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1 Supporting information Chojalactones A-C, Cytotoxic Butanolides Isolated from Streptomyces sp. Cultivated with Mycolic Acid Containing Bacterium Shotaro Hoshino, Toshiyuki Wakimoto, Hiroyasu Onaka, and Ikuro Abe * Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo , Japan Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo , Japan Table of contents General experimental procedure S3 Isolation and identification of Streptomyces sp. CJ-5 S3 Analysis of metabolic profiles of Streptomyces sp. CJ-5 S4 Isolation of chojalactone A-C S4 Synthetic procedures S5 Chiral HPLC analysis S7 Cytotoxicity assay to P388 cells S7 Antimicrobial screening S7 Figure S1 1 H NMR spectrum of 1 (CDCl 3, 500 MHz) S8 Figure S2 1 H- 1 H COSY spectrum of 1 (CDCl 3, 500 MHz) S8 Figure S3 HMQC spectrum of 1 (CDCl 3, 500 MHz) S9 Figure S4 HMBC spectrum of 1 (CDCl 3, 500 MHz) S9 Figure S5 13 C NMR spectrum of 1 (CDCl 3, 125 MHz) S10 Figure S6 1 H NMR spectrum of the mixture of 2 and 3 (CDCl 3, 500 MHz) S10 Figure S7 1 H- 1 H COSY spectrum of the mixture of 2 and 3 (CDCl 3, 500 MHz) S11 Figure S8 HMQC spectrum of the mixture of 2 and 3 (CDCl 3, 500 MHz) S11 Figure S9 HMBC spectrum of the mixture of 2 and 3 (CDCl 3, 500 MHz) S12 Figure S10 13 C NMR spectrum of the mixture of 2 and 3 (CDCl 3, 125 MHz) S12 Figure S11 1 H NMR spectrum of synthetic 2a (= 2) (CDCl 3, 500 MHz) S13 Figure S12 1 H- 1 H COSY spectrum of synthetic 2a (CDCl 3, 500 MHz) S13 Figure S13 HMQC spectrum of synthetic 2a (CDCl 3, 500 MHz) S14 Figure S14 HMBC spectrum of synthetic 2a (CDCl 3, 500 MHz) S14 Figure S15 13 C NMR spectrum of synthetic 2a (CDCl 3, 125 MHz) S15 Figure S16 1 H NMR spectrum of (3S)-7 (CDCl 3, 500 MHz) S15 S1

2 Figure S17 13 C NMR spectrum of (3S)-7 (CDCl 3, 125 MHz) S16 Figure S18 TOF-HRMS spectrum of 1 S16 Figure S19 TOF-HRMS spectrum of the mixture of 2 and 3 S16 Figure S20 Chiral-HPLC chromatograms of natural chojalactones (1-3) and synthetic standards (1a, 1b, 2a and 2b) S17 Table S1 NMR spectrum of 1 S18 Table S2 NMR spectrum of 2 S19 Table S3 NMR spectrum of 3 S20 S2

3 General experimental procedures Optical rotations were measured on a JASCO DIP-1000 digital polarimeter. The UV spectra of 1 and 2 were obtained with a Gene Spec III UV/vis spectrometer, and the UV spectrum of 3 was measured with a JASCO MD-2010 Plus Multiwavelength Detector. All NMR spectra were recorded on a JEOL ECX-500 or ECA-500 spectrometer ( 1 H NMR: 500 MHz, 13 C NMR: 125 MHz), using CDCl 3 as the solvent. The HRMS data of 1-3 were obtained with a JEOL AccuTOF LC-plus JMS-T 100LP mass spectrometer. Isolation and identification of Streptomyces sp. CJ-5 Streptomyces sp. CJ-5 was isolated from a soil sample collected at Isumi City (Chiba pref., Japan) by Hayakawa and Nonomura s protocol. 1 To identify its 16S rrna gene, Streptomyces sp. CJ-5 was cultured in tryptosoya broth (TSB, 100 ml) for 3 days, on a rotary shaker at 160 rpm and 30 C. The cell pellet was collected by centrifugation for genome extraction, and the 16S rrna gene region was amplified by PCR, using universal primers and its genomic DNA as the template. The PCR product was inserted into the pt7blue vector and sequenced. The following sequence (1,348 bp) was subjected to a BLAST search, and showed 99% identity to the 16S rrna gene of Streptomyces fradiae strain N10T-Cu-51. The sequence data have been deposited in DDBJ/EMBL/GenBank, under accession code LC Partial 16S rrna gene sequence of Streptomyces sp. CJ-5 for blast search: ATTAGTGGCGAACGGGCGAGTAACACGTGGGCAATCTGCCCTGCACTCTGGGACAAGCC CTGGAAACGGGGTCTAATACCGGATACGACACACTCAGGCATCTGATGTGTGTGGAAAG CTCCGGCGGTGCAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGAGGTAACGGCTCACC AAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACAC GGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGA TGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAA GAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGC GGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCG GCTTGTCACGTCGGATGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCATTCGATACGGGC AGGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATA TCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGACGCTGAGGAGTG AAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGC ACTAGGTGTGGGCGACATTCCACGTCGTCCGTGCCGCAGCTAACGCATTAAGTGCCCCGC CTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCG GCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATACA CCGGAAACGTCCAGAGATGGGCGCCCCCTCGTGGTCGGTGTACAGGTGGTGCATGGCTGT CGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCCG TGTTGCCAGCAGGCCCTTGTGGTGCTGGGGACTCACGGGAGACCGCCGGGGTCAACTCGG AGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCT ACAATGGCCGGTACAAAGAGCTGCGATACCGTGAGGTGGAGCGAATCTCAAAAAGCCGG TCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGC AGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCA CGAAAGTCGGTAACACCCGAAGCCGGTGGCCCAACCCCTTG S3

4 Analysis of the metabolic profile of Streptomyces sp. CJ-5 Streptomyces sp. CJ-5 and Tsukamurella pulmonis TP-B0596 (both strains were maintained on Bennetto maltose agar) were independently inoculated into 100 ml of V-22 medium (1.0% starch, 0.5% glucose, 0.5% Bacto Tryptone (Difco), 0.3% NZ case (Wako), 0.2% Yeast Extract (Difco), 0.1% K 2 HPO 4, 0.05% MgSO 4 7H 2 O and 0.3% CaCO 3, ph 7.0) in 500 ml Erlenmeyer flasks. Streptomyces sp. CJ-5 was cultured for 3 days, and T. pulmonis TP-B0596 was cultured for 2 days, on a rotary shaker at 160 rpm and 30 C. Aliquots of both culture broths (Streptomyces sp. CJ-5: 3 ml, T. pulmonis: 1 ml) were simultaneously added to 100 ml of A-3M medium (2.0% starch, 2.0% glycerol, 0.5% glucose, 1.5% Pharma media (Archer Daniels Midland Co.), 1.0% HP-20 (Nihon Rensui) and 0.3% yeast extract, ph 7.0) in a 500 ml baffled Erlenmeyer flask, and cultured for 6 days at 160 rpm and 30 C. As control cultures, each pre-culture was individually inoculated into 500 ml baffled Erlenmeyer flasks with 100 ml of A-3M medium, and cultured under the same conditions. The cell pellets together with HP-20 resins were collected by centrifugation (8,000 rpm, 5 min), lyophilized, extracted with 50 ml of 1:1 methanol-chloroform, and concentrated in vacuo. Each extract was dissolved in 1 ml of 1:1 methanol-chloroform, and aliquots of each sample (10 µl) were analyzed by HPLC with a Cosmosil 5C 18 -MS-II column ( mm, Nacalai Tesque, Japan) in a CH 3 CN (solvent A)/H 2 O with 0.1% trifluoroacetic acid (solvent B) gradient system (solvent A: 20 70% (15 min) 100% (20-22 min)), at 35 C and a flow rate of 1.0 ml min -1. All eluates were monitored at 315 nm, and compounds 1-3 were observed at 18.9 min, 18.2 min, and 18.3 min, respectively. Isolation of chojalactones A-C (1-3) The combined-culture broth (1.5 L, ml) of Streptomyces sp. CJ-5 was centrifuged (6,000 rpm, 10 min), and the cell pellet was collected. After lyophilization, the cell pellet was extracted with 800 ml of 1:1 methanol-chloroform and concentrated in vacuo. The residue (1.71 g, brown oil) was separated by silica gel column chromatography (methanol/chloroform = 0/100-20/80), to afford the 1:99 methanol-chloroform fraction (130 mg) containing compounds 1-3. Further purification was conducted by semi-preparative reverse-phase HPLC equipped with a Cosmosil cholester column ( mm, Nacalai Tesque, Japan) in a CH 3 CN (solvent A)/H 2 O with 1% acetic acid (solvent B) gradient system (solvent A: 47 54% (10 min) 100% (11-15 min)), at 35 C and a flow rate of 3.0 ml min -1, to yield 1 (0.6 mg, detected at 9.8 min) and the mixture of 2 and 3 (1.5 mg, both compounds detected at 9.0 min). Chojalactone A (1): pale yellow oil. [α] 25 D (c 0.184, CH 3 CN, obtained from synthetic product); UV (CH 3 CN) λ max (log ε) 223 nm (3.90), 337 nm (4.40); HR-TOFMS m/z (calcd for C 13 H 16 O 4, ); 1 H and 13 C NMR are listed in Table S1. S4

5 Chojalactone B (2): pale yellow oi. [α] 25 D (c 0.302, CH 3 CN, obtained from synthetic product); UV (CH 3 CN) λ max (log ε) 223 nm (3.93), 337 nm (4.37); HR-TOFMS m/z (calcd for C 13 H 16 O 4, ); 1 H and 13 C NMR are listed in Table S2. Chojalactone C (3): pale yellow oil (mixture with 2). UV (CH 3 CN) λ max 223, 339 nm; HR-TOFMS m/z (calcd for C 13 H 14 O 4, ); 1 H and 13 C NMR are listed in Table S3. Synthetic procedures Preparation of 6: (2E,4E,6E)-octa-2,4,6-trienoic acid (441 mg, 3.19 mmol) was suspended in anhydrous CH 2 Cl 2 (18 ml), and oxalyl chloride (600 µl, 6.99 mmol) was added to the suspension. After stirring for 10 min at room temperature, the solvent and excess oxalyl chloride were removed in vacuo. The resulting product 6 (brown oil, 512 mg) was used for the next reaction without purification. Ti-crossed-Claisen condensation: (S)-5 or (R)-5 (240 mg, 2.40 mmol) and N-methyl-imidazole (230 µl, 2.91 mmol) were dissolved in anhydrous CH 2 Cl 2 (18 ml). To the solution, crude 6 (512 mg) dissolved in anhydrous CH 2 Cl 2 (9 ml) was added, and the mixture was stirred for 15 min at -40 C under an argon atmosphere. Then TiCl 4 (in 8.4 ml of CH 2 Cl 2, 8.40 mmol) and tributylamine (2.30 ml, 9.68 mmol) were added, and the mixture was stirred for 30 min under the same conditions. The reaction was quenched by the addition of water (40 ml), and the aqueous phase was extracted with 1:1 hexane-acoet (3 40 ml). The combined organic layer was washed with brine, dried over MgSO 4, and concentrated in vacuo. The residue was purified by flash silica-gel column chromatography (hexane/acoet = 10/0-8/2) to yield 250 mg (1.14 mmol, 47%) of (3S) or (3R)-7 as a yellow oil. S5

6 [α] 25 D ((3S)-7, c 0.465, CHCl 3 ), ((3R)-7, c 1.10, CHCl 3 ). 1 H NMR (in CDCl 3 ) δ ppm of (3S)/(3R)-7 in keto form: 1.17 (d, 3H, J = 6.5 Hz, H-5), 1.85 (d, 3H, J = 6.0 Hz, H-8 ), 3.12 (m, 1H, H-3), 3.47 (d, 1H, J = 8.0 Hz, H-2), 3.88 (dd, 1H, J = 7.5, 9.0 Hz, H-4a), 4.48 (dd, 1H, J = 7.5, 9.0 Hz, H-4b), 6.02 (m, 1H, H-7 ), (2H, H-4 and H-6 ), 6.43 (d, 1H, J = 15.5 Hz, H-2 ), 6.65 (dd, 1H, J = 11.0, 15.0 Hz, H-5 ), 7.34 ppm (dd, 1H, J =11.5, 15.5 Hz, H-3 ). 1 H NMR (in CDCl 3 ) δ ppm of (3S)/(3R)-7 in enol form: 1.26 (d, 3H, J = 7.5, H-5), 1.83 (d, 3H, J = 7.5, H-8 ), 3.25 (dqui, 1H, J = 3.0, 7.5, H-3), 4.00 (dd, 1H, J = 3.0, 9.0 Hz, H-4a), 4.45 (dd, 1H, J = 7.5, 9.0, H-4b), 5.93 (m, 1H, H-7 ), 5.97 (d, 1H, J = 15.5 Hz, H-2 ), (2H, H-4 and H-6 ), 6.50 (dd, 1H, J = 11.5, 15.5 Hz, H-5 ), 7.15 (dd, 1H, J = 11.5, 15.5 Hz, H-3 ), ppm (s, 1H, -OH). 13 C NMR (in CDCl 3 ) δ ppm of (3S)/(3R)-7 in keto form: 17.4 (C-5), 18.8 (C-8 ), 31.4 (C-3), 58.5 (C-2), 73.6 (C-4), (C-2 ), (C-4 ), (C-6 ), (C-7 ), (C-5 ), (C-3 ), (C-1), (C-1 ). 13 C NMR (in CDCl 3 ) δ ppm of (3S)/(3R)-7 in enol form: 18.7 (C-8 ), 22.0 (C-5), 33.1 (C-3), 74.5 (C-4), (C-2), (C-2 ), (C-4 ), (C-6 ), (C-7 ), (C-3 ), (C-5 ), (C-1 ), (C-1). Oxidation of 7: (3S) or (3R)-7 (200 mg, mmol) was dissolved in EtOH (13 ml). To the solution, 10% Pd/C (200 mg) and Et 3 N (166 µl, 1.19 mmol) were added, and the mixture was stirred for 10 h at room temperature under an O 2 atmosphere. The reaction mixture was passed through a 0.22 µm membrane filter (Millipore, Millex -GP). The combined filtrate was concentrated in vacuo, and the residue was purified by flash silica-gel column chromatography (hexane/acoet = 10/0-5/5). As a result, the 8:2 hexane-acoet fraction contained 1a (30 mg, 14%) or 1b (27 mg, 12%), and the 1:1 hexane-acoet fraction contained 2a (37 mg, 17%) or 2b (33 mg, 15%). [α] 25 D (1a, c 0.267, CH 3 CN), (1b, c 0.184, CH 3 CN), (2a, c 0.302, CH 3 CN), (2b, c 0.142, CH 3 CN). 1 H and 13 C NMR assignment of 1a and 1b are listed in Table S1. 1 H and 13 C NMR assignment of 2a and 2b are listed in Table S2. S6

7 Chiral HPLC analysis Natural chojalactones (1 and an inseparable mixture of 2 and 3) and synthetic standards 1a, 1b, 2a, and 2b were dissolved in CH 3 CN, and each sample (5 µl) was analyzed by chiral-phase HPLC with a CHIRALPAK AS-RH column ( mm, Daicel Co., Ltd., Japan) using 34% aqueous CH 3 CN as a solvent at 35 C and a flow rate of 0.5 ml min -1. All elutes were monitored by UV absorption at 223 nm. The natural products 1, 2, and 3 were detected at 22.1 min, 18.9 min, and 17.9 min, whereas the synthetic standards 1a, 1b, 2a, and 2b were detected at 23.5 min, 22.1 min, 18.8 min, and 20.6 min, respectively (Figure S20 (i) ~ (viii)). Cytotoxicity assay to P388 cells The cytotoxicities of 1a, 1b, 2a, and 2b against P388 murine leukemia cells were tested, using the methyl thiazol tetrazolium (MTT) assay. 2 P388 murine leukemia cells were cultured in RPMI 1640 (Wako Chemicals) medium containing 10 µg/ml of penicillin/streptomycin and 10% fetal bovine serum (MP Biomedicals), at 37 C under a 5% CO 2 atmosphere. The test solutions (100 µl, final concentrations of each compound were 100 µm, 50 µm, 25 µm, 12.5 µm, 6.3 µm, 3.1 µm, 1.6 µm, and 0.78 µm, all samples were dissolved in CH 3 CN) were added to each well of a 96-well microplate containing 100 µl of cells/ml tumor cell suspension. The plates were incubated for 4 days at 37 C under a 5% CO 2 atmosphere, and then 50 µl of MTT solution (1 mg/ml, dissolved in DMSO) was added to each well. After an incubation for 4 h under the same conditions, the mixtures were centrifuged and the supernatants were removed. The precipitates were dissolved in 50 µl DMSO, and the absorbance at 570 nm was measured with a microplate reader. Antimicrobial assay For the disk diffusion assay, Candida albicans, Bacillus subtilis and Saccharomyces cerevisiae were pre-cultured in their respective broths (Candida albicans: 4.0% glucose, 1.0% Bacto peptone (Difco), ph 5.7, Bacillus subtilis: LB broth, Saccharomyces cerevisiae: 5.0% glucose, 0.5% Bacto peptone, 0.2% yeast extract, 0.4% K 2 HPO 4, 0.2% KH 2 PO 4 and 0.02% MgSO 4 7H 2 O, ph 5.6). The 6-mm paper disks containing each compound (10 µg, 5 µg, 2.5 µg, 1.25 µg/disk, all samples were dissolved in CH 3 CN) were placed on plates containing 5% pre-culture broth of each strain (the other compositions are the same as the media for pre-culture), and the plates were incubated at 37 C for 1-2 days to determine the presence of inhibition rings around the paper disks. S7

8 Figure S1. 1 H NMR spectrum of 1 (CDCl 3, 500 MHz) Figure S2. 1 H- 1 H COSY spectrum of 1 (CDCl 3, 500 MHz) S8

9 Figure S3. HMQC spectrum of 1 (CDCl 3, 500 MHz) Figure S4. HMBC spectrum of 1 (CDCl 3, 500 MHz) S9

10 Figure S5. 13 C NMR spectrum of 1 (CDCl 3, 125 MHz) Figure S6. 1 H NMR spectrum of the mixture of 2 and 3 (CDCl 3, 500 MHz) S10

11 Figure S7. 1 H- 1 H COSY spectrum of the mixture of 2 and 3 (CDCl 3, 500 MHz) Figure S8. HMQC spectrum of the mixture of 2 and 3 (CDCl 3, 500 MHz) S11

12 Figure S9. HMBC spectrum of the mixture of 2 and 3 (CDCl 3, 500 MHz) Figure S C NMR spectrum of the mixture of 2 and 3 (CDCl 3, 125 MHz) S12

13 Figure S11. 1 H NMR spectrum of synthetic 2a (= 2) (CDCl 3, 500 MHz) Figure S12. 1 H- 1 H COSY spectrum of synthetic 2a (CDCl 3, 500 MHz) S13

14 Figure S13. HMQC spectrum of synthetic 2a (CDCl 3, 500 MHz) Figure S14. HMBC spectrum of synthetic 2a (CDCl 3, 500 MHz) S14

15 Figure S C NMR spectrum of synthetic 2a (CDCl 3, 125 MHz) Figure S16. 1 H NMR spectrum of (3S)-7 (CDCl 3, 500 MHz) S15

16 Figure S C NMR spectrum of (3S)-7 (CDCl 3, 125 MHz) Figure S18. TOF-HRMS spectrum of Chojalactone A (1) Figure S19. TOF-HRMS spectrum of the mixture of chojalactone B (2) and C (3) S16

17 Figure S20. Chiral-HPLC chromatograms of natural chojalactones (1-3) and synthetic standards (1a, 1b, 2a and 2b) S17

18 Table S1. NMR spectrum of 1 (in CDCl 3 ) Position δ H, mult (J in Hz) δ C, type of carbon OH 4.32, s , C , C , m 39.1, CH 4a 4.08, t (8.5) 4b 4.54, dd (7.5, 8.5) 72.9, CH , d (7.0) 9.8, CH 3 1' , C 2' 6.32, d (15.0) 120.0, CH 3' 7.54, dd (12.0, 15.0) 147.9, CH 4' 6.23, dd (12.0, 15.0) 127.7, CH 5' 6.70, dd (11.5, 15.0) 145.7, CH 6' 6.20, dd (11.5, 15.5) 131.4, CH 7' 6.06, dq (7.0, 15.5) 138.2, CH 8' 1.86, d (7.0) 18.9, CH 3 S18

19 Table S2. NMR spectrum of 2 (in CDCl 3 ) Position δ H, mult (J in Hz) δ C, type of carbon OH 4.23 brs a , C , C , m 42.6, CH 4a 4.03, dd (9.0, 10.5) 4b 4.48, t (9.0) 71.0, CH , d (7.0) 10.7, CH 3 1' , C 2' 6.50, d (15.5) 121.2, CH 3' 7.46, dd (12.0, 15.5) 147.5, CH 4' 6.25, dd (12.0, 15.0 b ) 127.9, CH 5' 6.70, dd (10.5, 15.0) 145.9, CH 6' 6.20, dd (10.5, 14.5 b ) 131.4, CH 7' 6.05, dq (7.0, 14.5 b ) 138.1, CH 8' 1.85, d (7.0) 18.9, CH 3 a Observed only in the synthetic compound 2. b Coupling constants were determined by 1 H NMR spectrum of synthetic compound 2. S19

20 Table S3. NMR spectrum of 3 (in CDCl 3 ) Position δ H, mult (J in Hz) δ C, type of carbon OH not observed , C , C , C , t (2.0) 70.3, CH 2 5a 5.39, dt (1.0, 2.0) 5b 5.42,dt (1.0, 2.0) 113.8, CH 2 1' , C 2' 6.14, d (15.5) 118.8, CH 3' 7.54, dd (12.0, 15.5) 149.1, CH 4' , m 127.5, CH 5' 6.71, dd (12.0, 14.0) 146.4, CH 6' , m 131.4, CH 7' 6.08, m 138.8, CH 8' 1.87, d (7.0) 19.0, CH 3 Reference (1) Hayakawa, M.; Nonomura, H. J. Ferment. Technol. 1987, 5, (2) Mosmann, T. J. Immunol. Methods 1983, 65, S20