Supporting information
Oleamycins A and B: New Anti-bacterial cyclic hexadepsipeptides isolated from a Terrestrial Streptomyces sp.
Ritesh Rajua,b,Oleksandr Gromykoc, Butsiak Andriyc, Viktor Fedorenkoc,Andriy Luzhetskyya,b and Rolf Müller*a,b
aDepartment of Microbial Natural Products, Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Centre for Infection Research (HZI), Saarland University, Campus C2 3, 66123 Saarbrücken, Germany
bDepartment of Pharmaceutical Biotechnology, Saarland University, Campus C2 3, 66123 Saarbrucken, Germany.
cDepartment of Genetics and Biotechnology of Ivan Franko National University of L’viv, Grushevskogo st. 4, L’viv 79005, Ukraine.
Figure S1a1H NMR (500 MHz,CDCl3) spectrum ofoleamycin A(1) 2
Figure S1b. COSY (500 MHz, CDCl3) spectrum ofoleamycin A (1) 3
Figure S1c.HSQC (500 MHz, CDCl3) spectrum oleamycin A (1) 4
Figure S1d. HMBC (500 MHz, CDCl3) spectrum ofoleamycin A (1) 5
Figure S1e. ROESY (500 MHz, CDCl3) spectrum ofoleamycin A (1) 6
Figure S2a1H NMR (700 MHz, CDCl3) spectrum of oleamycin B (2) 7
Figure S2b. COSY (700 MHz, CDCl3) spectrum of oleamycin B (2) 8
Figure S2c. HSQC (700 MHz, CDCl3) spectrum oleamycin B (2) 9
Figure S2d. HMBC (700 MHz, CDCl3) spectrum of oleamycin B (2) 10
Table S1. NMR (700 MHz, CDCl3) data for oleamycin B (2) 11
Figure S3. HRMS spectrum of oleamycin A (1) 12
Figure S4. HRMS spectrum of oleamycin B (2) 13
Taxonomic Identification 14
Biological assay 15
Figure S1a.1H NMR (500 MHz, CDCl3) spectrum of oleamycin A (1a)
Figure S1b. COSY (500 MHz, CDCl3) spectrum of oleamycin A (1a)
Figure S1c. HSQC (500 MHz, CDCl3) spectrum of oleamycin A (1a)
Figure S1d. HMBC (500 MHz, CDCl3) spectrum of oleamycin A (1a)
Figure S1e. ROESY (500 MHz, CDCl3) spectrum of oleamycin A (1)
Figure S2a.1H NMR (700 MHz, CDCl3) spectrum of oleamycin B (2)
Figure S2b.COSY (700 MHz, CDCl3) spectrum of oleamycin B (2)
Figure S2c.HSQC (700 MHz, CDCl3) spectrum of oleamycin B (2)
Figure S2d. HMBC (700 MHz, CDCl3) spectrum of oleamycin B (2)
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Table S1. NMR (700 MHz, CDCl3) data for Oleamycin B (2)position / H, mult (J in Hz) (1) / C* / COSY / HMBC
3-hydroxyleucine
1 / 172.5
2 / 5.84, d (9.0) / 48.2 / 3, 2N-H
3 / 4.62a, d (7.0) / 82.1 / 2, 4
4 / 1.89, m / 29.7 / 3, 5, 6
5 / 1.02, d (7.0) / 19.5 / 4 / 3, 4, 6
6 / 0.94, d (7.0) / 18.2 / 4 / 3, 4, 5
2N-H / 8.00, d (9.8) / 2 / 23
Glycine (1)
7 / 170.6
8a / 4.28, d (17.0) / 52.1 / 8b / 7, 37
8b / 3.62 / 8a
Glycine (2)
9 / 169.2
10a / 5.31 / 50.2 / 10b / 9, 38
10b / 3.36, d (14.3) / 10a
Glycine (3)
11 / 169.2
12a / 4.54, dd (16.6, 9.5) / 40.6 / 12b, 12N-H / 11
12b / 3.70 / 12a, 12N-H
12N-H / 7.53, d (9.6) / 12a/b / 13
Piperazic acid (1)
13 / 169.7
14 / 5.15, m / 52.6 / 15a/b / 13
15a / 2.51, m / 23.5 / 14, 15b, 16
15b / 1.64, m / 14, 15a, 16
16 / 1.56, m / 22.1 / 15a/b, 17a/b
17a / 3.14b, m / 47.9 / 16, 17N-H, 17b
17b / 2.73, m / 16, 17N-H, 17a
17N-H / 5.18, d (12.7) / 17a/b
Piperazic acid (2)
18 / 174.8
19 / 5.50, dd (6.4, 5.9) / 50.6 / 20 / 18, 19, 20
20 / 1.96, m / 23.1 / 19, 21a/b / 18
21a / 1.77, m / 20 / 20, 21b, 22a/b
21b / 1.57, m / 20, 21a, 22a/b
22a / 3.16b, m / 46.0 / 21a/b, 22b, 22N-H
22b / 2.93, m / 21a/b, 22b, 22N-H
22N-H / 4.62a / 22a/b
Polyketide
23 / 176.8
24 / 77.3
25 / 99.1
26 / 1.61-1.76, m / 27.8 / 27
27 / 1.30-1.38 / 24.0 / 26, 28
28 / 1.17-1.29, m / 36.8 / 27, 29
29 / 3.53, dt (10.1, 2.5) / 76.1 / 28, 30
30 / 1.25-1.37, m / 25.5 / 29, 31
31 / 0.79, t (6.5) / 9.6 / 30
32 / 1.12, m / 39.3 / 28, 33
33 / 1.48, m / 28.2 / 32, 34, 35
34 / 0.84c, d (6.6) / 22.8 / 33 / 32, 33, 35
35 / 0.84c, d (6.6) / 22.8 / 33 / 32, 33, 34
36 / 1.26, s / 20.0 / 23, 24, 25
37 / 2.83, s / 34.3 / 8, 9
38 / 3.07, s / 33.2 / 10, 11
*assignments supported by HSQC and HMBC, a-coverlapping signals
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Figure S3. HRMS spectrum ofoleamycin A(1)
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Figure S4. HRMS spectrum ofoleamycin B (2)
Taxonomic identification of strain 20-58
16S rDNA gene sequence
ACGCTCCCTCCCCACAGGGGGTTGGGCCACCGGCTTCGGGTGTTACCGACTTTCGTGACGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGCAATGCTGATCTGCGATTACTAGCAACTCCGACTTCATGGGGTCGAGTTGCAGACCCCAATCCGAACTGAGACAGGCTTTTTGAGATTCGCTCCGCCTTGCGACATCGCAGCTCATTGTACCTGCCATTGTAGCACGTGTGCAGCCCAAGACATAAGGGGCATGATGACTTGACGTCGTCCCCACCTTCCTCCGAGTTGACCCCGGCAGTCTCCTGTGAGTCCCCACCATCCCCGAAGGGACGTGCTGGCAACACAGAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCACCCGACCACAAGGGGGGCACCATCTCTGATGCTTTCCGGGCGATGTCAAGCCTTGGTAAGGTTCTTCGCGTTGCGTCGAATTAAGCCACATGCTCCGCTGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGGAACTTAATGCGTTAGCTGCGGCACCGACGACGTGGAATGTCGCCAACACCTAGTTCCCACCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTAATGGCCCAGAGATCCGCCTTCGCCACCGGTGTTCCTCCTGATATCTGCGCATTTCACCGCTACACCAGGAATTCCGATCTCCCCTACCACACTCTANTCTGCCCGTATCGAATGCAGACCCGGGGGTTAAGCCCCCGGGCTTTCACATCCCGACGTTGACAGAACCGCCCTACNAAGCTCNTTTACGCCCCAATAAATTCCCGGA
LOCUS JN696642 1016 bp DNA linear BCT 30-SEP-2012
DEFINITION Streptomyces sp. K25-16L 16S ribosomal RNA gene, partial sequence.
ACCESSION JN696642
VERSION JN696642.1 GI:380704367
KEYWORDS .
SOURCE Streptomyces sp. K25-16L
ORGANISM Streptomyces sp. K25-16L
Bacteria; Actinobacteria; Actinobacteridae; Actinomycetales;
Streptomycineae; Streptomycetaceae; Streptomyces.
REFERENCE 1 (bases 1 to 1016)
AUTHORS Yang,A. and Lv,J.
TITLE Diversity of microorganisms in permafrost active layer soil of Hoh
Xil and Tanggula Mountains, Qinghai-Tibet Plateau
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 1016)
AUTHORS Yang,A. and Lv,J.
TITLE Direct Submission
JOURNAL Submitted (16-SEP-2011) Beijing University of Chemical Technology,
College of Life Science and Technology, 15 Beisanhuan East Road,
Chaoyang District, Beijing, Beijing 100029, China
Biological Assay
Human HCT-116 colon carcinoma cells (DSMZ, ACC 581)were seeded at 1.2 x 104 cells per well of 96-well plates (Corning, CellBind) in 180 μl complete medium and directly treated with 1 at 1 and 10 µg/ml to assess acute cytotoxicity. After 2 d incubation, 20 μl of 5 mg/ml MTT (thiazolyl blue tetrazolium bromide) in PBS was added per well and it was further incubated for 2 h at 37°C. The medium was then discarded and cells were washed with 100 μl PBS before adding 100 μl 2-propanol/10 N HCl (250:1) in order to dissolve formazan granules. The absorbance at 570 nm was measured using a microplate reader (EL808, Bio-Tek Instruments Inc.), and cell viability was expressed as percentage relative to the respective control. As a result, all tested derivatives are not cytotoxic up to a concentration of 10 µg/ml.
Microbial susceptibility was assessed by determination of the minimal inhibitory concentration (MIC). Therefore, microorganisms were incubated in EBS or Myc medium over night at 30 °C on a shaker. Microorganisms were seeded into 96-well plated, treated with different dilutions of the compound and incubated over night at 30°C on a shaker. The viability of microbial cells was analyzed by measurement of the absorbance at 600 nm on a plate reader.
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