Vanchrobactin and Anguibactin Siderophores produced by Vibrio sp.

DS40M4

Moriah Sandy†, Andrew Han‡, John Blunt§, Murray Munro§, Margo Haygood‡ and Alison Butler†*

†
Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106-

9510, ‡Division of Environmental and Biomolecular Systems, Oregon Health & Science University,

Portland, Oregon 97006-8921, §Department of Chemistry, University of Canterbury, Private Bag 4800,

Christchurch, New Zealand

List of Figures in the Supporting Information

Figure S1. RP-HPLC of the MeOH XAD-2 Extract of the Supernatant of Vibrio sp. DS40M4
Figure S2. ESI-MS/MS of vanchrobactin (3)
Figure S3. ESI-MS/MS of divanchrobactin (2)
Figure S4. Trivanchrobactin (1) 1H NMR Spectrum (800 MHz) in CD3OD
Figure S5. Divanchrobactin (2) 1H NMR Spectrum (500 MHz) CD3OD
Figure S6. Vanchrobactin (3) 1H NMR Spectrum (500 MHz) in CD3OD
Figure S7. Trivanchrobactin (1) 13C NMR Spectrum in CD3OD
Figure S8. 1H-13C HSQC Spectrum (800 MHz) of trivanchrobactin (1) in CD3OD
Figure S9. 1H-13C HMBC Spectrum (800 MHz) of trivanchrobactin (1) in CD3OD
Table S1. NMR Data (800 MHz) for trivanchrobactin (1) isolated from Vibrio sp. DS40M4 in CD3OD
Table S2. NMR Data (500 MHz) for vanchrobactin (3) isolated from Vibrio sp. DS40M4 in CD3OD
Figure S10. 1H-1H- TOCSY Spectrum (500 MHz) of vanchrobactin (3) in CD3OD
Figure S11. 1H-13C HSQC Spectrum (500 MHz) of vanchrobactin (3) in CD3OD
Figure S12. 1H-13C HMBC Spectrum (500 MHz) of vanchrobactin (3) in CD3OD
Table S3. Comparison of 1H NMR of anguibactin (4) isolated from Vibrio sp. DS40M4 to that of anguibactin
(4) isolated from Vibrio anguillarum
Figure S13A. 1H NMR Spectrum of anguibactin (4) (500 MHz) in CD3OD
Figure S13B. ESI-MS of anguibactin (4)
Figure S14. ESI-Tandem mass spectrum of the di-tyrosine-methine-bridged compound isolated from the
growth medium.
Table S4. NMR Data (500 MHz) for the di-tyrosine-methine-bridged compound isolated from ASG-Fe Media
Figure S15. 1H Spectrum (500 MHz) of the di-tyrosine methine bridged compound
Figure S16. 1H-1H COSY Spectrum (500 MHz) of the di-tyrosine methine bridged compound
Figure S17. 1H-13C HSQC Spectrum (500 MHz) of the di-tyrosine methine bridged compound
Figure S18. 1H-13C HMBC Spectrum (500 MHz) of the di-tyrosine methine bridged compound
1
 di-tyrosine methine bridged compound from media

trivanchrobactin (1)

anguibactin (4)
vanchrobactin (3)

divanchrobactin (2)
compound from media
compound from XAD

Figure S1. RP-HPLC of the MeOH XAD-2 Extract of the Supernatant of Vibrio sp. DS40M4

262
msa2-27-1 peak 2 msms
BUT102807D 53 (1.016) Cm (33:53)
262
TOF MSMS 398.18ES+
87
262.24 1.24e3
100

311
311.19
137 311
%

398.19

175.30
276.17
293.19 398 [M+H]+
112.36
137 251.20

245.22
137.25
154.25 244.24
157.31

158.29 203.25 233.21 380.18

0 m/z
120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420

Figure S2. ESI-MS/MS of vanchrobactin (3)

2
butler/sandy M4 021507 peak 4 msms
BUT022707MK 43 (0.837) Cm (17:93)
100
398.19 398 TOF MSMS 777.36ES+
125

398

380
380.18

380
%

293.14

262.16 485.23

777 [M+H]+
244.14 399.19
336.18 777.34
467.20
200.16 641.31

0 m/z
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900

Figure S3. ESI-MS/MS of divanchrobactin (2)

3
 4
with an X
Figure S4. Trivanchrobactin (1) 1H NMR Spectrum (800 MHz) in CD3OD. Impurities are indicated

7, 7’, 7”(3H)

5, 5’, 5”(3H)
6, 6’,6” (3H)

9, 15’, 15”(3H)
9’,9”,16’ (3H)
15, 16” (2H)
16’, 16” (2H)

16(1H)
16(1H)

12, 12’, 12” (6H)

11, 11’, 11” (3H)

11, 11’, 11” (3H)

10, 10’, 10”(6H)
 5
an X.
Figure S5. Divanchrobactin (2) 1H NMR Spectrum (500 MHz) CD3OD. Impurities are indicated with

7, 7’(2H)

5, 5’(2H)

6, 6’(2H)

9, 9’(2H)
15, 15’(2H)
16’(2H)

16(1H)
16(1H)

12, 12’(4H)

11, 11’(2H)
11, 11’(2H)
10, 10’(4H)
 10(1H)
11(1H)
11(1H)

12(2H)

16(1H)
16(1H)

15(1H)
9(1H)

6(1H)
5(1H)

7(1H)

Figure S6. Vanchrobactin (3) 1H NMR Spectrum (500 MHz) in CD3OD. Impurities are indicated with
an X.
6
 7
Figure S7. Trivanchrobactin (1) 13C NMR Spectrum in CD3OD. Impurities are indicated with an X.

8, 8’, 8”
1, 1’, 1“
14, 14’, 14”
13, 13’, 13”

3, 3’, 3”
4, 4’, 4”

7, 7’, 7”
6, 6’, 6”
5, 5’, 5”

16”
16’ 16
15
9, 9’, 9”
15’, 15”

12, 12’, 12”

10, 10’, 10”

11, 11’, 11”
Figure S8: 1H-13C HSQC Spectrum (800 MHz) of trivanchrobactin (1) in CD3OD

Figure S9: 1H-13C HMBC Spectrum (800 MHz) of trivanchrobactin (1) in CD3OD

8
Table S1. NMR Data (500 MHz) for trivanchrobactin (1) isolated from Vibrio sp. DS40M4 in CD3OD
Position δC δH (J in Hz) HMBC
DHBA
C1, C1’, C1” 170.73, 170.69, 170.65
C2, C2’, C2” 117.59, 117.49, 117.37
C3, C3’, C3” 149.27, 149.11, 149.07
C4, C4’, C4” 147.16, 147.14
C5, C5’, C5” 119.86, 119.83, 119.81 6.96, d (8.0), [3H] 3,4,6
C6, C6’, C6” 120.04, 120.00, 119.99 6.76, m, [3H] 1,2,3,4,5
C7, C7’, C7” 120.08, 120.04 7.33, d (8.0), [1H] 1,3,4,5,6
7.36, d (8.0), [2H]
Arginine
C8 174.38 -
C8’ 174.19
C8” 173.95
C9 54.35 4.79, m, [1H] 1,8,10,11
C9’ 54.31 4.74, m, [1H] 1’, 8’,10’, 11’
C9” 54.21 4.71, m, [1H] 1”, 8”, 10”,
11”
C10 30.50 2.03, m, [2H] 8,9,11,12
1.89, m, [2H]
C10’ 30.36
C10” 30.34
C11 26.34 1.71, m, [6H] 12
C11’ 26.29
C11” 26.27
C12 41.98 3.23, m, [6H] 11,10,13
C12’ 41.95
C12” 41.59
C13 158.61 -
C13’ 158.57
C13” 158.53
Serine
C14 171.32 -
C14’ 169.85
C14” 171.95
C15 56.35 4.58, t (4.8), [1H] 8,14
C15’ 53.22 4.80, d (4.8), [1H] 1’, 8’, 14’
C15” 53.19 4.80, d (4.8), [1H] 1”, 8”, 14”
C16 62.62 3.84, dd (4.0, 11.2), 14, 15
[1H] 14, 15
3.96, dd (4.8, 11.2 )
[1H]
C16’ 64.73 4.43, m, [1H] 14,14’
4.59, m, [1H] 8,14,14’
C16” 65.62 4.43, m, [1H] 14’,14”
4.69, m, [1H] 8,14’,14”
9
Table S2. NMR Data (500 MHz) for vanchrobactin (3) isolated from Vibrio sp. DS40M4 in CD3OD

Position δH (J in Hz) HSQC HMBC TOCSY COSY

DHBA
1 (C) 169.9
2 (C) 116.7
3 (C) 151.3
4 (C) 146.8
5 (CH) 6.96 (1H, dd), 121.6 3,4,6 6 6
J=1.5, 8
6 (CH) 6.76 (1H, t), 121.3 2,4,5 5 5,7
J=8
7 (CH) 7.33 (1H, dd), 122.9 1,3,5 5,7 6
J=1.5, 8
Arginine
8 (C) - 172.7
9 (CH) 4.78 (1H, dd), 52.8 10, 9, 8 12,10,11 15,12
J=5.5, 8.5
10 (CH2) 2.04 (1H, m) 28.7 8 10,11,15
1.87 (1H, m) 12 10,12
11 (CH2) 1.70 (2H, m) 27.0 12, 10, 9 10 (1.87)
12 (CH2) 3.23 (2H, t), 40.8 13 9,10,11 15,10 (2.04)
J=7
13 (C) - 157.7
Serine
14 (C) - 171.8
15 (CH) 4.52 (1H, t), 55.3 8 16 10(1.87),16,9
J=5
16 (CH2) 3.85 (1H, dd), 61.6 14 16 (3.96), 15 15,16
J=5, 11.5 14, 15 16 (3.92), 15
3.94 (1H, dd),
J=5.5, 11.5

10
Figure S10: 1H-1H- TOCSY Spectrum (500 MHz) of vanchrobactin (3) in CD3OD

Figure S11: 1H-13C HSQC Spectrum (500 MHz) of vanchrobactin (3) in CD3OD

11
Figure S12: 1H-13C HMBC Spectrum (500 MHz) of vanchrobactin (3) in CD3OD

12
Table S3: Comparison of 1H NMR of anguibactin (4) isolated from Vibrio sp. DS40M4 to that of
anguibactin isolated from Vibrio anguillarum (see 1H NMR spectrum in Figure S13).

Positiona Group 1
H (ppm) (500 MHz), J(Hz) 1
H (ppm) (300 MHz) , J(Hz)
Vibrio sp. DS40M4 Vibrio anguillarum
in CD3OD 775(pJM1)*

3 Imidazole CH 7.35 (1H, s) 6.90 (1H, s)

5 Imidazole CH 8.76 (1H, s) 7.61 (1H, s)
6 CH2 3.12 (2H, m) 2.97 (2H, t), J=6.9
7 CH2 3.92 (1H, m) 3.94 (2H, m)
4.14 (1H, m) J=6.5, 11.0
4’ Thiazoline CH 5.85 (1H, t), J=9 5.88 (1H, t), J=9.1
5’ Thiazoline CH2 3.45 (1H, t), J=10 3.41 (1H, q)
3.61 (1H, t), J=10 3.66 (1H, q), J=9.1, 11
4” Aromatic CH 6.94 (1H, d), J=8 6.94 (1H, d)
5” Aromatic CH 6.74 (1H, t), J=8 6.74 (1H, t)
6” Aromatic CH 6.92 (1H, d), J=8 6.92 (1H, d), J=7.9
a. See numbering above
*Jalal, M. A. F.; Hossain, M. B.; Vanderhelm, D.; Sandersloehr, J.; Actis, L. A.; Crosa, J. H., J. Am.
Chem. Soc. 1989, 111, 292-296

13
 6(2H)
5’(1H)
5’(1H)
7(1H)
7(1H)
4’(1H)
6”(1H)
4”(1H), 5”(1H)
3(1H)
5(1H)
Figure S13a. 1H NMR spectrum of anguibactin (4) (500 MHz) in CD3OD
14
 m4 peak 36min 040907 msms
BUT042007MI 20 (0.389) Cm (19:34) 194 TOF MSMS 349.11ES+
100
194.04
155 36

H
127 N
HO OH O
N N N
222
222.04
OH
S
110.07

% 128 128.09

128.08
240.03 194
192.02 220.03

136.05
220.01 222
349[M+H]+
153.02 349.09
166.05

213.09
165.00 167.03 239.40
122.08 207.10 332.12

0 m/z
110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350

Figure S13b. ESI-MS of 4 indicates a molecular ion peak [M+H]+ at m/z 349.1, consistent with the
reported molecular weight of 348 for anguibactin. The fragmentation pattern observed by ESI-MSMS
of 4 is consistent with anguibactin, as is the NMR (Figure S13a, Table S3).

163 181 46

343
m s a 2 -2 7 -1 p e a k 3 m s m s
B U T 1 0 28 0 7F 50 (0 .96 0 ) C m (3 3:8 4 ) T O F M S M S 3 8 9.1 9E S +
343 .20 3.3 1e 3
100

208 343
126 208
162 .29

163 20 8.2 3

1 47.3 0

%
191 .23

29 7.2 3

2 82.22
389 (M+H)
126 2 98.21

13 6.3 1
181 3 26.1 9
34 4.1 9 3 89.20

14 9.2 7 1 82.25
16 3.2 7 28 0.2 1
173 .24
311 .18 328 .20
26 5.2 1

0 m /z
11 0 1 20 130 1 40 150 16 0 1 70 18 0 1 90 200 21 0 220 23 0 2 40 25 0 2 60 270 28 0 290 30 0 3 10 320 3 30 34 0 3 50 36 0 3 70 380 39 0

Figure S14. ESI-Tandem mass spectrum of the di-tyrosine-methine-bridged compound isolated from the
growth medium.

15
Table S4. NMR Data (500 MHz) for the di-tyrosine-methine-bridged compound isolated from ASG-Fe
Media

1
Position H[ppm] HSQC HMBC COSY
Tyrosine
1 171.2
2 4.05 (2H) 54.7 1, 4, 3 2,3
3 3.17 35.7 1, 5, 9, 4, 2 2,3
2.99
4 125.0
5 7.01 128.9 7,9,3,1” 9
6 132.8
7 154.1
8 6.73 115.4 7,6,4,1” 9
9 6.93 127.7 7,5,3 8
Methine
1” 4.79 30.9 7,7’,6,6’,5’,1’” 1’”
Methyl
1’” 1.53 19.0 6,6’,1” 1”
Tyrosine’
1’ 171.0
2’ 4.00 (2H) 54.7 1’,4’,3’ 2’,3’
3’ 3.13 35.5 1’, 5’, 9’, 4’, 2’ 2’,3’
3.08
4’ 125.2
5’ 7.15 129.1 7’,9’,3’,1” 9’
6’ 133.3
7’ 154.1
8’ 6.73 115.4 7’,6’,4’,1” 8’
9’ 6.93 127.7 7’,5’,3’ 9’

16
 Aug 17 2007 g51967h MSA1-12-2 Department of Chemistry, University of Canterbury
8,8’ 1”’
9,9’
5’ 5

2,2’
3,3’
1”

7 6 5 4 3 2 1 ppm
1
Figure S15: H Spectrum (500 MHz) of the di-tyrosine methine bridged compound, impurities are
indicated with an X.

Figure S16: 1H-1H COSY Spectrum (500 MHz) of the di-tyrosine methine bridged compound

17
Figure S17: 1H-13C HSQC Spectrum (500 MHz) of the di-tyrosine methine bridged compound

Figure S18: 1H-13C HMBC Spectrum (500 MHz) of the di-tyrosine methine bridged compound

18