supplementary data amycolatopsins a - c: anti ... · supplementary data amycolatopsins a - c:...
TRANSCRIPT
SUPPLEMENTARY DATA
Amycolatopsins A - C: Anti-Mycobacterial Glycosylated Polyketide Macrolides from the Australian Soil Amycolatopsis sp. MST-108494
Zeinab G. Khalil,1,3 Angela A. Salim,1 Daniel Vuong,2 Andrew Crombie,2 Ernest Lacey,2 Antje Blumenthal3 and Robert J. Capon1
1Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD, 4072, Australia
2Microbial Screening Technologies, Smithfield, NSW, 2164, Australia
3The University of Queensland Diamantina Institute, The University of Queensland, Brisbane, QLD, 4102, Australia
*Corresponding author. Email: [email protected]
2
Table of Contents
1 Experimental ................................................................................................................................... 4 1.1 Bacterial taxonomy and gene sequence of Amycolatopsis sp. (MST-108494) ......................... 4 1.2 BLAST search (closest match) .................................................................................................. 4 1.3 Phylogenetic tree ....................................................................................................................... 5 1.4 Media composition .................................................................................................................... 7 1.5 HPLC-DAD chromatogram of MST-108494 ........................................................................... 9
2 Biological Data for 1-3 ................................................................................................................. 19 2.1 Anti-microbial assays .............................................................................................................. 19 2.2 Cytotoxicity assay ................................................................................................................... 19 2.3 Anti-mycobacterial assay ........................................................................................................ 20
3 References ..................................................................................................................................... 20
3
List of Tables
Table S1. 1D and 2D NMR (600 MHz, MeOH-d4) data for amycolatopsin A (1) ......................... 10Table S2. 1D and 2D NMR (600 MHz, MeOH-d4) data for amycolatopsin B (2) ......................... 12Table S3. 1D and 2D NMR (600 MHz, MeOH-d4) data for amycolatopsin C (3) ......................... 14
List of Figure
Figure S1. Blast search (closest match) for MST-108494 .................................................................. 5Figure S2. Phylogenetic tree of MST-108494. ................................................................................... 6Figure S3. HPLC-DAD chromatogram of the crude extract of Amycolatopsis sp. (MST-108494) ... 9Figure S4. 1H NMR (600 MHz, MeOH-d4) spectrum of amycolatopsin A (1) ................................ 15Figure S5. UV-vis (HPLC-DAD, H2O/MeCN plus HCO2H) spectrum of amycolatopsin A (1) ..... 15Figure S6. 1H NMR (600 MHz, MeOH-d4) spectrum of amycolatopsin B (2) ................................ 16Figure S7. UV-vis (HPLC-DAD, H2O/MeCN plus HCO2H) spectrum of amycolatopsin B (2) ..... 16Figure S8. 1H NMR (600 MHz, MeOH-d4) spectrum of amycolatopsin C (3) ................................ 17Figure S9. UV-vis (HPLC-DAD, H2O/MeCN plus HCO2H) spectrum of amycolatopsin C (3) ..... 17Figure S10. 1H NMR (600 MHz, MeOH-d4) spectra comparison for amycolatopsin A – C (1 – 3). 18Figure S11. Anti-microbial assay data for amycolatopsins A - C (1 - 3) .......................................... 19Figure S12. Cytotoxicity assay data for amycolatopsins A - C (1 - 3) .............................................. 19Figure S13. Anti-mycobacterial assay data for amycolatopsins A - C (1 - 3) ................................... 20
4
1 Experimental
1.1 Bacterial taxonomy and gene Sequence of Amycolatopsis sp. (MST-108494)
16S rRNA sequence AGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGATGAAGCCCTTCGGGGTGGATTAGTGGCGAACGGGTGAGTAACACGTGGGTAATCTGCCCTGTACTTTGGGATAAGCCCTGGAAACGGGGTCTAATACCGGATATGACCGCGCATCGCATGGTGTGTGGTGGAAAGCTCCGGCGGTATGGGATGAACCCGCGGCCTATCAGCTTGTTGGTGGGGTAAAAGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGGGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCGACAGGGACGAAGGGTAACTGACGGTACCTGTAGAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCGGCCGTGAAAACTGGAGGCTTAACCTCCAGCTTGCGGTCGATACGGGCAGACTTGAGTTCGGTAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCCGATACTGACGCTGAGGAGCGAAAGCGTGCGGAGCGAACA
1.2 BLAST search (closest match)
An NCBI-BLAST database search of 16s rRNA sequence established a 99% similarity for MST-
108494 with Amycolatopsis magusensis (Figure S1).
5
Figure S1. Blast search (closest match) for MST-108494
1.3 Phylogenetic tree
Phylogenetic tree obtained by PhyML Maximum Likelihood analysis was constructed using the top
similar 16S rRNA sequences displayed after BLAST on Refseq RNA NCBI database using MST-
108494 16S rRNA as queries. The JC69 model was used to infer phylogeny sequences.1 Sequences
alignments were produced with the MUSCLE program.2 Phylogenetic tree was constructed using
the UGENE program using the aforementioned models and visualized using Ugene’s tree view.3
6
Figure S2. Phylogenetic tree by PhyML Maximum Likehood analysis of 16s rRNA sequences showing the relationship of MST-108494 among selected reference strains (RefSeq GenBank) with their accession numbers.
NR_116263.1 Actinophytocola xinjiangensis strain QAIII60
NR_133956.1 Actinophytocola sediminis strain YIM M13705
NR_109530.1 Amycolatopsis cihanbeyliensis strain BNT52
NR_109638.1 Amycolatopsis jiangsuensis strain KLBMP 1262 70
NR_116718.1 Amycolatopsis xylanica strain CPCC 202699
NR_109060.1 Amycolatopsis magusensis strain KT2025
MST_108494100
NR_044465.1 Amycolatopsis marina strain Ms392A
NR_025231.1 Amycolatopsis palatopharyngis strain 97
NR_109305.1 Amycolatopsis ruanii strain NMG112
NR_109504.1 Amycolatopsis dongchuanensis strain YIM 75904
NR_112696.1 Amycolatopsis sacchari strain NBRC
NR_028774.1 Amycolatopsis sacchari strain K24 69
91
NR_109300.1 Amycolatopsis bartoniae strain SF26
NR_116742.1 Amycolatopsis halophila strain YIM 93223
NR_115661.1 Amycolatopsis jejuensis strain N7-3 11
7
5
1
0
0
1
NR_117350.1 Amycolatopsis samaneae strain RM287
NR_115160.1 Amycolatopsis mediterranei strain NRRL B-3240 66
NR_114859.1 Amycolatopsis coloradensis strain DSM 44225
NR_115688.1 Amycolatopsis umgeniensis strain UM16 83
7
NR_114905.1 Amycolatopsis lurida strain DSM 43134
NR_042040.1 Amycolatopsis lurida strain IMSNU 20057
NR_042103.1 Amycolatopsis azurea strain IMSNU 20053
NR_036923.1 Amycolatopsis keratiniphila strain D2
NR_025563.1 Amycolatopsis keratiniphila subsp. nogabecina strain DSM 44586
NR_025562.1 Amycolatopsis decaplanina strain DSM 44594
NR_042104.1 Amycolatopsis orientalis strain IMSNU 20058
NR_025561.1 Amycolatopsis japonica strain MG 417-CF 17 89
34
19
35
28
41
69
25
NR_043528.1 Amycolatopsis minnesotensis strain 32U-2
NR_043880.1 Amycolatopsis nigrescens strain CSC17Ta-90 43
3
0
NR_134686.1 Actinokineospora mzabensis strain PAL84
NR_134687.1 Actinokineospora spheciospongiae strain EG49 93
NR_040873.1 Actinokineospora auranticolor strain IFO 16518 G
NR_024962.2 Actinokineospora diospyrosa strain NRRL B-24047
NR_112847.1 Actinokineospora cianjurensis strain BTCC B-558
NR_024963.1 Actinokineospora globicatena strain NRRL B-24048
NR_024774.1 Actinokineospora terrae strain IFO 15668 84
67
45
39
NR_041734.1 Actinokineospora riparia strain NRRL B-16432
NR_040872.1 Actinokineospora enzanensis strain IFO 16517 34
67
56
NR_133693.1 Alloactinosynnema iranicum strain Chem10
NR_041737.1 Lechevalieria flava strain NRRL B-16131
NR_109528.1 Lechevalieria nigeriaca strain NJ2035
NR_144590.1 Lentzea guizhouensis strain DHS C013
NR_108456.1 Lentzea jiangxiensis strain FXJ1.034
NR_043694.1 Lentzea kentuckyensis strain NRRL B-24416 61
NR_024686.1 Lentzea waywayandensis strain JCM 9114
NR_024649.1 Lentzea albida strain IFO 1610282
59
68
25
72
NR_108468.1 Labedaea rhizosphaerae strain RS-49
NR_116313.1 Actinophytocola oryzae strain GMKU 367
NR_112955.1 Actinophytocola corallina strain NBRC 105525
NR_112954.1 Actinophytocola timorensis strain NBRC 105524 71
94
27
8
5
4
6
100
7
1.4 Media composition
Glycerol Casein Agar (CGA) Quantity Glycerol (g) 30 Casein peptone (g) 2 K2HPO4 (g) 1 NaCl (g) 1 MgSO4.7H2O (g) 0.5 Trace element solution (mL)* 5 Distilled H2O (mL) 1000 Bacteriological agar (g) 20 *Trace element solution CaCl2.2H2O (g) 3 FeC6O7H5 (g) 1 MnSO4 (g) 0.2 ZnCl2 (g) 0.1 CuSO4.5H2O (g) 0.025 Na2B4O7.10H2O (g) 0.02 CoCl2 (g) 0.004 Na2MoO4.2H2O (g) 0.01 Distilled H2O (mL) 1000
Glucose Yeast Extract Starch (GYA) Quantity Yeast extract (g) 4 Malt extract (g) 10 Glucose (g) 4 CaCO3 (g) 2 Soluble starch (g) 20 Bacteriological agar (g) 20 Distilled water (mL) 1000 Adjust pH to 7.3
ISP2 agar (I2A) Quantity Yeast extract (g) 4 Malt extract (g) 10 Glucose (g) 4 Bacteriological agar (g) 20 Distilled water (mL) 1000 Adjust pH to 7.3
8
ISP2 + Mannitol agar (IMA) Quantity Yeast extract (g) 4 Malt extract (g) 10 Glucose (g) 4 Mannitol (g) 40 Bacteriological agar (g) 20 Distilled water (mL) 1000 Adjust pH to 7.3
Soybean Beef Extract agar (LUA) Quantity Soluble starch (g) 30 Soya bean meal (g) 7 Dry yeast (g) 10 Mannitol (g) 40 CaCO3 2 Bacteriological agar (g) 20 Distilled water (mL) 900 Beef extract (g) 2 Distilled water (mL) 100 Filter sterilize into autoclaved media
Modified Bennett’s Agar (MSA) Quantity Yeast extract (g) 2 Beef extract (g) 1.6 Casitone (g) 4 Glycerol (g) 20 Mannitol (g) 10 Soytone (g) 10 Bacteriological agar (g) 20 Distilled water (mL) 1000 pH adjust to 7.0
Molasses Peptone Glucose Agar (MOA) Quantity Bacteriological peptone (g) 3 Molasses (g) 60 Bacteriological glucose (g) 60 Bacteriological agar (g) 20 Distilled water (mL) 1000 pH adjust to 5.5
9
1.5 HPLC-DAD chromatogram of MST-108494
Figure S3. Expansion (4–8 min) of the HPLC-DAD chromatogram of the crude extract of Amycolatopsis sp. (MST-108494) showing amycolatopsins A - C.
4 5 6 7 80
100
200
300
400
500
Retention time (min)
Abs
orba
nce
at 2
10 n
m (m
AU
) A
B
C
10
Table S1. 1D and 2D NMR (600 MHz, MeOH-d4) data for amycolatopsin A (1) # δH, mult, (J in Hz) δC
COSY 1H – 13C HMBC ROESY 1 171.2 2 126.3 3 7.00, q (1.3) 137.6 2-Me 1, 2, 2-Me, 4, 5 3-OMe, 5 4 152.8 5 5.74, s 131.5 2-Me, 7 3, 4, 4-OMe, 6, 6-CH2, 7 2-Me, 3, 4-OMe, 7 6 135.7 7 5.50, d (10.6) 144.0 5, 8 5, 6, 6-CH2, 8-Me, 9 5, 9, 8-Me 8 2.92, ddq (10.6, 9.1, 6.6) 37.1 7, 8-Me, 9 8-Me, 9, 11 6a, 6b, 8-Me, 10-Me 9 3.80, d (9.1) 87.8 8 1ʹ, 7, 8, 8-Me, 10, 10-Me 7, 8-Me, 11, 1ʹ 10 136.6e 11 5.94, br s 136.8 e 10-Me, 12-Me, 13 9, 10-Me, 12,12-Me 7, 9, 12-Me, 13, 1ʹ 12 134.4
13 5.60, s 133.8 10-Me, 11, 12-Me, 14-Me, 15, 16 12-Me, 14, 15 10-Me, 11, 14-Me, 15
14 136.6e 15 5.22, br d (7.8) 129.6 13, 14-Me, 16 13, 14-Me, 16 13, 17, 18a, 19 16 4.81, br d (7.8) 67.9 14-Me, 15, 13, 17 14, 15, 17, 18 14-Me, 17-OMe 17 2.66, br d (10.1) 83.0 16, 18a, 18b 16, 17-OMe, 18, 19 15, 18a, 19 18 a 1.91b, m 36.0 17, 18b, 19 16, 17, 19 b 1.63, dd (14.5, 10.1) 17, 18a, 19 16, 17, 19 19 5.53, br d (10.8) 72.5 18a, 18b, 20 1, 17, 20-Me, 21 15, 17, 17-OMe, 20-Me 20 2.03, dd (7.2, 1.6) 45.1 19, 20-Me 21, 22 22, 22-OMe 21 100.0 22 3.04, d (9.5) 82.0 23 21, 22-OMe, 23 20, 20-Me 23 3.91, d (9.5) 77.0 22, 24-Me 22, 24, 24-Me 25 24 82.5 25 3.63, dd (9.5, 1.9) 74.0 26a, 26b 24-Me, 26, 27 23, 26, 27 26 a 1.57, m 25.9 25, 26b, 27b 25, 28 25 b 1.21c, m 25, 26a, 27b 25, 28 25 27 a 1.51, m 28.0 26a, 27b, 28 25, 28 28 b 1.32, m 26a, 27a, 28 25, 28 28 28 3.28a, m 74.1 27a, 27b 26, 27, 28-OMe 2-Me 2.09, br s 13.4 3, 5 1, 2, 3, 4 4-OMe 6-CH2OH a 4.49, d (12.1) 58.1 6b 5, 6, 7 7, 8 b 4.39, d (12.1) 6a 5, 6, 7 7, 8 8-Me 1.18, d (6.6) 19.1 8 7, 8, 9 7, 8, 9 10-Me 1.60, d (1.2) 12.2 11 9, 11, 12 8, 13 12-Me 1.88, br s 18.4 11, 13 11, 12 11 14-Me 1.73, br s 17.7 13, 15, 16 12, 14, 15 17 20-Me 1.12d, d (7.2) 9.4 20 19, 21 22-OMe, 23 24-Me 1.13d, s 10.8 23, 25 1ʹʹ 4-OMe 3.60, s 61.7 4, 5 2-Me 17-OMe 3.34, s 57.7 17 16, 19 22-OMe 3.53, s 61.6 22 20, 20-Me 28-OMe 3.24, s 58.8 28 1ʹ 4.64, d (3.8) 96.0 2ʹ 3ʹ, 5ʹ, 9 2ʹ, 9, 10-Me, 11 2ʹ 3.39, dd (9.7, 3.8) 73.5 1ʹ, 3ʹ 3ʹ, 5ʹ 1ʹ, 4ʹ, 6ʹ 3ʹ 3.75, dd (9.7, 9.7) 75.0 2ʹ, 4ʹ 1ʹ, 2ʹ, 4ʹ 1ʹ, 4ʹ 4ʹ 2.72, dd (9.7, 9.7) 87.7 3ʹ, 5ʹ 3ʹ, 4ʹ-OMe, 5ʹ, 6ʹ 2ʹ, 5ʹ, 4ʹ-OMe, 6ʹ 5ʹ 3.73, dq (9.7, 6.1) 68.3 4ʹ, 6ʹ-Me 1ʹ, 2ʹ, 4ʹ 4ʹ, 6ʹ, 8-Me 6ʹ 1.25c, d (6.1) 18.4 5ʹ 4ʹ 4ʹ, 4ʹ-OMe, 5ʹ 4ʹ-OMe 3.58, s 61.2 4ʹ 4ʹ, 6ʹ-Me 1ʹʹ 5.08, dd (9.5, 1.9) 92.7 2ʹʹa, 2ʹʹb 4ʹʹ, 24 2ʹʹa, 5ʹʹ, 25, 24-Me, 28 2ʹʹ a 1.92b, m 39.0 1ʹʹ, 2ʹʹb, 3ʹʹ 1ʹʹ, 4ʹʹ 3ʹʹ b 1.71, m 1ʹʹ, 2ʹʹa, 3ʹʹ 1ʹʹ, 4ʹʹ 3ʹʹ 4.24, ddd (2.8, 2.8, 2.8) 68.5 2ʹʹa, 2ʹʹb, 4ʹʹ 1ʹʹ 1ʹʹʹ, 2ʹʹa, 6ʹʹ 4ʹʹ 3.28a, m 83.4 3ʹʹ, 5ʹʹ 1ʹʹʹ, 5ʹʹ, 6ʹʹ 5ʹʹ 3.89, dq (9.6, 6.5) 69.6 4ʹʹ, 6ʹʹ 4ʹʹ 1ʹʹ, 1ʹʹʹ, 6ʹʹ 6ʹʹ 1.26c, d (6.5) 19.1 5ʹʹ 4ʹʹ
O
OMe
O
OH
OMe
OOH
OHOMeO
O
OH
MeOOH
OMe
1
1'
5'
2120
19
25
22
28
6
9 16
OH
O O OOH
OH
OHO1"
4"
1"' 5"'23
45
78
1011
1213
1415 17
18
11
Continued,
# δH, mult, (J in Hz) δC COSY 1H – 13C HMBC ROESY
1ʹʹʹ 4.69, dd (9.9, 1.9) 102.0 2ʹʹʹa, 2ʹʹʹb 4ʹʹ, 2ʹʹʹ, 5ʹʹʹ 3ʹʹ, 4ʹʹ, 5ʹʹʹ 2ʹʹʹ 1.93b, m 46.7 1ʹʹʹ, 2ʹʹʹa, 3ʹʹʹ-Me 1ʹʹʹ, 3ʹʹʹ, 4ʹʹʹ, 3ʹʹʹ-Me 4ʹʹʹ 1.70, m 1ʹʹʹ, 2ʹʹʹb, 3ʹʹʹ-Me 1ʹʹʹ, 3ʹʹʹ, 4ʹʹʹ, 3ʹʹʹ-Me 4ʹʹʹ 3ʹʹʹ 72.7 4ʹʹʹ 3.09, d (9.6) 80.3 5ʹʹʹ, 6ʹʹʹ 2ʹʹʹ, 3ʹʹʹ/5ʹʹʹ, 3ʹʹʹ-Me, 6ʹʹʹ-Me 2ʹʹʹb, 3ʹʹʹ/6ʹʹʹ 5ʹʹʹ 3.37, dq (9.6, 6.0) 72.5 4ʹʹʹ, 6ʹʹʹ 6ʹʹʹ 1ʹʹʹ, 3ʹʹʹ-Me, 6ʹʹʹ 6ʹʹʹ 1.27c, d (6.0) 19.1 5ʹʹʹ 5ʹʹʹ 5ʹʹʹ 3ʹʹʹ-Me 1.24c, s 20.5 2ʹʹʹ, 4ʹʹʹ 5ʹʹʹ
(a − d) overlapping resonances. (e) assignments are interchangeable within the same letter
12
Table S2. 1D and 2D NMR (600 MHz, MeOH-d4) data for amycolatopsin B (2)
# δH, mult, (J in Hz) δC COSY 1H – 13C HMBC ROESY
1 171.0 2 125.2 3 6.96, q (1.2) 138.2 2-Me 1, 2-Me, 4, 5 2-Me, 4-OMe, 5, 17 4 153.6 5 5.75, s 133.6 2-Me, 7 3, 4, 6-Me, 7 3, 6-Me, 7, 13, 14-OMe 6 133.8 7 5.31, br d (10.5) 142.0 5, 6-Me, 8 5, 6-Me, 8-Me 5, 9, 8-Me, 11 8 2.84, ddq (10.5, 9.1, 6.5) 36.8 7, 8-Me, 9 8-Me, 9 6-Me, 8-Me, 10-Me 9 3.79, d (9.1) 87.8 8, 11 1ʹ, 8-Me, 8, 10-Me, 11 1ʹ, 6-Me, 7, 8-Me,10-Me, 11 10 134.5 11 5.90, br s 136.3 9, 10-Me, 12-Me, 13 9, 13, 10-Me, 12-Me 1ʹ, 7, 9, 12-Me, 13 12 133.8 13 5.57, br s 133.6 11, 12-Me, 14-Me, 15 11, 14-Me, 15 5, 10-Me, 11, 15 14 135.6 15 5.21, br d (7.6) 129.4 13, 14- Me, 16 13, 14-Me, 16 13, 17, 18a, 19 16 4.81, br d (7.6) 67.9 14-Me, 15, 17 14, 15, 17, 18 14-Me, 17, 17-OMe 17 2.67, br d (10.7) 83.0 16, 18a, 18b 16, 17-OMe 3, 15, 16, 17-OMe, 18a, 19 18 a 1.90c, m 35.9 17, 18b, 19 16, 19 15, 17 b 1.63, dd (14.3, 10.7) 17, 18a, 19 16, 19 19, 20, 20-Me 19 5.51, br d (10.7) 72.2 18a, b, 20 20-Me 15, 17, 18b, 20, 20-Me, 22 20 2.03, qd (7.2, 1.7) 45.0 19, 20-Me 20-Me, 21 18b, 19, 22-OMe, 22 21 99.7 22 3.04, d (9.6) 81.7 23 22-OMe, 23 19, 20, 20/24-Me, 22-OMe, 25 23 3.91, d (9.6) 76.8 22, 24-Me 22, 23, 24-Me 22-OMe, 24-Me, 25 24 82.4 25 3.63, dd (9.6, 1.9) 73.8 26a, b 24 1ʹʹ, 22, 23, 27a, b 26 a 1.58e, m 25.9 25, 27 b 1.22g, m 25, 27 27 a 1.50, m 27.9 26a, 26b, 27b, 28 b 1.30, m 26a, 26b, 27a, 28 28 3.27a, m 74.0 27a, 27b 28-OMe 2-Me 2.08, br s 13.1 3, 5 1, 2, 3, 4 3, 4-OMe 6-Me 2.06, d (0.9) 13.8 7 5, 7 4-OMe, 5, 8, 9, 10-Me 8-Me 1.13f, d (6.5) 18.5 8 7, 8, 9 7, 8, 9 10-Me 1.57e, d (1.1) 12.0 11 9, 10, 11 1ʹ, 6-Me, 8, 9, 13 12-Me 1.88, br s 18.1 11, 13 11, 12, 13 11 14-Me 1.72, br s 17.8 13, 15, 16 13, 14, 15 16, 17, 17-OMe 20-Me 1.12f, d (7.2) 9.2 20 19, 20, 21 22-OMe 24-Me 1.13f, s 10.7 23 23, 24, 25 1ʹʹ, 22-OMe, 23 4-OMe 3.51, s 61.4d 4 2-Me, 3, 5, 6-Me 17-OMe 3.34, s 57.6 20-Me 17 14-Me, 16, 17 22-OMe 3.53, s 61.3d 22 20, 20-Me, 22, 23, 24-Me 28-OMe 3.24, s 58.6 28 1ʹ 4.64, d (3.8) 95.8 2ʹ 5ʹ, 9 2ʹ, 9, 10-Me, 11 2ʹ 3.39, dd (9.8, 3.8) 73.4 1ʹ, 3ʹ 1ʹ, 4ʹ-OMe, 4ʹ 3ʹ 3.75b, dd (9.8, 9.3) 74.8 2ʹ, 4ʹ 4ʹ 4ʹ, 4ʹ-OMe, 6ʹ-Me, 8-Me 4ʹ 2.72, dd (9.3, 9.3) 87.6 3ʹ/5ʹ 3ʹ, 4ʹ-OMe, 5ʹ, 6ʹ-Me 3ʹ, 4ʹ-OMe 5ʹ 3.73b, dq (9.3, 6.1) 68.1 4ʹ, 6ʹ 1ʹ, 4ʹ 4ʹ 6ʹ 1.26g, d (6.1) 18.0 5ʹ 4ʹ, 5ʹ 4ʹ, 4ʹ-OMe 4ʹ-OMe 3.58, s 61.0 4ʹ 2ʹ, 3ʹ, 4ʹ, 6ʹ 1ʹʹ 5.08, dd (9.5, 1.8) 92.6 2ʹʹa, 2ʹʹb 3ʹʹ 2ʹʹa, 5ʹʹ, 24-Me, 25, 28 2ʹʹ a 1.93c, m 38.9 1ʹʹ, 2ʹʹb, 3ʹʹ 3ʹʹ b 1.72d, m 1ʹʹ, 2ʹʹa, 3ʹʹ 3ʹʹ 4.24, ddd (2.9, 2.9, 2.9) 68.3 2ʹʹa, 2ʹʹb, 4ʹʹ 2ʹʹa, b 4ʹʹ 3.28a, m 83.3 3ʹʹ, 5ʹʹ 1ʹʹʹ 1ʹʹʹ, 6ʹʹ 5ʹʹ 3.89, dq (9.5, 6.3) 69.4 4ʹʹ, 6ʹʹ 1ʹʹ, 1ʹʹʹ 6ʹʹ 1.24g, d (6.3) 18.4 5ʹʹ 4ʹʹ
O
OMe
O
OH
OMe
O
OHOMeO
O
OH
MeOOH
OMe
1
1'
5'
2120
19
25
22
28
6
916
OH
O O OOH
OH
OHO1"
4"
1"' 5"'23
45
78
1011
1213
1415 17
18
13
Continued,
# δH, mult, (J in Hz) δC COSY 1H – 13C HMBC ROESY
1ʹʹʹ 4.69, dd (9.8, 1.9) 101.9 2ʹʹʹa, b 2ʹʹʹ, 4ʹʹʹ 2ʹʹʹa, 3ʹʹʹ-Me, 5ʹʹ, 5ʹʹʹ 2ʹʹʹ a 1.93c, m 46.7 1ʹʹʹ, 2ʹʹʹa, 3ʹʹʹ 1ʹʹʹ, 3ʹʹʹ-Me, 4ʹʹʹ 1ʹʹʹ, 4ʹʹʹ b 1.70d, m 1ʹʹʹ, 2ʹʹʹb, 3ʹʹʹ 1ʹʹʹ, 3ʹʹʹ-Me, 4ʹʹʹ 3ʹʹʹ 72.3h 4ʹʹʹ 3.09, d (9.5) 80.1 3ʹʹʹ/5ʹʹʹ 1ʹʹʹ, 3ʹʹʹ/5ʹʹʹ, 6ʹʹʹ 2ʹʹʹb, 3ʹʹʹ/6ʹʹʹ 5ʹʹʹ 3.37, dq (9.5, 6.1) 72.5h 4ʹʹʹ, 6ʹʹʹ 3ʹʹʹ-Me 1ʹʹʹ, 3ʹʹʹ/6ʹʹʹ 6ʹʹʹ 1.26g, d (6.1) 19.5 5ʹʹʹ 4ʹʹʹ, 5ʹʹʹ 4ʹʹʹ 3ʹʹʹ-Me 1.23g, s 18.5 2ʹʹʹ, 3ʹʹʹ, 4ʹʹʹ 1ʹʹʹ
(a − g) overlapping resonances within the same letter. (h - i) assignments are interchangeable within the same letter
14
Table S3. 1D and 2D NMR (600 MHz, MeOH-d4) data for amycolatopsin C (3)
# δH, mult, (J in Hz) δC COSY 1H – 13C HMBC ROESY
1 171.3 2 126.2 3 7.01, q (1.1) 137.6 2-Me 1, 2, 2-Me, 5 5, 13, 15 4 152.8 5 5.75, s 131.5 2-Me, 7 3, 4, 6-CH2, 7 3, 7 6 135.6 7 5.51c, br d (10.0) 143.9 5, 8, 10-Me 5, 9, 19 5, 9 8 2.93, m 37.0 7, 8-Me, 9 8-Me, 9 6a, b 9 3.81, d (8.9) 87.8e 8, 11 1ʹ, 7, 8, 8-Me, 10-Me, 11 7, 11 10 134.5 11 5.94, s 136.8 9, 10-Me, 12-Me, 13 9, 10-Me, 12-Me 1ʹ, 9, 12-Me 12 134.3 13 5.60, s 133.8 11, 12-Me, 14-Me, 15, 16 12-Me, 15 3, 10-Me, 14-Me, 15 14 136.4 15 5.22, br d (7.8) 129.6 13, 14-Me, 16 13, 14-Me, 16 3, 12-Me, 13, 17 16 4.81, br d (7.8) 67.9 13, 15, 17 14, 15, 17, 18 14-Me, 17, 17-OMe 17 2.66, d (8.2) 83.0 16, 18a, 18b 16, 17-OMe, 19 15, 16, 19 18 a 1.91, m 36.0 17, 18b, 19 16, 19 b 1.63a, m 17, 18a, 19 16, 19 17, 20 19 5.52c, d (10.5) 72.7 18a, 18b, 20 7, 17, 18, 20, 20-Me, 21 19 20 2.03, dd (6.9, 1.5) 45.1 19a, 19b 19, 20-Me, 21, 22 21 100.4 22-OMe 22 3.01, d (9.6) 82.4 23 20-Me, 22-OMe, 23 23 3.78, d (9.6) 78.4 22 22, 25 24 83.4 25 3.55, m 75.4 26a, 26b 23, 28 26 a 1.64a, m 25.8 26b, 27a, 27b 28 b 1.27b, m 26a, 27a, 27b 28 27 a 1.52, m 28.0 26a, b, 27b, 28 28 b 1.34, m 26a, b, 27a, 28 28 28 3.29, m 73.9 27a, b 25, 26, 27, 28-OMe 2-Me 2.10, s 13.3 3, 5 1, 2, 4 6-CH2OH a 4.49, d (12.1) 58.1 6b 5, 6 8 b 4.39, d (12.1) 6a 5, 6 8-Me 1.18, d (6.5) 19.1 8 7, 8, 9 1ʹ, 13 10-Me 1.60, s 12.6 7, 11 9, 11 11, 15 12-Me 1.89, s 18.4f 11, 13 11, 13 13, 16 14-Me 1.73, s 17.7 13, 15 15 20-Me 1.12, d (7.5) 9.3 20 19, 20 24-Me 1.04, s 13.9 4-OMe 3.60, s 61.6g 4 17-OMe 3.34, s 57.7 17 16 22-OMe 3.54, s 61.6g 22 22 28-OMe 3.24, s 58.8 28 1ʹ 4.63, d (3.8) 96.0 2ʹ 3ʹ, 5ʹ, 9 2ʹ, 10-Me, 11 2ʹ 3.39, dd (9.7, 3.8) 73.8 1ʹ, 3ʹ 3ʹ 1ʹ 3ʹ 3.74d, dd (9.7, 9.2) 75.1 2ʹ, 4ʹ 1ʹ 4ʹ 2.72, dd (9.2, 9.2) 87.7e 3ʹ/5ʹ 4ʹ-OMe, 5ʹ, 6ʹ 4ʹ-OMe 5ʹ 3.73d, m 68.5 4ʹ 1ʹ, 4ʹ 6ʹ 1.26b, m 18.5f 4ʹ 4ʹ-OMe 3.58, s 61.2g 4ʹ 4ʹ
(a − d) overlapping resonances within the same letter. (e − g) assignments are interchangeable within the same letter
O
OMe
O
OH
OMe
O
OHOMeO
O
OH
MeOOH
OMe
1
1'
5'
2120
19
25
22
28
6
916
OH
OH2
34
5
78
1011
1213
1415 17
18
OH
15
Figure S4. 1H NMR (600 MHz, MeOH-d4) spectrum of amycolatopsin A (1)
Figure S5. UV-vis (HPLC-DAD, H2O/MeCN plus HCO2H) spectrum of amycolatopsin A (1)
O
OMe
O
OH
OMe
OOH
OHOMeO
O
OH
MeOOH
OMe
1
1'
5'
2120
19
25
22
28
6
9 16
OH
O O OOH
OH
OHO1"
4"
1"' 5"'23
45
78
1011
1213
1415 17
18
1.01.52.02.53.03.54.04.55.05.56.06.57.0 ppm6.84
2.21
17.13
0.98
2.19
2.61
5.03
2.58
3.48
0.83
2.56
1.05
1.82
0.70
0.68
1.65
2.43
1.99
2.62
1.69
8.56
2.65
0.90
2.61
0.93
2.65
1.78
0.91
0.86
0.88
1.69
0.87
1.84
0.92
1.77
0.92
0.94
0.95
0.87
1H NMR spectrum (600 MHz) of MST.108494.LA5.121-39/40 in MeOH-d4
5.25.45.65.86.0 ppm
4.44.64.85.0 ppm
3.63.73.83.9 ppm
3.13.23.33.4 ppm
1.82.0 ppm
3
115
1319 7 15 1’’
161’’’ 1’ 6a 6b 3’’
23 5’’ 9 3’ 5’ 254-OMe
4’-OMe 22-OMe
2’, 5’’’
17-OMe4’’, 28
28-OMe
4’’’ 22
8
4’
17
2-Me
20
2’’’a,2’’a18a 12-Me
14-Me2’’’b2’’b
18b
10-Me
26a
27a27b
6’’6’6’’’
3’’’-Me26b
8-Me
20-Me24-Me
!2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 5 5 0
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0
16
Figure S6. 1H NMR (600 MHz, MeOH-d4) spectrum of amycolatopsin B (2)
Figure S7. UV-vis (HPLC-DAD, H2O/MeCN plus HCO2H) spectrum of amycolatopsin B (2)
O
OMe
O
OH
OMe
O
OHOMeO
O
OH
MeOOH
OMe
1
1'
5'
2120
19
25
22
28
6
916
OH
O O OOH
OH
OHO1"
4"
1"' 5"'23
45
78
1011
1213
1415 17
18
1.52.02.53.03.54.04.55.05.56.06.57.0 ppm
9.03
18.05
13.10
7.03
8.02
1.00
1.12
1.03
1.07
1.16
3.06
2.99
3.09
2.06
3.15
4.01
4.01
1.02
2.09
1.01
2.10
1.28
0.96
0.97
1.11
0.75
1.22
1.05
1.09
1.06
1.05
1.05
1.06
1.05
5.25.45.65.8 ppm
4.64.74.8 ppm
3.63.73.83.94.0 ppm
3.13.23.33.4 ppm
1.61.82.0 ppm
3
11 5 1319 7 15 1’’
161’’’ 1’
3’’
235’’
9 3’ 5’ 254-OMe
4’-OMe22-OMe
2’ 5’’
17-OMe 4’’28 4’’’
22
8
28-OMe
4’17
2-Me 6-Me
20
2’’a2’’’a18a
12-Me 14-Me
2’’b2’’’b
18b 26a27a
6’’, 6’, 6’’’3’’’-Me
26b
20-Me24-Me8-Me
1.28 1.22 1.16 1.10
20-Me24-Me8-Me
6’’, 6’, 6’’’3’’’-Me
26b
!
!2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 5 5 0
0
1 0 0
2 0 0
3 0 0
4 0 0
17
Figure S8. 1H NMR (600 MHz, MeOH-d4) spectrum of amycolatopsin C (3)
Figure S9. UV-vis (HPLC-DAD, H2O/MeCN plus HCO2H) spectrum of amycolatopsin C (3)
O
OMe
O
OH
OMe
O
OHOMeO
O
OH
MeOOH
OMe
1
1'
5'
2120
19
25
22
28
6
916
OH
OH2
34
5
78
1011
1213
1415 17
18
OH
1.01.52.02.53.03.54.04.55.05.56.06.57.0 ppm3.31
4.16
3.72
7.84
1.62
2.31
8.14
5.67
6.91
1.09
4.24
1.40
1.54
1.37
1.17
4.28
2.66
3.48
3.07
5.37
4.21
3.55
5.67
1.62
1.54
2.00
2.08
1.81
3.20
1.47
1.38
1.51
1.00
5.45.65.8 ppm
4.44.54.64.74.8 ppm
3.63.73.8 ppm
3.33.4 ppm
2.83.0 ppm
1.21.41.61.82.0 ppm
3
115
1319 7 15
161’ 6a 6b
9 23 3’ 5’
4-OMe4’-OMe 22-OMe 2’
17-OMe
28
28-OMe
22
8
4’ 17
2-Me
20 18a
12-Me 14-Me 10-Me
18b26a 27a 27b
6’
26b 8-Me20-Me
24-Me
!
!2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 5 5 0
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
1 2 0 0
1 4 0 0
18
Figure S10. 1H NMR (600 MHz, MeOH-d4) spectra comparison for amycolatopsin A – C (1 – 3). The red arrows are pointing to missing resonances in 3 compared to 1 and 2.
[rel
]-
0.0
0.2
0
.4
0.6
0
.8
MST_108494_AL26_16_17_19 1 1 /Volumes/Group-Capon/Instrument/Data/Bruker/data/z.khalil_600/nmr
amycolatopsin A (1)
amycolatopsin B (2)
amycolatopsin C (3)
3
311
513
19 7 15 1’’ 16 1’’’1’
3’’23
5’’9
3’5’
25
eM
O-’4 eM
O-22e
MO-4
2’5’’’
eM
O-71
4’’28
28-OMe
4’’’22
8
4’17
2-Me6-Me
20
18a2’’a2’’’a
12-Me 14-Me
2’’b2’’’b
18b
10-Me
26a27a27b
3’’’-Me
6’’6’’’26b
8-Me20-Me24-Me
115
1319 7 15 1’’ 161’’’ 1’
6a 6b 3’’ 235’’9
3’5’
25
4’-OMe
eM
O-4
eM
O-22
2’5’’’
eM
O-71
4’’28
28-OMe
4’’’
22
8
4’17
2-Me
20
18a2’’a2’’’a
12-Me14-Me
2’’b2’’’b
18b
10-Me
26a27a 27b
x
3’’’-Me
6’’6’’’26b
eM-8
20-Me24-Me
3 115
1319 7 15 161’ 6a 6b 9, 23, 3’, 5’
25
eM
O-4
4’-OMe
eM
O-22
2’
eM
O-71
28
28-OMe
228
4’ 17
2-Me
2018a
12-Me14-Me
26a18b
10-Me
27a 27b
6’26b
eM-8
eM-02
24-Me
19
2 Biological Data for 1 -3
2.1 Anti-microbial assays
Figure S11. Anti-microbial assay data for amycolatopsins A - C (1 - 3)
2.2 Cytotoxicity assay
Figure S12. Cytotoxicity assay data for amycolatopsins A - C (1 - 3) against (a) NCIH-460 (human lung cancer cell line) IC50 1.2 µM for (1), 0.28 µM for (2), 5.88 µM for (3) and (b) SW620 (human colon cancer cell line) IC50 0.08 µM for (1), 0.14 µM for (2) and 10 µM for (3)
0.001 0.01 0.1 1 10 1000.0
0.1
0.2
0.3
0.4
0.5
conc [µM]
OD
600
nm
B. subtilis ATCC 6633
123
0.001 0.01 0.1 1 10 1000.0
0.1
0.2
0.3
0.4
0.5
E. coli ATCC 25922
conc [µM]
OD
600
nm
1
2
3
0.001 0.01 0.1 1 10 1000.0
0.2
0.4
0.6
0.8
S. aureus ATCC 25923
conc [µM]
OD
600
nm
1
23
0.001 0.01 0.1 1 10 1000
1
2
3
4
C. albicans ATCC 90028
conc [µM]
OD
600
nm
123
0.001 0.01 0.1 1 10 1000.0
0.1
0.2
0.3
0.4
0.5
P. aeruginosa ATCC 27853
conc [µM]
OD
600
nm
123
0.01 0.1 1 10 1000.0
0.1
0.2
0.3
conc [µM]
OD
600
nm
1
2
3
Vinblastine
DMSO
(a)
0.0
0.1
0.2
0.3
0.01 0.1 1 10 100conc [µM]
OD
600
nm
1
2
3
Vinblastine
DMSO
(b)
20
2.3 Anti-mycobacterial assay
Figure S13. Growth inhibition of M. tuberculosis (H37Rv) and M. bovis (BCG) by amycolatopsins A - C (1 - 3) against (a) Mtb H37Rv IC50 4.4 µM for (1), >30 µM for (2), 5.74 µM for (3) and (b) BCG IC50 0.44 µM for (1), >30 µM for (2) and 2.74 µM for (3). Isoniazid (INH; 20 µg/mL) and DMSO served as controls.
3 References 1. Jukes, T. H.; Cantor, C. R.; New York: Academic Press.1969, 21. 2. Edgar, R. C.; BMC Bioinformatics.2004, 5, 113. 3. Okonechikov, K.; Golosova, O.; Fursov, M.; Bioinformatics.2012, 28, 1166.
0.01 0.1 1 10 1000.00
0.05
0.10
0.15
0.20
conc [µM]
OD
600
nm
123INHDMSO
(a)
0.01 0.1 1 10 1000.0
0.1
0.2
0.3
conc [µM]
OD
600
nm
1
2
3INH
DMSO
(b)