Supporting information

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1 Supporting information Rhabdopeptide/Xenortide-like Peptides from Xenorhabdus innexi with Terminal Amines Showing Potent Anti-protozoal Activity Lei Zhao,, Marcel Kaiser,, Helge B. Bode *,, Molekulare Biotechnologie, Fachbereich Biowissenschaften, Goethe Universität Frankfurt, Frankfurt am Main, Germany Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, China Parasite Chemotherapy, Swiss Tropical and Public Health Institute, 4051 Basel, Switzerland University of Basel, 4003 Basel, Switzerland Buchmann Institute for Molecular Life Sciences (BMLS), Goethe Universität Frankfurt, Frankfurt am Main, Germany * Corresponding author: Tel: Fax: h.bode@bio.unifrankfurt.de

2 Experimental Section General experimental procedures. NMR spectra were recorded on a Bruker AV 500 spectrometer at 500 MHz ( 1 H) and 125 MHz ( 13 C) using deuterated MeOH or DMSO as solvent. ESI-HPLC-MS analysis was carried out on a Dionex UltiMate 3000 system coupled to a Bruker AmaZonX mass spectrometer. Compounds were eluted on an ACQUITY UPLC BEH C 18 column (130 Å, 2.1 mm 100 mm, 1.7 μm) using a CH 3 CN/H 2 O gradient containing 0.1% HCOOH (5-95% CH 3 CN for 16 min) at a flow rate of 0.6 ml/min. A scan range of m/z in positive ionization mode was performed to detect compounds. HR-ESI-HPLC-MS were recorded on a Dionex UltiMate 3000 system coupled to a Bruker Impact II QTOF mass spectrometer using an ACQUITY UPLC BEH C 18 RP Column (130 Å, 2.1 mm 50 mm, 1.7 µm) and a same gradient at a flow rate of 0.4 ml/min for 16 min. Strain cultivation. Xenorhabdus innexi DSM was cultivated in a LB medium at 30 C. For HPLC-MS analysis, 100 µl of overnight culture was inoculated to 10 ml of fresh LB medium containing 2% of Amberlite XAD-16. The cultures were grown at 30 C and 200 rpm. After 72 h, the XAD beads were separated and extracted with 10 ml of MeOH for 1 h. For isolation of RXPs, 4 L of cultures were cultivated using above culture conditons. Extraction and purification. After 72 h incubation, the XAD-16 beads from 4 L of cultures were extracted with 300 ml of MeOH three times. The solvent was removed to obtain the MeOH extract. The crude extract was dissolved in H 2 O and extracted with EtOAc. The EtOAc fraction (865.7 mg) was fractionated by Sephadex LH-20 chromatography using MeOH as the eluent. The fractions were analyzed by HPLC-MS, and the enriched fractions containing RXPs were used for further purification. The purification was carried out on an Agilent preparative HPLC system and a Cholester column (10 ID 250 mm). The compounds were eluted by using a CH 3 CN/H 2 O

3 gradient containing 0.1% HCOOH (25-45% CH 3 CN for 40 min) at a flow rate of 3 ml/min to yield 1 (19.4 mg), 2 (21.5 mg), 3 (4.7 mg), 4 (3.0 mg), 5 (3.3 mg), 6 (7.2 mg) and 7 (3.0 mg). Determination of amino acid configuration. Amino acid configuration of 1-7 was determined using the advanced Marfey's method as described previously. 1,2 Bioactivity tests. Compounds 1-7 were tested against the parasites Trypanosoma brucei rhodesiense STIB900, Trypanosoma cruzi Tulahuen C4, Leishmania donovani MHOM-ET- 67/L82, and Plasmodium falciparum NF54 in duplicates as described previously. 3 Their cytotoxic activities against the rat skeletal myoblasts (L6 cells) were conducted also in duplicates as described previously. 3

4 Supplementary Tables Table S1. HR-MS data of 1-7 m/z [M+2H] 2+ m/z [M+H] + compound sum formula calcd found calcd found Δ ppm 1 C 49 H 87 N 9 O C 45 H 78 N 8 O C 50 H 89 N 9 O C 46 H 80 N 8 O C 54 H 96 N 10 O C 55 H 98 N 10 O C 51 H 89 N 9 O

5 Table S2. 1 H (600 MHz) and 13 C (150 MHz) NMR Data of 2 in DMSO-d 6 (δ in ppm, J in Hz) subunit position δ c δ H N-Me-L-Val (a) , d (5.9) , m , overlap , overlap HN-CH , s L-Val (b) , d (8.3) , m , overlap , overlap NH 8.07, d (8.6) N-Me-L-Val (c) , d (10.7) , m , overlap , overlap N-CH , s N-Me-L-Val (d) , d (11.3) , m , overlap , overlap N-CH , s N-Me-L-Val (e) , d (11.1) , m , overlap , d (6.5) N-CH , s N-Me-L-Phe (f) , d (7.7) , d (7.8) , overlap , overlap , overlap , overlap , overlap N-CH , s N-Me-L-Val (g) , d (10.9) , m , d (6.3) , overlap N-CH , s NH 2 (B) 7.31, s; 7.06, s

6 Supplementary Figures 1 -V----mF--Put +MS 2 ( [M+2H] 2+ ) Put mf m/z 2 -V----mF--NH 3 +MS 2 ( [M+H] + ) mf NH m/z

7 mf--put +MS 2 ( [M+2H] 2+ ) Put mf m/z mf--nh 3 +MS 2 ( [M+H] + ) mf NH m/z

8 5 -V---V--mF--Put +MS 2 ( [M+2H] 2+ ) V 99 Put mf m/z 6 -V-----mF--Put +MS 2 ( [M+2H] 2+ ) Put mf m/z

9 7 -V-----mF--NH 3 +MS 2 ( [M+H] + ) mf NH m/z Figure S1. MS 2 fragmentation pattern of 1-7

10 Figure S2. Determination of amino acid configuration for 1-7 using the advanced Marfey's method. (a)-(g) HPLC-MS analysis of hydrolyzed 1-7 and derivatized with L-FDLA (upper) and L-/D-FDLA (lower) in comparison to (h) standard amino acids. Depicted are extracted ion chromatogram (EIC) traces for valine (Val, m/z 412 [M+H] + ), N-methylvaline (N-MeVal, m/z 426 [M+H] + ), and N-methylphenylalanine (N-MePhe, m/z 474 [M+H] + ). The configuration is determined by the elution order and L-FDLA derivatized amino acids elute prior to its D- enantiomer.

11 Figure S3. 1 H NMR (500 MHz, methanol-d 4 ) spectrum of compound 1 Figure S4. 13 C NMR (125 MHz, methanol-d 4 ) spectrum of compound 1

12 Figure S5. COSY (methanol-d 4 ) spectrum of compound 1 Figure S6. HSQC (methanol-d 4 ) spectrum of compound 1

13 Figure S7. HMBC (methanol-d 4 ) spectrum of compound 1

14 Figure S8. 1 H NMR(500 MHz, methanol-d 4 ) spectrum of compound 2 Figure S9. 13 C NMR (125 MHz, methanol-d 4 ) spectrum of compound 2

15 Figure S10. COSY (methanol-d 4 ) spectrum of compound 2 Figure S11. HSQC (methanol-d 4 ) spectrum of compound 2

16 Figure S12. HMBC (methanol-d 4 ) spectrum of compound 2

17 Figure S13. 1 H NMR (600 MHz, DMSO-d 6 ) spectrum of compound 2 Figure S C NMR (150 MHz, DMSO-d 6 ) spectrum of compound 2

18 Figure S15. COSY (DMSO-d 6 ) spectrum of compound 2 Figure S16. HSQC (DMSO-d 6 ) spectrum of compound 2

19 Figure S17. HMBC (DMSO-d 6 ) spectrum of compound 2

20 Figure S18. 1 H NMR (500 MHz, methanol-d 4 ) spectrum of compound 3 Figure S C NMR (125 MHz, methanol-d 4 ) spectrum of compound 3

21 Figure S20. COSY (methanol-d 4 ) spectrum of compound 3 Figure S21. HSQC (methanol-d 4 ) spectrum of compound 3

22 Figure S22. HMBC (methanol-d 4 ) spectrum of compound 3

23 Figure S23. 1 H NMR (500 MHz, methanol-d 4 ) spectrum of compound 4 Figure S C NMR (125 MHz, methanol-d 4 ) spectrum of compound 4

24 Figure S25. COSY (methanol-d 4 ) spectrum of compound 4 Figure S26. HSQC (methanol-d 4 ) spectrum of compound 4

25 Figure S27. HMBC (methanol-d 4 ) spectrum of compound 4

26 Figure S28. 1 H NMR (500 MHz, methanol-d 4 ) spectrum of compound 5 Figure S C NMR (125 MHz, methanol-d 4 ) spectrum of compound 5

27 Figure S30. COSY (methanol-d 4 ) spectrum of compound 5 Figure S31. HSQC (methanol-d 4 ) spectrum of compound 5

28 Figure S32. HMBC (methanol-d 4 ) spectrum of compound 5

29 Figure S33. 1 H NMR (methanol-d 4, 500 MHz) spectrum of compound 6 Figure S C NMR (methanol-d 4, 125 MHz) spectrum of compound 6

30 Figure S35. COSY (methanol-d 4 ) spectrum of compound 6 Figure S36. HSQC (methanol-d 4 ) spectrum of compound 6

31 Figure S37. HMBC (methanol-d 4 ) spectrum of compound 6

32 Figure S38. 1 H NMR (500 MHz, methanol-d 4 ) spectrum of compound 7 Figure S C NMR (125 MHz, methanol-d 4 ) spectrum of compound 7

33 Figure S40. COSY (methanol-d 4 ) spectrum of compound 7 Figure S41. HSQC (methanol-d 4 ) spectrum of compound 7

34 Figure S42. HMBC (methanol-d 4 ) spectrum of compound 7

35 References (1) Reimer, D.; Cowles, K. N.; Proschak, A.; Nollmann, F. I.; Dowling, A. J.; Kaiser, M.; Ffrench-Constant, R.; Goodrich-Blair, H.; Bode, H. B. ChemBiochem 2013, 14, (2) Grundmann, F.; Kaiser, M.; Kurz, M.; Schiell, M.; Batzer, A.; Bode, H. B. RSC Adv. 2013, 3, (3) Orhan, I.; Sener, B.; Kaiser, M.; Brun, R.; Tasdemir, D. Mar. Drugs 2010, 8,