Identification of novel endophenaside antibiotics produced by Kitasatospora sp. MBT66

SUPPORTING INFORMATION belonging to the manuscript: Identification of novel endophenaside antibiotics produced by Kitasatospora sp. MBT66 by Changsheng Wu 1, 2, Gilles P. van Wezel 1, *, and Young Hae Choi 2,* Molecular Biotechnology, Institute of Biology, Leiden University, Sylviusweg 72, 2333 BE, The Netherlands Natural Products Laboratory, Institute of Biology, Leiden University, Sylviusweg 72, 2333 BE, The Netherlands * shared senior authors. g.wezel@biology.leidenuniv.nl; y.choi@chem.leidenuniv.nl

CONTENTS Figure S1. Instability of endophenaside E in methanol. Figure S2. UHPLC-Q-TOF analysis of Kitasatospora sp. MBT66. Table S1. Extracted peaks and ions of UHPLC-Q-TOF chromatogram for the confirmation of previously identified endophenazines. Supplemental data file 1. Structure elucidation of compounds 2 4, and 6. Supplemental data file 2. Physical properties of new compounds Supplemental data file 3. Spectra of all new compounds, including NMR, MS, UV, and IR.

N N OH HO HO O O OH N O CH 3 OH O O N HO HO O OH 1-carbomethoxy phenazine Endophenaside E, day 7 Endophenaside E, day 0 8.60 8.55 8.50 8.45 8.40 8.35 8.30 8.25 8.20 8.15 8.10 8.05 8.00 7.95 7.90 7.85 7.80 40 4.35 4.30 4.25 4.20 4.15 4.10 4.05 4.00 3.95 3.90 3.85 3.80 3.75 3.70 3.65 3.60 3.55 3.50 3.45 f1 (ppm) f1 (ppm) Figure S1. Instability of endophenaside E in methanol. Endophenaside E was unstable in methanol, which was prone to nucleophilic reaction (methanolysis) and degraded into 1-carbomethoxy phenazine and rhamnose. The reaction was observed in deuterated methanol when measuring NMR data in one week. The product identification was done in comparison with the 1 H NMR of purified 1-carbomethoxy phenazine.

Figure S2. UHPLC-Q-TOF analysis of Kitasatospora sp. MBT66. UHPLC-Q-TOF profiling of secondary metabolites produced by kitasatospora sp. MBT66. A) mass detection, and B) UV detection at 254 nm. Table S1. Extracted peaks and ions of UHPLC-Q-TOF chromatogram for the confirmation of previously identified endophenazines. Retention Detected mass Deduced chemical Corresponding compound time (m/z) formula described by Heine D. et al 8.5 min 285.0879 [M + H] + C 15 H 13 N 2 O 4 5-(2-Hydroxyacetyl)-5,10-dihydrophenazine-1-carboxylic acid (2) 8.8 min 309.1231 [M + H] + C 18 H 17 N 2 O 3 (E)-9-(4-Hydroxy-3-methylbut-2-en-1-yl)phenazine-1-carboxylic acid (3) 9.4 min 309.1234 [M + H] + C 18 H 17 N 2 O 3 (E)-6-(3-Hydroxy-3-methylbut-1-en-1-yl)phenazine-1-carboxylic acid (5) 14.1 min 377.1853 [M + H] + C 23 H 25 N 2 O 3 5,9-bis(3-Methylbut-2-en-1-yl)-7-oxo-5,7-dihydrophenazine-1-carboxylic acid (4)

Supplemental data file 1. Structure elucidation of compounds 2 4, and 6. Compound 2 gave a molecular formula of C 24 H 26 N 2 O 7 established from a high-resolution mass measurement of the [M + H] + peak at m/z 455.18187. 1 H NMR (500 MHz, CD 3 OD) comparison with compound 1 indicated the presence of a disubstituted phenazine ring, as well as the same existence of deoxyhexopyranose moiety in compound 2. In contrast to compound 1, the key HMBC correlation from H-1' ( H 6.41) to the carbonyl C-11 ( C 166.2) established the linkage of 6-deoxyhexopyranose moiety with phenazine ring through ester bond rather than ether bond. Moreover, an additional hydroxylated prenyl chain in 2 was established by intensive 2D NMR spectral analysis (Figure 2). NOESY experiments assigned the trans-position of the hydroxymethyl group owing to coupling signals for 2'' and 5'' in combination with lacking signals for 2'' and 4''. The difference between endophenaside B (2) and the known compound phenazoviridin is the terminal hydroxylation in prenyl substitute. 30 The structure identification of compound 3 (endophenaside C) was effortless, since both high-resolution mass and 1 H NMR demonstrated an additional methyl group was added to 2. Methylation pattern of the sugar moiety as seen for 1 was confirmed by HMBC correlation from H 3-7' to C-2' ( C 80.2). Endophenaside C (3) was a new glycosylated phenazine derivative, possessing the methylated sugar structural feature of compound 1, as well as the hydroxylation attribute in the isoprenyl chain as in compound 2. Compound 5 shared the same molecular formula C 24 H 26 N 2 O 7 with 2, as deduced from [M + H] + peak at m/z 455.18243. Though disubstituted phenazine and 6-deoxyhexopyranose feature were found in 1 H NMR spectrum (500 MHz, CD 3 OD), difference was obviously observed as well: a trans double bond, H 7.89 (d, J = 16.0 Hz, H-1''), 7.12 (d, J = 16.0 Hz, H-2''), and two methyls linked to saturated carbon, H 1.54 (s, H 3-4''), 1.53 (s, H 3-5''). The double bond of the isoprenyl group was assumed to be rearranged from C-2''=C-3'' to C-2''=C-1'' due to the oxidation of C-3'', which was ultimately verified by the HSQC and HMBC spectral analysis (Figure 2).

Remarkably, the 6-deoxyhexopyranose sugar of compound 4 (endophenaside D) was located at prenyl side chain through 3''-OH instead of 11-COOH, based on the key HMBC correlation from sugar anomeric proton H-1' ( H 6.43) to the oxygenated quaternary carbon C-3'' ( C 71.7). Compound 6 was isolated as a violet amorphous powder, whose molecular formula was deduced as C 23 H 24 N 2 O 4 by experimental high resolution mass [M + H] + peak at m/z 393.18115. 1 H NMR spectrum (600 MHz, CD 3 OD) of 6 indicated its attribution to phenazine type. Both 1 H NMR and APT spectra showed two prenyl side chains, but without the 6-deoxyhexopyranose sugar moiety. These two prenyl substitutents were respectively positioned at nitrogen atom 5 and carbon atom 9 by intensive 2D NMR analysis. The distinctive downfield carbonyl signal at C 185.8 (C-7) in APT spectrum in combination with the two high field shifted narrow doublets in 1 H NMR spectrum, via H 6.19 (d, J = 2.4 Hz, H-6) and 6.88 (d, J = 2.4 Hz, H-8), revealed oxidation pattern happening at C-7 of phenazine scaffold to form a conjugated ketone like chromophenazine C, 31 which was further reflected by additional UV/vis absorption at 518 nm and finally confirmed by the HMBC experiment. Also, the geometry of the double bond of the prenyl group was assigned as trans based on the NOESY correlation between H-2'' ( H 5.45) and H-5'' ( H 4.00). Supplemental data file 2. Physical properties of the new compounds Endophenaside A (1): yellowish, amorphous powder, UV (MeOH) max (log ) 251 (4.79), 366 (4.17) nm; IR 3352, 2926, 2855, 1674, 1597, 1560, 1456, 1204, 16, 1067, 958, 748 ν max cm 1 ; 1 H and 13 C NMR data, see Table 1 and 2; HRMS (positive mode) m/z 400.15076 [M + H] + (calcd for C 20 H 22 N 3 O 6 400.15031), 422.13339 [M + Na] + (calcd for C 20 H 21 N 3 O 6 Na 422.13281). Endophenaside B (2): yellowish, amorphous powder, UV (MeOH) max (log ) 252 (4.72), 365 (4.10) nm; IR ν max 3350, 2920, 1732, 1672, 1531, 85, 1263, 1123, 1061, 978, 947, 760cm 1 ; 1 H and 13 C NMR data, see Table 1 and 2; HRMS (positive mode) m/z 455.18187 [M + H] + (calcd for C 24 H 27 N 2 O 7 455.18128), 477.16375 [M + Na] + (calcd for C 24 H 27 N 2 O 7 Na 477.16377).

Endophenaside C (3): yellowish, amorphous powder, UV (MeOH) max (log ) 251 (4.50), 367 (3.97) nm; IR ν max 3341, 2926, 2853, 1682, 1456, 1418, 1271, 1203, 18, 1067, 841, 764 cm 1 ; 1 H and 13 C NMR data, see Table 1 and 2; HRMS (positive mode) m/z 469.19641 [M + H] + (calcd for C 25 H 29 N 2 O 7 469.19693), 491.18021 [M + Na] + (calcd for C 25 H 28 N 2 O 7 Na 491.17942). Endophenaside D (4): yellowish, amorphous powder, UV (MeOH) max (log ) 251 (4.58), 368 (4.05) nm; IR ν max 3400, 2926, 2855, 1734, 1717, 1682, 1456, 77, 1277, 1126, 1069, 976, 762 cm 1 ; 1 H and 13 C NMR data, see Table 1 and 2; HRMS (positive mode) m/z 455.18243 [M + H] + (calcd for C 24 H 27 N 2 O 7 455.18128), 477.16458 [M + Na] + (calcd for C 24 H 26 N 2 O 7 Na 477.16377), 493.13879 [M + K] + (calcd for C 24 H 26 N 2 O 7 K 493.13771). Endophenaside E (5): yellowish, amorphous powder, UV (MeOH) max (log ) 248 (5.53), 365 (3.91) nm; IR ν max 3358, 2924, 2853, 1721, 1670, 1522, 1422, 1277, 1125, 1059, 941, 831, 750 cm 1 ; 1 H and 13 C NMR data, see Table 1 and 2; HRMS (positive mode) m/z 371.1233 [M + H] + (calcd for C 19 H 19 N 2 O 6 371.1238), 393.1062 [M + Na] + (calcd for C 19 H 18 N 2 O 6 Na 393.1063), 225.0657 [M + H - sugar] + (calcd for C 13 H 9 N 2 O 2 225.0659). Endophenazine F1 (6): purple, amorphous powder, UV/vis (MeOH) max (log ) 283 (4.33), 376 (3.83), 518 (3.79) nm; IR ν max 3348, 2920, 2855, 1732, 1717, 1684, 1624, 1539, 1456, 96, 1233, 1206, 745 cm 1 ; 1 H and 13 C NMR data, see Table 1 and 2; HRMS (positive mode) m/z 393.18115 [M + H] + (calcd for C 23 H 25 N 2 O 4 393.18088). Supplemental data file 3. Spectra list of new compounds S1. 1 H NMR spectrum (600 MHz) of endophenaside A (1) in CD 3 OD. S2. APT spectrum of endophenaside A (1) in CD 3 OD. S3. HSQC spectrum of endophenaside A (1) in CD 3 OD. S4. HMBC spectrum of endophenaside A (1) in CD 3 OD. S5. 1 H- 1 H COSY spectrum of endophenaside A (1) in CD 3 OD. S6. 1 H- 1 H NOSEY spectrum of endophenaside A (1) in acetone-d 6. S7. HRMS spectrum of endophenaside A (1). S8. IR spectrum of endophenaside A (1).

S9. UV spectrum of endophenaside A (1). S10. 1 H NMR spectrum (500 MHz) of endophenaside B (2) in CD 3 OD. S11. APT spectrum of endophenaside B (2) in CD 3 OD. S12. HSQC spectrum of endophenaside B (2) in CD 3 OD. S13. HMBC spectrum of endophenaside B (2) in CD 3 OD. S14. 1 H- 1 H COSY spectrum of endophenaside B (2) in CD 3 OD. S15. 1 H- 1 H NOSEY spectrum of endophenaside B (2) in acetone-d 6. S16. HRMS spectrum of endophenaside B (2). S17. IR spectrum of endophenaside B (2). S18. UV spectrum of endophenaside B (2). S19. 1 H NMR spectrum (500 MHz) of endophenaside C (3) in CD 3 OD. S20. HSQC spectrum of endophenaside C (3) in CD 3 OD. S21. HMBC spectrum of endophenaside C (3) in CD 3 OD. S22. 1 H- 1 H COSY spectrum of endophenaside C (3) in CD 3 OD. S23. HRMS spectrum of endophenaside C (3). S24. IR spectrum of endophenaside C (3). S25. UV spectrum of endophenaside C (3). S26. 1 H NMR spectrum (500 MHz) of endophenaside D (4) in CD 3 OD. S27. HSQC spectrum of endophenaside D (4) in CD 3 OD. S28. HMBC spectrum of endophenaside D (4) in CD 3 OD. S29. 1 H- 1 H COSY spectrum of endophenaside D (4) in CD 3 OD. S30. HRMS spectrum of endophenaside D (4). S31. IR spectrum of endophenaside D (4). S32. UV spectrum of endophenaside D (4). S33. 1 H NMR spectrum (500 MHz) of endophenaside E (5) in CD 3 OD. S34. HSQC spectrum of endophenaside E (5) in CD 3 OD.

S35. HMBC spectrum of endophenaside E (5) in CD 3 OD. S36. 1 H- 1 H COSY spectrum of endophenaside E (5) in CD 3 OD. S37. HRMS spectrum of endophenaside E (5). S38. IR spectrum of endophenaside E (5). S39. UV spectrum of endophenaside E (5). S40. 1 H NMR spectrum (600 MHz) of endophenazine F1(6) in CD 3 OD. S41. APT spectrum of endophenazine F1(6) in CD 3 OD. S42. HSQC spectrum of endophenazine F1(6) in CD 3 OD. S43. HMBC spectrum of endophenazine F1(6) in CD 3 OD. S44. 1 H- 1 H COSY spectrum of endophenazine F1 (6) in CD 3 OD. S45. 1 H- 1 H NOSEY spectrum of endophenazine F1 (6) acetone-d 6.. S46. HRMS spectrum of endophenazine F1(6). S47. IR spectrum of endophenazine F1(6). S48. UV spectrum of endophenazine F1(6).

S1. 1 H NMR spectrum (600 MHz) of endophenaside A (1) in CD 3 OD. S2. APT spectrum of endophenaside A (1) in CD 3 OD.

S3. HSQC spectrum of endophenaside A (1) in CD 3 OD. S4. HMBC spectrum of endophenaside A (1) in CD 3 OD.

S5. 1 H- 1 H COSY spectrum of endophenaside A (1) in CD 3 OD. S6. 1 H- 1 H NOSEY spectrum of endophenaside A (1) in acetone-d 6.

S7. HRMS spectrum of endophenaside A (1). Hm125-10 #464 RT: 8.02 AV: 1 NL: 8.18E5 T: FTMS + c ESI Full ms [100.00-1000.00] 800000 400.15076 C 20 H 22 O 6 N 3 1.11202 ppm Intensity 750000 700000 650000 600000 550000 500000 450000 400000 350000 300000 250000 200000 150000 100000 50000 0 214.08600 C 13 H 12 O 2 N -1.19870 ppm 240.07686 C 13 H 10 O 2 N 3 0.43911 ppm 279.15939 C 16 H 23 O 4 1.10195 ppm 327.07660 C 20 H 11 O 3 N 2 0.55153 ppm 371.10226 C 22 H 15 O 4 N 2-0.99446 ppm 422.13339 C 22 H 20 O 6 N 3-3.00726 ppm 200 250 300 350 400 450 m/z

S8. IR spectrum of endophenaside A (1).

S9. UV spectrum of endophenaside A (1). 1.4 1.2 1.0 0.8 Abs. 0.6 0.4 0.2 0.0-0.2 200 250 300 350 400 450 500 wavelength (nm) S10. 1 H NMR spectrum (500 MHz) of endophenaside B (2) in CD 3 OD.

S11. APT spectrum of endophenaside B (2) in CD 3 OD. S12. HSQC spectrum of endophenaside B (2) in CD 3 OD.

S13. HMBC spectrum of endophenaside B (2) in CD 3 OD. S14. 1 H- 1 H COSY spectrum of endophenaside B (2) in CD 3 OD.

S15. 1 H- 1 H NOSEY spectrum of endophenaside B (2) in acetone-d 6. S16. HRMS spectrum of endophenaside B (2).

Hm125-7 #555-592 RT: 9.61-10.24 AV: 38 NL: 1.30E6 T: FTMS + c ESI Full ms [100.00-1000.00] 1300000 455.18187 C 24 H 27 O 7 N 2 1.29463 ppm 1200000 1100000 1000000 900000 800000 Intensity 700000 600000 500000 400000 300000 158.02697 H 6 O 6 N 4-7.66074 ppm 309.12314 C 18 H 17 O 3 N 2-0.73835 ppm 200000 100000 212.07402 C 4 H 12 O 6 N 4-5.25350 ppm 391.28487 C 24 H 39 O 4 1.49525 ppm 477.16375 C 38 H 21-0.06409 ppm 0 150 200 250 300 350 400 450 500 550 600 m/z

S17. IR spectrum of endophenaside B (2).

S18. UV spectrum of endophenaside B (2). 1.0 0.8 0.6 Abs. 0.4 0.2 0.0 200 250 300 350 400 450 500 wavelength (nm) S19. 1 H NMR spectrum (500 MHz) of endophenaside C (3) in CD 3 OD.

S20. HSQC spectrum of endophenaside C (3) in CD 3 OD.

S21. HMBC spectrum of endophenaside C (3) in CD 3 OD. S22. 1 H- 1 H COSY spectrum of endophenaside C (3) in CD 3 OD.

S23. HRMS spectrum of endophenaside C (3). Hm125-11 #588 RT: 10.17 AV: 1 NL: 4.68E5 T: FTMS + c ESI Full ms [100.00-1000.00] Intensity 460000 440000 420000 400000 380000 360000 340000 320000 300000 280000 260000 240000 220000 200000 180000 160000 140000 120000 100000 80000 60000 40000 20000 212.07405 C 4 H 12 O 6 N 4-5.11452 ppm 235.20593 C 16 H 27 O 1.23554 ppm 293.09229 C 17 H 13 O 3 N 2 0.73822 ppm 391.28445 C 24 H 39 O 4 0.42980 ppm 356.13293 C 14 H 20 O 7 N 4 0.79793 ppm 454.17520 C 27 H 24 O 4 N 3-2.05054 ppm 469.19641 C 25 H 29 O 7 N 2-1.10090 ppm 491.18021 C 25 H 25 O 6 N 5 0.55244 ppm 200 250 300 350 400 450 500 m/z

S24. IR spectrum of endophenaside C (3).

S25. UV spectrum of endophenaside C (3). 1.0 0.8 0.6 Abs. 0.4 0.2 0.0 200 250 300 350 400 450 500 wavelength (nm) S26. 1 H NMR spectrum (500 MHz) of endophenaside D (4) in CD 3 OD.

S27. HSQC spectrum of endophenaside D (4) in CD 3 OD.

S28. HMBC spectrum of endophenaside D (4) in CD 3 OD. S29. 1 H- 1 H COSY spectrum of endophenaside D (4) in CD 3 OD.

S30. HRMS spectrum of endophenaside D (4). Hm125-12 #554-578 RT: 9.62-10.03 AV: 25 NL: 1.68E4 T: FTMS + c ESI Full ms [100.00-1000.00] 19000 18000 17000 455.18243 Intensity 16000 15000 14000 13000 12000 11000 10000 9000 8000 7000 6000 5000 457.19880 477.16458 4000 3000 2000 1000 0 445.12127 493.13879 479.17395 460.24161 447.34793 451.64261 463.22922 471.17744 483.38229 490.11026 495.30093 445 450 455 460 465 470 475 480 485 490 495 m/z

S31. IR spectrum of endophenaside D (4).

S32. UV spectrum of endophenaside D (4). 1.2 1.0 0.8 Abs. 0.6 0.4 0.2 0.0 200 250 300 350 400 450 500 wavelength (nm) S33. 1 H NMR spectrum (500 MHz) of endophenaside E (5) in CD 3 OD.

S34. HSQC spectrum of endophenaside E (5) in CD 3 OD. S35. HMBC spectrum of endophenaside E (5) in CD 3 OD.

S36. 1 H- 1 H COSY spectrum of endophenaside E (5) in CD 3 OD.

S37. HRMS spectrum of endophenaside E (5). Intens. [%] 100 1+ 225.0650 80 60 40 20 1+ 207.0546 1+ 371.1231 271.1294 321.0827 351.1772 393.1088 162.0644 0 100 200 300 400 500

S38. IR spectrum of endophenaside E (5).

S39. UV spectrum of endophenaside E (5). 1.0 0.8 0.6 Abs. 0.4 0.2 0.0 200 250 300 350 400 450 500 wavelength (nm)

S40. 1 H NMR spectrum (600 MHz) of endophenazine F1 (6) in CD 3 OD. S41. APT spectrum of endophenazine F1 (6) in CD 3 OD.

S42. HSQC spectrum of endophenazine F1 (6) in CD 3 OD. S43. HMBC spectrum of endophenazine F1 (6) in CD 3 OD.

S44. 1 H- 1 H COSY spectrum of endophenazine F1 (6) in CD 3 OD. S45. 1 H- 1 H NOSEY spectrum of endophenazine F1 (6) in CD 3 OD.

S46. HRMS spectrum of endophenazine F1 (6).

Hm125-8 #673 RT: 11.64 AV: 1 NL: 4.98E5 T: FTMS + c ESI Full ms [100.00-1000.00] 393.18115 C 23 H 25 O 4 N 2 0.68313 ppm Intensity 480000 460000 440000 420000 400000 380000 360000 340000 320000 300000 280000 260000 240000 220000 200000 180000 160000 140000 120000 100000 80000 60000 40000 20000 0 214.08606 C 13 H 12 O 2 N -0.91360 ppm 279.15939 C 16 H 23 O 4 1.10195 ppm 327.07886 C 23 H 9 N 3-0.73820 ppm 371.10181 C 20 H 13 O 3 N 5 1.39005 ppm 410.18573 C 26 H 24 O 2 N 3-1.39824 ppm 475.23804 C 32 H 31 O 2 N 2 0.06813 ppm 200 250 300 350 400 450 500 m/z

S47. IR spectrum of endophenazine F1 (6).

S48. UV spectrum of endophenazine F1 (6). 1.6 1.4 1.2 1.0 Abs. 0.8 0.6 0.4 0.2 0.0 200 300 400 500 600 700 wavelength (nm)