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Supplementary Information Vitroprocines, new antibiotics against Acinetobacter baumannii, discovered from marine Vibrio sp. QWI 6 using mass spectrometry based metabolomics approach Chih Chuang Liaw 1,2,3 *, Pei Chin Chen 1, Chao Jen Shih 4, Sung Pin Tseng 5, Ying Mi Lai 4, Chi Hsin Hsu 1,2, Pieter C. Dorrestein 6, and Yu Liang Yang 2,3,4 * 1 Department of Marine Biotechnology and Resources, National Sun Yat sen University, Kaohsiung 8424, Taiwan 2 Doctoral Degree Program in Marine Biotechnology, National Sun Yat sen University, Kaohsiung 8424, Taiwan 3 Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 87, Taiwan 4 Agricultural Biotechnology Research Center, Academia Sinica, Taipei 11529, Taiwan 5 Department of Medical Laboratory Sciences and Biotechnology, Kaohsiung Medical University, Kaohsiung 87, Taiwan 6 Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 9293 636, United States *Corresponding. ccliaw@mail.nsysu.edu.tw, ylyang@gate.sinica.edu.tw 1

Contents of Supplementary Information Contents Pages Experimental Section 5 Table S1 1 H (5 MHz) and 13 C NMR (125 MHz) Data of 1-3 and 8 in [D 4 ]MeOH 6 Figure S1. Anti-microbial assay of Vibrio sp. QWI-6 crude extract (1, 1 g) and 7 compound 1 (2-7, 4 g) Figure S2. Phylogenetic analysis of Vibrio sp. QWI-6 7 Figure S3 Intact-cell MALDI-MS analysis the colonies of Vibrio sp. QWI-6 (red) and 8 V. proteolyticus ATCC 15338 (blue) Figure S4 Full molecular networking of Vibrio sp. QWI-6 8 Figure S5 Mass vs. error distribution of vitroprocines nodes selected from molecular 9 networking Table S2 Summary of vitroprocines (I): subgroups A-D and F 1 Table S3 Summary of vitroprocines (II): subgroups E and G 12 Figure S6 HR-ESIMS/MS spectra of subgroup A 13 Figure S7 HR-ESIMS/MS spectra of subgroup B 14 Figure S8 HR-ESIMS/MS spectra of subgroup C 15 Figure S9 HR-ESIMS/MS spectra of subgroup D 16 Figure S1 HR-ESIMS/MS spectra of subgroup E 17 Figure S11 HR-ESIMS/MS spectra of subgroup F 18 Figure S12 HR-ESIMS/MS spectra of subgroup G 19 Figure S13 UV spectrum of 1 2 Figure S14 FTIR spectrum of 1 2 Figure S15 1 H-NMR spectrum of 1 21 Figure S16 13 C NMR spectrum of 1 21 Figure S17 HSQC spectrum of 1 22 Figure S18 HMBC spectrum of 1 22 Figure S19 1 H- 1 H COSY spectrum of 1 23 Figure S2 1 H NMR Spectra of 1 and its R and S Mosher derivatives in CDCl 3 23 Figure S21 EIMS spectrum and fragmentation of the dimethyl disulfide derivative of 1 24 Figure S22 UV spectrum of 2 25 Figure S23 FTIR spectrum of 2 25 Figure S24 1 H NMR spectrum of 2 26 Figure S25 13 C NMR spectrum of 2 26 Figure S26 HSQC spectrum of 2 27 Figure S27 HMBC spectrum of 2 27 Figure S28 1 H- 1 H COSY spectrum of 2 28 2

Figure S29 EIMS spectrum and fragmentation of the dimethyl disulfide derivative of 2 28 Figure S3 UV spectrum of 3 29 Figure S31 FTIR spectrum of 3 29 Figure S32 1 H NMR spectrum of 3 3 Figure S33 13 C NMR spectrum of 3 3 Figure S34 HSQC spectrum of 3 31 Figure S35 HMBC spectrum of 3 31 Figure S36 1 H- 1 H COSY spectrum of 3 32 Figure S37 UV spectrum of 4 33 Figure S38 FTIR spectrum of 4 33 Figure S39 1 H NMR spectrum of 4 34 Figure S4 13 C NMR spectrum of 4 34 Figure S41 HSQC spectrum of 4 35 Figure S42 HMBC spectrum of 4 35 Figure S43 1 H- 1 H COSY spectrum of 4 36 Figure S44 EIMS spectrum and fragmentation of the dimethyl disulfide derivative of 4 36 Figure S45 UV spectrum of 5 37 Figure S46 FTIR spectrum of 5 37 Figure S47 1 H NMR spectrum of 5 38 Figure S48 13 C NMR spectrum of 5 38 Figure S49 UV spectrum of 6 39 Figure S5 FTIR spectrum of 6 39 Figure S51 1 H NMR spectrum of 6 4 Figure S52 13 C NMR spectrum of 6 4 Figure S53 EIMS spectrum and fragmentation of the dimethyl disulfide derivative of 6 41 Figure S54 UV spectrum of 7 42 Figure S55 FTIR spectrum of 7 42 Figure S56 1 H NMR spectrum of 7 43 Figure S57 13 C NMR spectrum of 7 43 Figure S58 EIMS spectrum and fragmentation of the dimethyl disulfide derivative of 7 44 Figure S59 UV spectrum of 8 45 Figure S6 FTIR spectrum of 8 45 Figure S61 1 H NMR spectrum of 8 46 Figure S62 13 C NMR spectrum of 8 46 Figure S63 HSQC spectrum of 8 47 Figure S64 HMBC spectrum of 8 47 Figure S65 1 H- 1 H COSY spectrum of 8 48 3

Figure S66 EIMS spectrum and fragmentation of the dimethyl disulfide derivative of 8 48 Figure S67 UV spectrum of 9 49 Figure S68 FTIR spectrum of 9 49 Figure S69 1 H NMR spectrum of 9 5 Figure S7 13 C NMR spectrum of 9 5 Figure S71 UV spectrum of 1 51 Figure S72 FTIR spectrum of 1 51 Figure S73 1 H NMR spectrum of 1 52 Figure S74 13 C NMR spectrum of 1 52 Figure S75 EIMS spectrum and fragmentation of the dimethyl disulfide derivative of 1 53 Figure S76 Quantitation results of 1, 3, and 8 from Vibrio sp. QWI-6 under different 54 cultural condition Figure S77 HR-ESIMS/MS spectrum and fragmentation of 1 from Vibrio sp. QWI-6 55 feed with 13 C 9 -tyrosine Figure S78 13 C NMR spectra of 1 and 1a 56 4

Experimental Section Instrumentation. Optical rotations were measured on a JASCO P12 digital polarimeter at 25 C. UV spectra were recorded on a JASCO V-65 spectrophotometer. IR spectra were obtained on a JASCO FT/IR-41 infrared spectrophotometer. NMR spectra were recorded with Bruker 5 AVII and Varian Unity 4 FT NMR spectrometers. HPLC-ESIMS data were processed using an AmaZon SL ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany) and UPLC-HR-ESIMS and MS/MS data were acquired on a Thermo Orbitrap Elite system and a Thermo TSQ Quantum Access MAX Triple Quadrupole system. IMS data were acquired in a Bruker autoflex TM speed MALDI-TOF/TOF system. Extracts were chromatographed on Sephadex LH-2 (Amersham Biosciences) and purified using a semi-preparative RP HPLC column (Supelco Discovery C18, 25 x 1 mm, 5 μm). HPLC was performed on a Hitachi L-213 apparatus equipped with a Hitachi L-242 UV-VIS detector. Minimum inhibitory concentration (MIC) assay. The MICs of compounds against A. baumannii ATCC 1966 were determined using the broth microdilution method as described previously. [1] Briefly, A. baumannii was inoculated in BHI agar at 37 C overnight then diluted with CAMHB broth to adjust the turbidity approximately to the standard McFarland.5 (2 1 8 CFU/mL). [2] Then the bacterial suspension was further diluted by 2 times with CAMHB broth to achieve an initial loading of 1 1 6 CFU/mL. Compounds resolved in DMSO were series diluted in H 2 O at various concentrations. The bacterial suspension (1 μl) was mixed with an equal volume of sample solution at various concentrations in each well of a 96-well plate, and incubated for 2~24 h at 37 C. The MIC was defined as the lowest concentration, which no visible turbidity was detected with unaided eyes and a microplate reader (Bio-Rad) in OD6. Broth with bacteria only was used as a negative control, ceftazidime and ticarcillin were used as positive control (MICs of both drugs are 8 μg/ml), and all the data were measured in triplicate. Biosynthetic studies. 1-13 C-sodium acetate and 13 C 9 -tyrosine were used (99% of 13 C, Cambridge Isotope Laboratories, Isotec, and Aldrich) for the isotope labeling experiments. Vibrio sp. QWI-6 was cultivated in Difco marine agar. For each experiment,.1 mm of 1-13 C-sodium acetate and 13 C 9 -tyrosine were respectively added in the sterile agar medium for 5 Petri dish plates (9 15 mm). Following the inoculum, all the cultures were grown for three days at 3 C. The isolation of labeled vitroprocine A (1) was carried out according to the aforementioned procedure and the compound was subjected to NMR and LC-HR-ESIMS/MS analysis (Supporting information Figures S77 and S78). The yields of 1-13 C-sodium acetate labeled vitroprocine A (1a) were approximately.2 mg/plate. References [1] Performance standards for antimicrobial susceptibility testing: twenty-third informational supplement, Vol. 33, Clinical and Laboratory Standards Institute, Wayne, PA, 213. [2] a) I. Wiegand, K. Hilpert, R. E. Hancock, Nat. Protoc. 28, 3, 163-175; b) Y. Huang, N. Wiradharma, K. Xu, Z. Ji, S. Bi, L. Li, Y. Y. Yang, W. Fan, Biomaterials 212, 33, 8841-8847. 5

Table S1. 1 H (5 MHz) and 13 C NMR (125 MHz) Data of 1-3 and 8 in [D 4 ]MeOH. 1 2 3 8 H (J in Hz) C [a] [b] H (J in Hz) C H (J in Hz) C H (J in Hz) C 1 2.92, dd (14.4, 5.5) 33.8 CH 2 2.92, dd (14.5, 5.6) 33.9 CH 2 2.92, dd (14.5, 5.6) 33.8 CH 2 3.1, dd (14.3, 5.3) 35.7 CH 2 2.72, dd (14.4, 9.) 2.72, dd (14.5, 9.2) 2.73, dd (14.5, 9.) 2.76, dd (14.3, 9.1) 2 3.38, ddd (9., 5.5, 3.2) 59.2 CH 3.38, ddd (9.2, 5.6, 3.1) 59.2 CH 3.39, ddd (9., 5.6, 3.2) 59.2 CH 3.37, m 58.9 CH 3 3.74, dt (9., 3.2) 71.5 CH 3.73, dt (9.2, 3.1) 71.5 CH 3.74, m 71.4 CH 3.7, m 72.3 CH 4 1.25~1.6, m 33.1 CH 2 2.4, m 28.3 CH 2 1.25~1.4, m 3.8 CH 2 1.25~1.6, m 33.1 CH 2 5 2.7, m 28.4 CH 2 5.38, m 131.2 CH 1.25~1.6, m 27.2 CH 2 1.25~1.4, m 31.1 CH 2 6 5.4, m 131.6 CH 5.34, m 13.7 CH 1.25~1.4, m 3.9 CH 2 1.25~1.4, m 3.2 CH 2 7 5.36, m 13.4 CH 2.4, m 28.3 CH 2 1.25~1.4, m 3.9 CH 2 1.25~1.6, m 27.5 CH 2 8 2.7, m 28.1 CH 2 1.25~1.6, m 27.1 CH 2 1.25~1.4, m 3.8 CH 2 2.5, m 28.4 CH 2 9 1.25~1.6, m 27.4 CH 2 1.25~1.4, m 31. CH 2 1.25~1.4, m 3.8 CH 2 5.4, m 131.6 CH 1 1.25~1.4, m 31. CH 2 1.25~1.4, m 3.9 CH 2 1.25~1.4, m 3.7 CH 2 5.36, m 13.4 CH 11 1.25~1.4, m 3.2 CH 2 1.25~1.4, m 3.3 CH 2 1.25~1.4, m 3.6 CH 2 2.5, m 28.2 CH 2 12 1.25~1.6, m 33. CH 2 1.25~1.4, m 3.2 CH 2 1.25~1.6, m 33.2 CH 2 1.25~1.6, m 33.1 CH 2 13 1.25~1.4, m 23.9 CH 2 1.25~1.6, m 33.3 CH 2 1.25~1.4, m 23.9 CH 2 1.25~1.4, m 23.9 CH 2 14.9, t (7.) 14.6 CH 3 1.25~1.6, m 33.1 CH 2.9, t (6.9) 14.6 CH 3.9, t (6.5) 14.6 CH 3 15 1.25~1.4, m 23.9 CH 2 16.9, t (6.8) 14.6 CH 3 Ar 1 128.1 qc 128.1 qc 128.2 qc 138.5 qc 2 7.1, d (8.5) 131.4 CH 7.1, d (8.5) 131.4 CH 7.1, d (8.5) 131.5 CH 7.28, d (6.8) 13.4 CH 3 6.78, d (8.5) 117. CH 6.79, d (8.5) 117. CH 6.78, d (8.5) 117.3 CH 7.35, t (7.5) 13.2 CH 4 158. qc 158.2 qc 158.1 qc 7.28, m 128.3 CH 5 6.78, d (8.5) 117. CH 6.79, d (8.5) 117. CH 6.78, d (8.5) 117.3 CH 7.35, t (7.5) 13.2 CH 6 7.1, d (8.5) 131.4 CH 7.1, d (8.5) 131.4 CH 7.1, d (8.5) 131.5 CH 7.28, d (6.8) 13.4 CH [a] Data were recorded at 4 MHz and [b] 1 MHz. 6

Figure S1. Anti-microbial assay of Vibrio sp. QWI-6 crude extract (1, 1 g) and compound 1 (2-7, 4 g). 1 and 2: Acinetobacter baumanni; 3: Klebsiella pneumonia; 4: Escherichia coli; 5: Staphylococcus aureus; 6: Bacillus cereus; 7: Candida albicans. Scale bar:.5 cm. 94 45 64 58 Vibrio campbellii strain 4 Vibrio rotiferianus strain CAIM 577 Vibrio owensii strain DY5 Vibrio azureus strain LC2-5 65 92 Vibrio alginolyticus strain NBRC 1563 Vibrio natriegens strain ATCC 1448 Vibrio sp. QWI6 Vibrio proteolyticus strain ATCC 15338 Vibrio parahaemolyticus strain ATCC 1782.2 Figure S2. Phylogenetic analysis of Vibrio sp. QWI-6. The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length =.54964 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. The analysis involved 9 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1395 positions in the final dataset. Evolutionary analyses were conducted in MEGA5. The accession numbers of Vibrio type strains used for analysis: V. alginolyticus NBRC 1563 (NR_1226), V. parahaemolyticus ATCC 1782 (NR_118928), V. campbellii strain 4 (NR_29222), V. azureus LC2-5 (NR_117997), V. natriegens ATCC 1448 (NR_11789), V. rotiferianus CAIM 577 (NR_4281), V. proteolyticus ATCC 15338 (NR_118927), V. owensii DY5 (NR_117424). 7

Figure S3. Intact-cell MALDI-MS analysis the colonies of Vibrio sp. QWI-6 (red) and V. proteolyticus ATCC 15338 (blue). vitroprocines diketopiperazines Figure S4. Full molecular networking of Vibrio sp. QWI-6. 8

Figure S5. Mass vs. error distribution of vitroprocines nodes selected from molecular networking 9

Table S2. Summary of vitroprocines (I): subgroups A-D and F [M+H] + Found Calcd. Error (Da) Isolated Networking Double bond A1 C 2 H 34 NO 2 32.257 32.259.2 1 V 6,7 A2 C 21 H 36 NO 2 334.2725 334.2746.21 - V DNC* A3 C 22 H 38 NO 2 348.2882 348.293.21 2 V 5,6 *Does not confirm (DNC) [M+H] + Found Calcd. Error (Da) Isolated Networking B1 C 16 H 28 NO 2 266.212 266.212.18 - V B2 C 17 H 3 NO 2 28.2258 28.2277.19 - V B3 C 18 H 32 NO 2 294.2415 294.2433.18 - V B4 C 19 H 34 NO 2 38.2571 38.259.19 - V B5 C 2 H 36 NO 2 322.2726 322.2746.2 3 V B6 C 21 H 38 NO 2 336.2884 336.293.19 - V [M+H] + Found Calcd. Error (Da) Isolated Networking Double bond C1 C 18 H 3 NO 3 38.228 38.2226.18 - V - C2 C 2 H 34 NO 3 336.2518 336.2539.21 - V - C3 C 22 H 36 NO 3 362.2675 362.2695.2 6 V 6,7 C4 C 22 H 38 NO 3 364.2831 364.2852.21 5 V - C5 C 24 H 4 NO 3 39.2986 39.38.22 4 V 6,7 C6 C 26 H 44 NO 3 418.3299 418.3321.22 7 -* 6,7 *C6 was detected in LC-MS but the intensity was too low to be selected for data-dependent LC-MS/MS analysis 1

[M+H] + Found Calcd. Error (Da) Isolated Networking Double bond D1 C 2 H 34 NO 34.2623 34.264.17 8 V 9,1 D2 C 22 H 36 NO 33.2774 33.2797.23 - V DNC* D3 C 24 H 4 NO 358.388 358.311.22 - V DNC* *Two double bonds are on the aliphatic chain but the positions are unidentified. [M+H] + Found Calcd. Error (Da) Isolated Networking Double bond F1 C 18 H 3 NO 2 292.2259 292.2277.18 - V - F2 C 22 H 36 NO 2 346.2726 346.2746.2 1 -* 9,1 F3 C 22 H 38 NO 2 348.2882 348.293.21 9 V - *F2 was detected in LC-MS but the intensity was too low to be selected for data-dependent LC-MS/MS analysis 11

Table S3. Summary of vitroprocines (II): subgroups E and G. The structures of E1 and G1 were proposed based on the HR-ESIMS/MS fragmentations. The stereochemistry of C-2 in subgroup E and C-2, C-3 in subgroup G were proposed based on the same biosynthetic origin of vitroprocines A-J (1-1). E1 [M+H] + Found Calcd. Error (Da) E1 C 16 H 28 NO 25.2155 25.2171.16 E2 C 17 H 3 NO 264.2311 264.2327.16 E3 C 18 H 32 NO 278.2467 278.2484.17 E4 C 19 H 34 NO 292.2623 292.264.17 *The stereochemistry is unidentified. G1 [M+H] + Found Calcd. Error (Da) Double bond G1 C 18 H 32 NO 3 31.2362 31.2382.2 - G2 C 19 H 34 NO 3 324.2519 324.2539.2 - G3 C 2 H 36 NO 3 338.2675 338.2695.2 - G4 C 21 H 38 NO 3 352.283 352.2852.22 - G5 C 22 H 38 NO 3 364.2827 364.2852.25 DNC G6 C 22 H 4 NO 3 366.2987 366.38.21 - *The stereochemistry is unidentified. 12

1 p @ [ ] 32.247 8 6 4 2 17.488 197.1894 112.413 136.754 179.1789 285.224 133.644 95.852 159.8 21.1266 228.2315 243.1742 267.298 32.2591 8 1 12 14 16 18 2 22 24 26 28 3 32 34 349.1729 A1 1 316.263 8 6 4 17.487 2 211.247 136.754 299.2364 193.1943 91.8193 123.1162 148.741 179.1792 214.9985 247.624 269.8528 322.2486 8 1 12 14 16 18 2 22 24 26 28 3 32 34 A2 1 33.2784 8 6 334.558 4 224.2368 17.488 2 136.754 27.214 313.2519 95.852 121.645 159.82 187.1114 231.1738 261.1291 289.1627 348.2889 8 1 12 14 16 18 2 22 24 26 28 3 32 34 Figure S6. HR-ESIMS/MS spectra of subgroup A A3 13

8 6 248.25 142.1588 4 242.736 2 15.912 86.9823 97.953 121.644 136.755 181.755 195.1844 213.1648 231.1738 265.2631 8 1 12 14 16 18 2 22 24 26 28 3 1 17.488 x2 B1 8 6 4 2 15.914 95.854 121.647 136.756 171.1166 186.1851 29.211 219.1717 245.192 279.2792 291.2447 8 1 12 14 16 18 2 22 24 26 28 3 1 17.49 113.32 x2 156.1746 262.2165 B2 1 276.2322 8 17.49 6 258.2216 4 15.914 17.193 2 98.995 91.547 117.699 133.65 157.113 185.1331 2.21 223.2171 241.195 293.2947 8 1 12 14 16 18 2 22 24 26 28 3 B3 1 17.486 29.2469 8 6 4 184.252 272.2365 15.98 2 133.643 214.2158 91.534 117.693 171.116 199.1474 243.1915 255.211 37.2879 8 1 12 14 16 18 2 22 24 26 28 3 32 34 B4 p @ [ ] 34.2627 1 8 17.488 6 286.2523 4 15.91 198.2212 2 188.1147 117.695 133.645 322.2733 95.852 157.11 171.1163 24.1473 228.2316 257.213 269.226 8 1 12 14 16 18 2 22 24 26 28 3 32 34 B5 1 318.2783 8 6 17.488 322.2615 4 212.2368 3.2678 2 117.693 15.911 93.164 133.643 171.1161 184.256 243.255 27.233 283.2413 335.2663 8 1 12 14 16 18 2 22 24 26 28 3 32 34 Figure S7. HR-ESIMS/MS spectra of subgroup B B6 14

p @ [ ] 29.2111 8 184.1693 6 4 2 17.489 95.852 133.646 17.1537 157.646 197.1533 233.198 255.1742 273.1846 38.2222 8 1 12 14 16 18 2 22 24 26 28 3 32 34 1 C1 8 6 4 p @ [ ] 2 17.487 31.2154 336.2527 136.753 157.653 181.754 198.1848 224.23 237.177 283.251 8 1 12 14 16 18 2 22 24 26 28 3 32 34 1 8 6 4 2 1 1 p @ [ ] 192.113 212.23 344.2576 318.2418 26.1171 238.216 17.488 136.754 221.1895 39.226 327.2312 157.646 199.1113 362.2684 256.2268 27.2412 321.9673 8 1 12 14 16 18 2 22 24 26 28 3 32 34 36 38 4 p @ [ ] x5 346.2729 5 226.216 24.2315 17.488 136.753 27.125 329.2463 173.959 253.2163 281.986 311.2366 364.2625 8 1 12 14 16 18 2 22 24 26 28 3 32 34 36 38 4 C2 C3 C4 1 5 1 p @ [ ] x5 266.2476 372.2893 136.755 157.647 252.232 355.2628 39.3 17.441 173.958 217.1946 275.1638 298.2365 337.2521 8 1 12 14 16 18 2 22 24 26 28 3 32 34 36 38 4 5 @ [ ] x1 294.2799 4.3219 145.652 178.867 341.2848 358.3114 17.45 21.1282 235.2436 252.2692 288.2589 36.2792 376.3218 419.2776 8 1 12 14 16 18 2 22 24 26 28 3 32 34 36 38 4 42 Figure S8. HR-ESIMS/MS spectra of subgroup C C5 C6 15

8 6 4 2 1 91.539 117.696 12.85 x2 157.19 171.1165 129.696 185.1321 269.2256 213.1629 247.6125 286.2523 34.2634 8 1 12 14 16 18 2 22 24 26 28 3 32 34 36 D1 1 12.85 8 6 4 2 81.693 198.2213 15.696 117.697 123.1163 171.1167 13.539 143.854 17.489 157.19 196.251 131.854 95.852 165.1638 185.1324 212.2371 133.647 147.1168 173.1321 21.1636 8 9 1 11 12 13 14 15 16 17 18 19 2 21 D1* x5 312.2682 32.2471 8 6 4 12.87 265.2332 295.2426 33.2788 2 15.697 129.699 169.112 191.1793 252.2698 143.854 269.2952 211.1479 238.2174 287.345 8 1 12 14 16 18 2 22 24 26 28 3 32 34 36 1 D2 8 6 4 2 1 12.85 123.1165 117.696 15.695 143.852 157.19 171.1165 91.539 19.18 129.696 185.1321 191.1789 13.537 165.1631 133.18 147.1166 173.1323 199.148 213.1629 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 D2* x1 34.299 8 6 12.85 4 129.696 143.853 219.212 2 236.2366 323.2727 358.394 15.695 157.18 183.1163 197.1317 241.1945 281.2265 294.286 8 1 12 14 16 18 2 22 24 26 28 3 32 34 36 1 D3 1 8 12.85 123.1167 6 4 117.696 129.696 143.853 219.212 2 15.695 131.851 157.18 171.1164 183.1163 19.11 149.132 91.55 13.543 163.1474 177.164 197.1317 211.148 8 9 1 11 12 13 14 15 16 17 18 19 2 21 Figure S9. HR-ESIMS/MS spectra of subgroup D (*expanded spectra) D3* 16

8 6 131.853 117.696 4 215.1791 2 91.537 15.696 145.112 159.1167 236.1127 173.132 25.2171 8 1 12 14 16 18 2 22 24 26 28 3 1 x5 232.256 E1 8 131.852 6 12.84 4 229.1944 2 15.695 252.6272 91.536 145.19 173.1317 22.1111 215.1143 264.2311 8 1 12 14 16 18 2 22 24 26 28 3 1 x5 246.229 E2 x5 26.2361 8 6 131.849 12.82 4 243.298 2 91.535 15.693 145.15 156.1741 173.1314 189.1647 24.594 278.2464 289.6844 8 1 12 14 16 18 2 22 24 26 28 3 1 E3 x5 274.2524 8 6 131.852 4 12.84 2 257.2259 15.695 91.536 145.19 156.1743 17.19 187.1476 21.1635 292.2628 8 1 12 14 16 18 2 22 24 26 28 3 1 Figure S1. HR-ESIMS/MS spectra of subgroup E E4 17

8 6 4 2 1 12.85 17.1537 157.19 131.854 183.1165 15.695 197.1326 223.865 x1 239.1792 257.1897 274.2162 291.1849 8 1 12 14 16 18 2 22 24 26 28 3 32 34 36 F1 17.1537 1 12.85 8 117.697 6 157.19 4 131.854 155.854 115.542 143.853 183.1165 2 15.695 129.695 197.1326 95.853 133.643 166.1592 178.853 19.1221 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 x5 328.2625 1 293.2256 5 311.2361 95.852 12.83 133.644 165.1632 183.1163 26.1897 224.23 24.2317 282.2422 346.2727 8 1 12 14 16 18 2 22 24 26 28 3 32 34 36 p @ [ ] 1 5 133.644 12.83 141.695 165.1632 26.1897 117.695 2.1429 19.17 183.1163 129.695 163.1474 197.1319 29.1317 95.852 143.852 167.849 195.1162 155.851 213.1636 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 F1* F2 F2* 8 6 4 2 1 226.2161 12.86 211.1478 232.1692 141.697 169.19 183.1166 197.1322 15.697 25.2525 267.213 x1 295.2415 312.2683 33.2785 334.371 348.289 8 1 12 14 16 18 2 22 24 26 28 3 32 34 36 F3 1 12.86 8 117.696 141.697 183.1166 6 129.697 143.854 157.19 169.19 197.1322 4 19.18 131.854 191.1791 211.1478 2 15.697 133.647 171.1165 199.1479 91.499 13.537 151.148 163.1473 216.1741 8 9 1 11 12 13 14 15 16 17 18 19 2 21 Figure S11. HR-ESIMS/MS spectra of subgroup F (*expanded spectra) F3* 18

1 292.2268 8 6 274.2163 4 17.489 166.861 2 169.1585 257.1898 95.853 133.646 147.84 195.624 223.939 239.1797 31.246 8 1 12 14 16 18 2 22 24 26 28 3 32 34 G1 1 36.242 8 288.2314 6 17.487 166.858 4 182.1898 324.232 117.691 159.81 271.249 2 95.85 133.644 147.8 187.119 215.1428 245.1881 26.1973 8 1 12 14 16 18 2 22 24 26 28 3 32 34 G2 1 32.257 8 6 4 2 32.2466 166.856 17.486 196.252 159.798 285.221 95.849 133.643 145.642 179.1786 2.21 243.1745 259.244 338.2683 8 1 12 14 16 18 2 22 24 26 28 3 32 34 G3 1 334.2735 8 6 4 2 166.86 21.2213 17.489 299.2365 133.646 149.593 187.1115 214.2162 265.149 288.2295 316.263 352.292 8 1 12 14 16 18 2 22 24 26 28 3 32 34 36 G4 x2 346.2733 8 6 4 328.2628 2 166.86 17.488 222.2212 133.646 15.911 176.165 25.195 24.2319 311.2363 364.287 8 1 12 14 16 18 2 22 24 26 28 3 32 34 36 38 4 1 G5 1 348.2883 8 6 4 2 33.2778 166.856 224.2364 17.486 313.2514 228.2312 95.852 121.642 145.643 187.1111 27.298 271.263 295.2425 366.2969 8 1 12 14 16 18 2 22 24 26 28 3 32 34 36 38 4 Figure S12. HR-ESIMS/MS spectra of subgroup G G6 19

Figure S13. UV spectrum of 1 Figure S14. FTIR spectrum of 1 2

Figure S15. 1 H NMR spectrum of 1 Figure S16. 13 C NMR spectrum of 1 21

Figure S17. HSQC spectrum of 1 Figure S18. HMBC spectrum of 1 22

Figure S19. 1 H- 1 H COSY spectrum of 1 S R 1 Figure S2. 1 H NMR Spectra of 1 and its R and S Mosher derivatives in CDCl 3 23

Q11 #6-73 RT: 1.25-1.51 AV: 14 NL: 8.99E6 T: + c Full ms [5.-65.] 1 148.99 x5 8 6 4 2 57.5 253.81 167. 71.6 69.5 83.7 13.98 15.4 126.9 141.94 151.5 168.5 185.15 27.11 221.15 239.2 256.2 279.12 293.24 6 8 1 12 14 16 18 2 22 24 26 28 3 Figure S21. EIMS spectrum and fragmentation of the dimethyl disulfide derivative of 1 24

Figure S22. UV spectrum of 2 Figure S23. FTIR spectrum of 2 25

Figure S24. 1 H NMR spectrum of 2 Figure S25. 13 C NMR spectrum of 2 26

Figure S26. HSQC spectrum of 2 Figure S27. HMBC spectrum of 2 27

Figure S28. 1 H- 1 H COSY spectrum of 2 Q-14 #378 RT: 1.44 AV: 1 NL: 1.73E5 T: + c Full ms [5.-65.] 1 57.11 8 6 4 2 149.8 71.6 83.2 145.93 27.1 241.16 69. 111.1 97.1 67.7 113.2 95.4 129.3 185.9 15.9 165.2 28.14 281.18 239.2 197.16 242.31 27.41 294.39 6 8 1 12 14 16 18 2 22 24 26 28 3 Figure S29. EIMS spectrum and fragmentation of the dimethyl disulfide derivative of 2 28

Figure S3. UV spectrum of 3 Figure S31. FTIR spectrum of 3 29

Figure S32. 1 H NMR spectrum of 3 Figure S33. 13 C NMR spectrum of 3 3

Figure S34. HSQC spectrum of 3 Figure S35. HMBC spectrum of 3 31

Figure S36. 1 H- 1 H COSY spectrum of 3 32

Figure S37. UV spectrum of 4 Figure S38. FTIR spectrum of 4 33

Figure S39. 1 H NMR spectrum of 4 Figure S4. 13 C NMR spectrum of 4 34

Figure S41. HSQC spectrum of 4 Figure S42. HMBC spectrum of 4 35

Figure S43. 1 H- 1 H COSY spectrum of 4 Q22 #6-74 RT: 1.25-1.53 AV: 15 NL: 6.54E6 T: + c Full ms [5.-65.] 1 148.97 8 6 4 2 57.3 6.97 71.6 166.97 126.86 83.5 13.99 113.8 137.91 15.1 253.78 159.84 168.2 186.89 27.2 221.7 232.97 241.14 255.76 279.11 293.2 6 8 1 12 14 16 18 2 22 24 26 28 3 Figure S44. EIMS spectrum and fragmentation of the dimethyl disulfide derivative of 4 36

Figure S45. UV spectrum of 5 Figure S46. FTIR spectrum of 5 37

Figure S47. 1 H NMR spectrum of 5 Figure S48. 13 C NMR spectrum of 5 38

Figure S49. UV spectrum of 6 Figure S5. FTIR spectrum of 6 39

Figure S51. 1 H NMR spectrum of 6 Figure S52. 13 C NMR spectrum of 6 4

Q17 #65-68 RT: 1.35-1.41 AV: 4 NL: 1.17E5 T: + c Full ms [5.-65.] 1 57.5 x2 8 6 4 2 69.5 83.7 91.1 149.2 67.3 126.93 15.4 127.96 119.3 253.85 167.5 173.94 27.4 191.91 226.99 241.19 255.78 281.21 291.31 6 8 1 12 14 16 18 2 22 24 26 28 3 Figure S53. EIMS spectrum and fragmentation of the dimethyl disulfide derivative of 6 41

Figure S54. UV spectrum of 7 Figure S55. FTIR spectrum of 7 42

Figure S56. 1 H NMR spectrum of 7 Figure S57. 13 C NMR spectrum of 7 43

Q19 #64-89 RT: 1.33-1.84 AV: 26 NL: 8.61E5 T: + c Full ms [5.-65.] 1 148.98 8 6 4 2 57.4 73. 78.96 93.97 126.86 141.9 253.79 17.2 127.91 129.3 111.5 15.1 167. 18.98 27.2 221.6 239.14 256.7 281.7 295.14 6 8 1 12 14 16 18 2 22 24 26 28 3 Figure S58. EIMS spectrum and fragmentation of the dimethyl disulfide derivative of 7 44

Figure S59. UV spectrum of 8 Figure S6. FTIR spectrum of 8 45

Figure S61. 1 H NMR spectrum of 8 Figure S62. 13 C NMR spectrum of 8 46

Figure S63. HSQC spectrum of 8 Figure S64. HMBC spectrum of 8 47

Figure S65. 1 H- 1 H COSY spectrum of 8 Q-13 #316-48 RT: 1.21-1.56 AV: 93 NL: 2.57E7 T: + c Full ms [5.-65.] 1 148.97 x2 8 6 4 2 57.2 166.99 71.5 7.6 83.6 113.1 15.3 13.98 279.13 67.2 121.3 141.1 151.6 168.6 191.13 215.11 231.13 239.21 256.24 287.18 6 8 1 12 14 16 18 2 22 24 26 28 3 Figure S66. EIMS spectrum and fragmentation of the dimethyl disulfide derivative of 8 48

Figure S71. UV spectrum of 9 Figure S72. FTIR spectrum of 9 49

Figure S73. 1 H NMR spectrum of 9 Figure S74. 13 C NMR spectrum of 9 5

Figure S67. UV spectrum of 1 Figure S68. FTIR spectrum of 1 51

Figure S69. 1 H NMR spectrum of 1 Figure S7. 13 C NMR spectrum of 1 52

Q-25 #452-618 RT: 1.71-2.33 AV: 167 NL: 6.5E5 T: + c Full ms [5.-65.] 1 57.4 x2 8 6 4 2 71.6 85.8 27.6 69.5 83.7 97.8 73.1 96.8 6.1 111.1 129.1 28.1 281.7 149.9 163.1 171.18 191.7 221.19 239.29 256.29 267.17 283.18 6 8 1 12 14 16 18 2 22 24 26 28 3 Figure S75. EIMS spectrum and fragmentation of the dimethyl disulfide derivative of 1 53

Figure S76. Quantitation results of 1, 3, and 8 from Vibrio sp. QWI-6 under different cultural condition. Control is marine agar medium and tyrosine and phenylalanine were added for evaluating the production of vitroprocines. 54

1 9 8 p @ [ ] 31.2747 7 6 5 4 114.725 198.1933 3 144.126 2 195.1385 293.2481 124.122 155.171 18.1828 1 141.916 167.173 29.1537 223.1695 251.216 1 12 14 16 18 2 22 24 26 28 3 Figure S77. HR-ESIMS/MS spectrum and fragmentation of 1 from Vibrio sp. QWI-6 feed with 13 C 9 -tyrosine. 55

Figure S78. 13 C NMR spectra of 1 and 1a. 1a was isolated from Vibrio sp. QWI-6 feed with 1-13 C-sodium acetate. The enhanced carbon signals of 1a were marked in red. 56