon cancer cell line) 0 38

Absorbance at 600 nm Absorbance at 600 nm Absorbance at 600 nm Absorbance at 600 nm Absorbance at 600 nm Absorbance at 600 nm Absorbance at 600 nm 6. Antimicrobial assay Candida albicans ATCC 10231 Candida parapsilosis ATCC 22019 1.5 1.0 0.5 1 2 3 4 5 DMSO amphotericin B 1.5 1.0 0.5 1 2 3 4 5 DMSO amphotericin B 0.0 0.01 0.1 1 10 100 conc [µm] 0.0 0.01 0.1 1 10 100 conc [µm] Candida krusei ATCC 6258 Bacillus subtilis ATCC 6051 1.5 1.0 0.5 1 2 3 4 5 DMSO amphotericin B 0.8 0.7 0.6 0.5 0.4 0.3 0.2 1 2 3 4 5 DMSO rifampicin 0.1 0.0 0.01 0.1 1 10 100 conc [µm] 0.0 0.01 0.1 1 10 100 conc [µm] Staphylococcus aureus ATCC 25923 Escherichia coli ATCC 11775 1.5 1.0 0.5 1 2 3 4 5 DMSO rifampicin 3 2 1 1 2 3 4 5 DMSO rifampicin 0.0 0.01 0.1 1 10 100 conc [µm] 0 0.01 0.1 1 10 100 conc [µm] Pseudomonas aeruginosa ATCC 10145 4 1 3 2 1 2 3 4 5 DMSO rifampicin 0 0.01 0.1 1 10 100 conc [µm] Figure S35. Antimicrobial assay screening graphs of trichodermides A-E (1 5) and ampicillin and amphotericin served as controls for bacteria and fungi respectively, while DMSO served as positive control. Trichodermide A (1) showed MIC of 30 M. 39