Xentrivalpeptides A-Q: Depsipeptide diversification in Xenorhabdus

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1 Supplementary Material for Xentrivalpeptides A-Q: Depsipeptide diversification in Xenorhabdus Qiuqin Zhou, Andrea Dowling, Heinrich Heide, Jens Wöhnert, Ulrich Brandt, James Baum, Richard ffrench-constant, and Helge B. Bode, * Institut für Molekulare Biowissenschaften, Goethe Universität Frankfurt, Frankfurt am Main, Germany Biosciences, University of Exeter in Cornwall, Tremough Campus, Penryn, Cornwall TR10 9EZ, UK Zentrum für Biologische Chemie, Molekulare Bioenergetik, Klinikum der Goethe Universität Frankfurt, Frankfurt am Main, Germany Monsanto Company, Chesterfield, M 63017, USA 1

2 Table S1. Results from HRESIMS (exp.) and the resulting molecular formula with the tolerances in ppm. a) protonated xentrivalpeptides and the two main MS 2 fragments. b) MS 3 fragments using fragment B as precursor. Fragment A was fragmented to fragment B by losing a water molecule. Fragmentation of 17 was shown in Figure S8. a [M + H] + fragment A fragment B Nr. molecular formula exp. theo. ppm molecular formula m/z exp. ppm molecular formula m/z exp. ppm 1 C 46 H 66 9 N C 33 H 51 7 N C 33 H 49 6 N C 40 H 62 9 N C 33 H 51 7 N C 33 H 49 6 N C 41 H 64 9 N C 33 H 51 7 N C 33 H 49 6 N C 42 H 66 9 N C 33 H 51 7 N C 33 H 49 6 N C 43 H 68 9 N C 33 H 51 7 N C 33 H 49 6 N C 45 H 72 9 N C 33 H 51 7 N C 33 H 49 6 N C 44 H 70 9 N C 33 H 51 7 N C 33 H 49 6 N C 45 H 64 9 N C 33 H 51 7 N C 33 H 49 6 N C 47 H 68 9 N C 33 H 51 7 N C 33 H 49 6 N C 39 H 68 9 N C 30 H 53 7 N C 30 H 51 6 N C 40 H 70 9 N C 30 H 53 7 N C 30 H 51 6 N C 43 H 68 9 N C 30 H 53 7 N C 30 H 51 6 N C 42 H 74 9 N C 30 H 53 7 N C 30 H 51 6 N C 47 H 68 9 N C 34 H 53 7 N C 34 H 51 6 N C 45 H 64 9 N C 32 H 49 7 N C 32 H 47 6 N C 41 H 64 9 N C 33 H 51 7 N C 32 H 47 6 N C 41 H 57 8 N fragment A in figure S7 fragment B in figure S7 2

3 b fragment C fragment D fragment E fragment F Nr. precursor chemical chemical chemical chemical m/z exp. ppm m/z exp. ppm m/z exp. ppm formula formula formula formula m/z exp. ppm C 22 H 33 3 N C 23 H 31 4 N C 23 H 33 4 N C 20 H 35 4 N C 22 H 33 3 N C 23 H 31 4 N C 23 H 33 4 N C 20 H 35 4 N C 22 H 33 3 N C 23 H 31 4 N C 23 H 33 4 N C 20 H 35 4 N C 22 H 33 3 N C 23 H 31 4 N C 23 H 33 4 N C 20 H 35 4 N C 22 H 33 3 N C 23 H 31 4 N C 23 H 33 4 N C 20 H 35 4 N C 22 H 33 3 N C 23 H 31 4 N C 23 H 33 4 N C 20 H 35 4 N C 22 H 33 3 N C 23 H 31 4 N C 23 H 33 4 N C 20 H 35 4 N C 22 H 33 3 N C 23 H 31 4 N C 23 H 33 4 N C 20 H 35 4 N C 22 H 33 3 N C 23 H 31 4 N C 23 H 33 4 N C 20 H 35 4 N * * * C 20 H 33 4 N C 20 H 35 4 N C 20 H 35 4 N * * * C 20 H 33 4 N C 20 H 35 4 N C 20 H 35 4 N * * * C 20 H 33 4 N C 20 H 35 4 N C 20 H 35 4 N C 23 H 35 3 N C 23 H 31 4 N C 24 H 35 4 N C 21 H 37 4 N C 21 H 31 3 N C 23 H 31 4 N C 22 H 31 4 N C 19 H 33 4 N * not detected; MS 3 fragments of 13 and 16 were too weak. 3

4 Table S2. Results from the advanced Marfey s method: retention times (t R, minute) of the derivatized amino acids from the hydrolyzed 1 detected at negative ion mode by HPLC-MS. Commercial L-amino acids were used as standard. nly phenylalanine in 1 has D-configuration. m/z [M-H] - t R, L-FDLA a t R, LD-FDLA a Pro(4) Thr(2) Val(1)/(5)/(6)/(7) Phe(3) a : Marfey s reagents (N α -(5-fluoro-2,4-dinitrophenyl)-L-leucinamide, abbr. L-FDLA; N α -(5-fluoro-2,4- dinitrophenyl)-d-leucinamide, abbr. D-FDLA ); mixture of L-FDLA and D-FDLA, abbr. LD-FDLA. 4

5 Table S3. NMR Spectroscopic Data (500 MHz ( 1 H), 125 MHz ( 13 C) in CDCl 3 ) of 4, δ in ppm. Amino acid numbering from N- to C-terminus. subunit position δ C δ H, mult (J in Hz) Butyryl a , m 2b , m , m a 1.03, t (7.4) L-Val(1) C= α , m β , m γ 19.6 a 0.97, d (6.6) δ , d (6.4) NH 6.39, d (5.8) L-Thr(2) C= α , d (9.9) β , m γ , d (6.5) NH d (10.0) D-Phe(3) C= α , m β , m γ δ , m ε , m ζ , m NH 7.36, m L-Pro (4) C= α , m β , m β , m γ , m γ , m δ , m δ , m L-Val(5) C= α , m β , m γ 19.6 a 0.87, d, (7.0) δ , d, (6.9) NH 6.19, d, (10.1) L-Val(6) C= α , m β , m γ , d (6.8) δ 19.6 a 0.98, d (6.8) NH 8.04, d (6.9) L-Val(7) C= α , t (7.5) β , m γ , d (6.9) δ , d (6.7) NH 8.80, bs d: doublet; m: multiplet; a : same chemical shift 5

6 Figure S1. a) HRESIMS of 1. b) MS 2 -spectra of 1 after collision induced dissociation (CID) of [M+H] +. c) MS 3 -spectra of fragment B in selected rang. d) Schema for fragmentation path of 1 and the proposed fragment structures. The fragments A-F (Table S1) were selected for the structure elucidation. 6

7 Figure S2. Selected results of feeding experiments from 1-6, 8, and 9 which have the same ring structure. Proline and threonine were incorporated in all xentrivalpeptides (data not shown). Structures of R 1 and R 2 were shown in figure 4. Feeding experiments in nonlabled medium (LB medium, 12 C): incorporation of one L[2,3,3,5,5,6,6,7-2 H 8 ]phenylalanine ( 12 C H 8 -Phe), incorporation of two L[2,3,3,5,5,6,6,7-2 H]phenylalanine ( 12 C H 8 -Phe), incorporation of one DL[2,3,4,4,4,5,5,5-2 H 8 ]valine ( 12 C H 8 -Val), incorporation of two DL[2,3,4,4,4,5,5,5-2 H 8 ]valine ( 12 C H 8 -Val), incorporation of one L[2,3,3,4,5,5,5,5,5,5-2 H 10 ]leucine ( 12 C + 1 Leu), incorporation of one p-phenylacetic acid ( 12 C + 1 p-f- PAA), incorporation of one [U- 2 H 6 ]propionic acid ( 12 C H 6 -propionic acid). Feeding experiments in labled medium (ISGR - 13 C growth medium, 13 C): incorporation of one L-α-amino acid ( 13 C + 1 amino 7

8 acid), incorporation of two L-α-amino acids ( 13 C + 2 amino acids), incorporation of one butyric acid ( 13 C + 1 butyric acid). CID: collision induced dissociation. All xentrivalpeptides were fragmented to fragment A and B (Figure S1). Figure S3. Selected results of feeding experiments for 10-13, which have the same ring structure fragment B. Abbreviations see Figure S2. 8

9 Figure S4. Selected results of feeding experiments for 14. Abbreviations see Figure S2. 9

10 Figure S5. Selected results of feeding experiments for 15. Abbreviations see Figure S2. Fragment B of 15 and 16 indicated the same ring structure (Table S1). But, labeling of 16 was too weak for its characterization. 10

11 a N HN HN Pro(4) Thr(2) Val(1)/(5)/(6)/(7) NH Phe(3) Butyryl b H N N H H N H N N H N NH Figure S6. a) Subunits and selected CSY correlations (bond lines) for 4. b) Selected HMBC correlations (arrows from 1 H to 13 C) between subunits of 4. Amino acid numbering from N- to C-terminus. 11

12 a fragment A [M + H] + - Val fragment B fragment H fragment I fragment G fragment K fragment J m/z b c fragment formula m/z exp. ppm [M + H] + + -Val C 26 H 48 7 N fragment A C 28 H 42 6 N fragment B + C 28 H 40 5 N fragment G + C 26 H 32 5 N fragment H + C 24 H 37 5 N fragment I + C 26 H 30 4 N fragment J C 15 H 28 N fragment K C 17 H 21 N Figure S7. Structure elucidation of 17. a) HR-ESI MS 2 fragments. b) Proposed fragment structures. c) Molecular formula of the fragments. 12

13 Figure S8. Galleria mellonella hemocyte monolayers were incubated for 4h with (A) 2% DMS in Graces Insect Medium as a control, and (B) 100 µg/ml of 1. White arrows indicate regions of actin cytoskeletal ruffling caused by the compound. Green = FITC-phalloidin labeled f-actin, Red = polarized mitochondria, Blue = nuclei. 13

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