Natural Products Research Institute, College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea

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1 Supporting Information Nicrophorusamides A and B, Antibacterial Chlorinated Cyclic Peptides from a Gut Bacterium of the Carrion Beetle Nicrophorus concolor Yern-Hyerk Shin, Suhyun Bae, Jaehoon Sim,,ǁ Joonseong Hur, Shin-Il Jo, Jongheon Shin, Young-Ger Suh,,ǁ Ki-Bong Oh, Dong-Chan Oh *, Natural Products Research Institute, College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea Department of Agricultural Biotechnology, College of Agriculture & Life Sciences, Se oul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul , Republic of Korea ǁ College of Pharmacy, CHA University, 120 Haeryong-ro, Pocheon-si, Gyeonggi-do , Republic of Korea Animal Welfare Division, Seoul Zoo, Seoul Grand Park, 102 Daegongwongwangjangro, Gwacheon-si, Gyeonggi-do 13829, Republic of Korea Corresponding Author * D.-C Oh, Tel: , Fax: , dongchanoh@snu.ac.kr. S1

2 Table of Contents S3 : Figure S1. 1 H NMR spectrum of nicrophorusamide A (1) at 600 MHz in DMSO-d6. S4 : Figure S2. 13 C NMR spectrum of nicrophorusamide A (1) at 125 MHz in DMSO-d6. S5 : Figure S3. COSY NMR spectrum of nicrophorusamide A (1) at 600 MHz in DMSO-d6. Figure S4. TOCSY NMR spectrum of nicrophorusamide A (1) at 500 MHz in DMSO-d6. S6 : Figure S5. HSQC NMR spectrum of nicrophorusamide A (1) at 500 MHz in DMSO-d6. Figure S6. HMBC NMR spectrum of nicrophorusamide A (1) at 500 MHz in DMSO-d6. S7 : Figure S7. Expanded HMBC NMR spectrum of nicrophorusamide A (1) showing a correlation from 15-NH2b (δh 7.28) to C-15 (δc 173.2). Figure S8. ROESY NMR spectrum of nicrophorusamide A (1) at 500 MHz in DMSO-d6. S8 : Figure S9. 1 H NMR spectrum of nicrophorusamide B (2) at 500 MHz in DMSO-d6. S9 : Figure S C NMR spectrum of nicrophorusamide B (2) at 125 MHz in DMSO-d6. S10 : Figure S11. COSY NMR spectrum of nicrophorusamide B (2) at 500 MHz in DMSO-d6. Figure S12. TOCSY NMR spectrum of nicrophorusamide B (2) at 500 MHz in DMSO-d6. S11 : Figure S13. HSQC NMR spectrum of nicrophorusamide B (2) at 500 MHz in DMSO-d6. Figure S14. HMBC NMR spectrum of nicrophorusamide B (2) at 500 MHz in DMSO-d6. S12 : Figure S15. ROESY NMR spectrum of nicrophorusamide B (2) at 500 MHz in DMSO-d6. S13 : Figure S16. CD spectra of nicrophorusamides A and B (1 and 2) in MeOH. S14 : Table S1. LC/MS analysis of L- and D-FDAA derivatives of the amino acids in nicrophorusamide A (1) S15 : Figure S17. LC/MS analysis of L-FDAA derivatives of amino acids in nicrophorusamide A (1). Figure S18. LC/MS analysis of D-FDAA derivatives of amino acids in nicrophorusamide A (1). S16 : Synthesis of L-β-hydroxyaspartic acid for determination of the absolute configuration at the β-carbon of D-β-hydroxyasparagine. S17 : Scheme S1. Synthesis of L-erythro-β-hydroxyaspartic acid. S18 : Figure S19. Co-injection experiments to determinate the absolute configuration at the β- carbon of D-β-hydroxyasparagine. S19 : Figure S20. HPLC analysis of GITC derivatives of isoleucine unit in nicrophorusamide A (1) and authentic L- / L-allo-isoleucine. S20 : Table S2. Energy minimized coordinates of nicrophorusamide A (1) at the basis set def- SVP for all atoms (Å) S21 : Table S3. Energy minimized coordinates of nicrophorusamide B (2) at the basis set def- SVP for all atoms (Å) S22 : Figure S21. HR-FAB-MS data of nicrophorusamides A and B (1 and 2). S2

3 Figure S1. 1 H NMR spectrum of nicrophorusamide A (1) at 600 MHz in DMSO-d6. S3

4 Figure S2. 13 C NMR spectrum of nicrophorusamide A (1) at 125 MHz in DMSO-d6. S4

5 Figure S3. COSY NMR spectrum of nicrophorusamide A (1) at 600 MHz in DMSO-d6. Figure S4. TOCSY NMR spectrum of nicrophorusamide A (1) at 500 MHz in DMSO-d6. f1 (ppm) f1 (ppm) S5

6 Figure S5. HSQC NMR spectrum of nicrophorusamide A (1) at 500 MHz in DMSO-d6. Figure S6. HMBC NMR spectrum of nicrophorusamide A (1) at 500 MHz in DMSO-d6. f1 (ppm) f1 (ppm) S6

7 Figure S7. Expanded HMBC NMR spectrum of nicrophorusamide A (1) showing a correlation from 15-NH2b (δh 7.28) to C-15 (δc 173.2) δ C / δ H f2 (ppm) Figure S8. ROESY NMR spectrum of nicrophorusamide A (1) at 500 MHz in DMSO-d6. f1 (ppm) S7

8 Figure S9. 1 H NMR spectrum of nicrophorusamide B (2) at 500 MHz in DMSO-d6. S8

9 Figure S C NMR spectrum of nicrophorusamide B (2) at 125 MHz in DMSO-d6. S9

10 Figure S11. COSY NMR spectrum of nicrophorusamide B (2) at 500 MHz in DMSO-d f2 (ppm) Figure S12. TOCSY NMR spectrum of nicrophorusamide B (2) at 500 MHz in DMSO-d f1 (ppm) f2 (ppm) S10

11 Figure S13. HSQC NMR spectrum of nicrophorusamide B (2) at 500 MHz in DMSO-d f2 (ppm) Figure S14. HMBC NMR spectrum of nicrophorusamide B (2) at 500 MHz in DMSO-d f1 (ppm) f2 (ppm) S11

12 Figure S15. ROESY NMR spectrum of nicrophorusamide B (2) at 500 MHz in DMSO-d f1 (ppm) f2 (ppm) S12

13 Figure S16. CD spectra of nicrophorusamides A and B (1 and 2) in MeOH. S13

14 Table S1. LC/MS analysis of L- and D-FDAA derivatives of the amino acids in nicrophorusamide A (1) Amino acid trl (min) trd (min) Elution order Δt (min) 5-chloro-tryptophan L D 1.9 valine D L -4.0 leucine D L -3.8 isoleucine L D 4.2 ornithine D L -0.9 β-hydroxyaspartic acid L D 0.8 Δt = trd - trl S14

(a) valine, (b) isoleucine, (c) leucine, (d) ornithine, (e) β-hydroxyaspartic

LC/MS analysis of D-FDAA derivatives of amino acids in nicrophorusamide A (1):

15 Figure S17. LC/MS analysis of L-FDAA derivatives of amino acids in nicrophorusamide A (1): (a) valine, (b) isoleucine, (c) leucine, (d) ornithine, (e) β-hydroxyaspartic acid, and (f) 5- chloro-tryptophan. MS ion extraction (positive) Figure S18. LC/MS analysis of D-FDAA derivatives of amino acids in nicrophorusamide A (1): (a) valine, (b) isoleucine, (c) leucine, (d) ornithine, (e) β-hydroxyaspartic acid, and (f) 5- chloro-tryptophan. MS ion extraction (positive) S15

16 Synthesis of L-β-hydroxyaspartic acid for determination of the absolute configuration at the β-carbon of D-β-hydroxyasparagine. Synthesis of L-β-hydroxyaspartic acid was conducted according to Tang et al (J. Am. Chem. Soc. 2005, 127, ). A mixture of (+)- diethyl L-tartrate (1.0 g, 4.85 mmol) and anhydrous triethylamine (Et3N, 1.6 ml, 9.70 mmol) in 10 ml of dry CH2Cl2 was stirred, and thionyl chloride (SOCl2, 0.7 ml, 9.70 mmol) was added dropwise to the vial at 0 C. The reaction vial was gradually heated at room temperature, and the reaction was quenched by adding water. The product was extracted with CH2Cl2. The combined organic layer was dried over MgSO4 and concentrated in vacuo to acquire diethyl (2R, 3R)-2,3-O-sulfinyl-tartrate as a light-brown oil. The product was dissolved in 17 ml of dimethylformamide (DMF), and 347 mg of sodium azide (NaN3, 5.34 mmol) was added to the suspension. After stirring for 13 h, the reaction mixture was quenched with water, extracted with EtOAc and washed with water and brine. The mixed organic layer was dried over MgSO4 and condensed under low pressure. The reaction suspension was purified by flash column chromatography on silica gel (EtOAc:n-hexane = 1:3) to afford 785 mg (70%) of (2S, 3R)- diethyl 2-azido-3-hydroxysuccinate (3) as a colorless oil. Then, 352 mg of triphenylphosphine (PPh3, 1.34 mmol) was added to the solution of 3 (239 mg, 1.03 mmol) in tetrahydrofuran (THF)/water (THF:H2O = 5:1, 10 ml), and the reaction vial was heated at 70 C and stirred for 1.5 h. The reaction mixture was then cooled to ambient temperature and concentrated in vacuo. The residue was purified by flash column chromatography (CH2Cl2:MeOH = 9:1) to obtain 181 mg (86%) of (2S, 3R)-diethyl 2-amino-3-hydroxysuccinate (4) as a pale-yellow oil. Finally, L-erythro-β-hydroxyaspartic acid was obtained by acid hydrolysis of 4 with 6-N HCl at 115 C. S16

17 (2S, 3R)-diethyl 2-azido-3-hydroxysuccinate (3): 1 H NMR (in CDCl3 at 800 MHz) δ 4.60 (1H, dd, J = 5.4, 2.7 Hz), 4.29 (1H, d, J = 2.7 Hz), 4.26 (4H, m), 3.31 (1H, d, J = 5.4 Hz), 1.28 (3H, t, J = 7.2 Hz), 1.28 (3H, t, J = 7.2 Hz); 13 C NMR (in CDCl3 at 200 MHz) δ 170.7, 166.9, 72.0, 64.4, 62.7, 62.3, 14.0, 14.0; HR-ESI-MS [M + H] + m/z (calcd for C8H14N3O5, ). (2S, 3R)-diethyl 2-amino-3-hydroxysuccinate (4): 1 H NMR (in CDCl3 at 600 MHz) δ 4.47 (1H, d, J = 3.0 Hz), (4H, m), 3.88 (1H, d, J = 3.0 Hz), 1.24 (3H, t, J = 7.3 Hz), 1.22 (3H, t, J = 7.3 Hz); 13 C NMR (in CDCl3 at 150 MHz) δ 171.9, 171.7, 72.7, 61.8, 61.3, 57.5, 14.0; HR-ESI-MS [M + H] + m/z (calcd for C8H16NO5, ). Scheme S1. Synthesis of L-erythro-β-hydroxyaspartic acid. S17

18 Figure S19. Co-injection experiments to determinate the absolute configuration at the β- carbon of D-β-hydroxyasparagine. S18

19 Figure S20. HPLC analysis of GITC derivatives of isoleucine unit in nicrophorusamide A (1) and authentic L- / L-allo-isoleucine. UV detect: 210 nm S19

20 Table S2. Energy minimized coordinates of nicrophorusamide A (1) at the basis set def-svp for all atoms (Å) atom x y z atom x y z atom x y z N N C C O H C C H C H H N C C C H H C H H N C H C H H C C H N H H C H H C H H N C H C H H C C O N H O C H O C H O N H O C H O C C H C H H C C H C H C C H H C C C H H H H H H H H N Cl C H H H H C H H H H H C H H O C H H H H S20

21 Table S3. Energy minimized coordinates of nicrophorusamide B (2) at the basis set def-svp for all atoms (Å) atom x y z atom x y z atom x y z N O H C C H C H H C C C N H H C H H C C H N H H C C H C H H N H H C H H C C H N H H C C O C H O N H O C H O C H O N H O C C H C H H C C C C H H C H C C C H C H H H H N H H H H C H Cl H H H H H C H H H H H C C H H H H N C S21

22 Figure S21. HR-FAB-MS data of nicrophorusamides A and B (1 and 2). (a) HR-FAB-MS data of nicrophorusamide A (1). S22

23 S23

24 (b) HR-FAB-MS data of nicrophorusamide B (2). S24

25 S25

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