![AcAibAla AibAla Aib Gln AibVal Aib Gly LeuAib ProVal AibAib Glx Glx Fol
HR-ESI-MS
m/z 956.0242 [M+2Na]2+
Molecular formula:
C87H143N21O24
Aib = α-aminoisobutyric acid
O
N
H
H
N
N
H
H
N
N
H
O O O
O
O
N
H
O
O
N
N
H
O O
H
N
O
H
N
O
H
N
O
N
H
O
H
N
H2N O
H
N
HO
O
N
H
O
H
N
O
N
H
O
H
N
OHO
O
N
H
NH2O
Aib-3
Ala-2
Aib-1
Ala-4
Aib-5 Aib-7 Aib-9
Aib-12
Aib-15
Aib-16
Gln-6 Val-8
Val-14
Gly-10
Leu-11Pro-13Glu-17
Gln-18
Fol-19](https://image.slidesharecdn.com/4343ef44-3d58-4b47-b047-5c55b6ad427c-160613054805/85/Master_final-9-320.jpg)
![m/z 956 [M+2Na]2+
Molecular weight: 1867
2 Daughter ions
7751092
C-terminal
Pro Val Aib Aib Glu Gln Fol
624496367282197775
Ac Aib Ala Aib Ala Aib Gln Aib Val Aib Gly Leu Aib
440355284199 1007894837752653568 1092
_
N-terminal
MS/MS Fragmentation
AcAibAla AibAla Aib Gln AibVal Aib Gly LeuAib ProVal AibAib Glu Gln Fol](https://image.slidesharecdn.com/4343ef44-3d58-4b47-b047-5c55b6ad427c-160613054805/85/Master_final-10-320.jpg)
![Gly L-Glu L-Ala L-Pro Aib L-Val L-Fol L-Leu
R.T.(min)
Cosmosil MS-II [ϕ4.6 x 250mm]
UV: 340nm
Flow rate: 1.5 ml/min.
Linear gradient elution
MeCN/H2O (0.1%TFA)
10% à40% MeCN in 80 min.
D-Glu D-Pro D-Ala D-Val D-Fol D-Leu
40 50 60 70 80](https://image.slidesharecdn.com/4343ef44-3d58-4b47-b047-5c55b6ad427c-160613054805/85/Master_final-12-320.jpg)
![HR-ESI-MS
m/z 1097.6982 [M+H]+
Molecular formula:
C54H100N2O20
Erylusamine
Analogue
Fragment 1
N
H
N
O O
Fragment 3Fragment 2
Erylusamine B
N. Sata,et al., Tetrahedron, 1994, 50, 1105-1110.](https://image.slidesharecdn.com/4343ef44-3d58-4b47-b047-5c55b6ad427c-160613054805/85/Master_final-16-320.jpg)
Master_final
- 1. Ahmed Mohamed Natural Products Chemistry Search for anti-microbial substances against dormant mycobacterial bacilli from marine medicinal resources
- 2. SEM of Mycobacterium tuberculosis Surviving inside granuloma in dormant state (hypoxic condition) Cited from MedicineWorld Org. MTB Granuloma of Tuberculosis (Adapted from Johns Hopkins University) Challenges: • Infecting 32% of world population • Causing 2 million deaths annually. • Concurrent infection with HIV promotes its spread. • Worldwide emergence of Multi-Drug resistant strains. • Treatment requires protracted drug regimens (6-9 months).
- 3. Picture of low O2 chamber M. smegmatis MIC of isoniazid (µg/mL) Strain Aerobic Hypoxic 2.5 25 Isoniazid M. bovis BCG 0.03 >100 Pre-cultured M. smegmatis (1x104 cfu/ 0.1mL) or other strains (1x105 cfu/ 0.1mL) under aerobic or� hypoxic (0.2% O2) conditions 1) added serial diluted sample 2) incubated for 96 hr (for M. smegmatis) or 14 days (for other strains) under 0.2% O2 condition determination of MIC by MTT method incubated for 36 hr (for M. smegmatis) or 7 days (for other strains) under aerobic condition or M. tuberculosis H37Ra 0.05 >100
- 4. Isolation of anti-mycobacterial peptides from marine-derived fungi. Isolation of a glycolipid from a marine sponge.
- 5. Isolation of anti-mycobacterial peptides from marine-derived fungi. Isolation of a glycolipid from a marine sponge.
- 6. Strains 1 2 3 4 5 Aerobic Hypoxic Aerobic Hypoxic Aerobic Hypoxic Aerobic Hypoxic Aerobic Hypoxic M. smegmatis >200 >200 >200 >200 >200 >200 >200 >200 200 200 M. bovis BCG 25 50 12.5 12.5 12.5 12.5 25 50 3.1 3.1 MIC µg/mL Ac = acetyl, U = α-aminoisobutyric acid, Fol = phenylalaninol Compounds 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Alamethicin F50/5 (5) Ac U P U A U A Q U V U G L U P V U U Q Q Fol Trichokonin Va (1) Ac U A U A U -‐-‐ Q U V U G L U P V U U E Q Fol Trichokonin V (2) Ac U A U A U -‐-‐ Q U V U G L U P V U U Q Q Fol Longibranchin AI (3) Ac U A U A U A Q U V U G L U P V U U Q Q Fol Longibranchin BII (4) Ac U A U A U A Q U V U G L U P V U U E Q Fol From fungus KuF46 From fungus KuF61
- 7. Sephadex Column CH3Cl/MeOH 1:1 Fungus KuF46 Rice medium (250g) 30˚C, 1 week Solid Culture - Organic solvent extraction - 2-butatnone/H2O partition 2-butanone extract (4.5g) 200 200 >200 >200 SiO2 Open Column …A1 2.6g A4 550mg A5 320mg A6 192mg 50 50 B1 26mg B2 250mg B3 190mg A4 550mg 25 12.5 25 25 >200 >200 C2 13mg C3 38mg C4 70mg C1 31mg 50 25 50 25 12.5 12.5 12.5 12.5 Trichokonin V (2) Longibranchin AI (3) Longibranchin BII (4) Trichokonin Va(1) (New) HPLC PE column 45% aq.MeCN/0.1%TFA isocratic Hypoxic Aerobic Hypoxic Aerobic M.smeg BCG MIC µg/mL
- 8. Fungus KuF61 Rice medium (250g) 30˚C, 1 week Solid Culture -Organic solvent extraction -2-butatnone/H2O partition -n-hexane/90%MeOH partition Hypoxic Aerobic Hypoxic Aerobic M.smeg BCG MIC µg/mL 90%MeOH (8g) 6.25 6.25 12.5 12.5 A1 (2g) A3 (5g) A2 (390mg) 50 12.5 50 50 3.1 3.1 12.5 12.5 ODS MPLC A3 (5g)Sephadex LH-20 (CHCl3:MeOH 1:1) B1 (210) B2 (366) B3 (908) B4 (459) B5 (144) B6 (2660) (mg) 6.25 6.25 200 200 6.25 3.1 200 100 6.25 3.1 50 25 3.1 0.78 6.25 6.25 25 0.78 12.5 12.5 200 200 >200 >200 HPLC C8 column 85% aq.MeOH isocratic C1 (40) C2 (24.2) C3 (97.6) C4 (26.5) C5 (197) (mg) C6 (12.5) 1.5 0.02 0.78 0.78 12.5 1.5 25 25 3.1 3.1 200 200 3.1 1.5 200 200 12.5 12.5 >200 >200 200 100 >200 >200 Alamethicin F50/5 (5)
- 9. AcAibAla AibAla Aib Gln AibVal Aib Gly LeuAib ProVal AibAib Glx Glx Fol HR-ESI-MS m/z 956.0242 [M+2Na]2+ Molecular formula: C87H143N21O24 Aib = α-aminoisobutyric acid O N H H N N H H N N H O O O O O N H O O N N H O O H N O H N O H N O N H O H N H2N O H N HO O N H O H N O N H O H N OHO O N H NH2O Aib-3 Ala-2 Aib-1 Ala-4 Aib-5 Aib-7 Aib-9 Aib-12 Aib-15 Aib-16 Gln-6 Val-8 Val-14 Gly-10 Leu-11Pro-13Glu-17 Gln-18 Fol-19
- 10. m/z 956 [M+2Na]2+ Molecular weight: 1867 2 Daughter ions 7751092 C-terminal Pro Val Aib Aib Glu Gln Fol 624496367282197775 Ac Aib Ala Aib Ala Aib Gln Aib Val Aib Gly Leu Aib 440355284199 1007894837752653568 1092 _ N-terminal MS/MS Fragmentation AcAibAla AibAla Aib Gln AibVal Aib Gly LeuAib ProVal AibAib Glu Gln Fol
- 11. Y7P Y6 B11 B10 B9 B8B7 B6 B12 B5B4B3B2 DiagnoNc ions B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 MS Peaks N.D 199 284 355 440 568 653 752 837 894 1007 1092 Amino Acid Ac-‐Aib Ala Aib Ala Aib Gln Aib Val Aib Gly Leu Aib DiagnoNc ions Y7 Y6 Y5 Y4 Y3 Y2 MS Peaks 775 624 496 367 282 197 Amino Acid Fol Gln Glu Aib Aib Val-‐Pro Y5 Y4 Y3 Y2
- 12. Gly L-Glu L-Ala L-Pro Aib L-Val L-Fol L-Leu R.T.(min) Cosmosil MS-II [ϕ4.6 x 250mm] UV: 340nm Flow rate: 1.5 ml/min. Linear gradient elution MeCN/H2O (0.1%TFA) 10% à40% MeCN in 80 min. D-Glu D-Pro D-Ala D-Val D-Fol D-Leu 40 50 60 70 80
- 13. Compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Trichokonin Va (1) Ac U A U A U -‐-‐ Q U V U G L U P V U U E Q Fol Trichokonin V (2) Ac U A U A U -‐-‐ Q U V U G L U P V U U Q Q Fol Longibranchin AI (3) Ac U A U A U A Q U V U G L U P V U U Q Q Fol Longibranchin BII (4) Ac U A U A U A Q U V U G L U P V U U E Q Fol Alamethicin F50/5 (5) Ac U P U A U A Q U V U G L U P V U U Q Q Fol Ac = acetyl, U = α-aminoisobutyric acid, Fol = phenylalaninol Strains 1 2 3 4 5 Aerobic Hypoxic Aerobic Hypoxic Aerobic Hypoxic Aerobic Hypoxic Aerobic Hypoxic M. smegmatis >200 >200 >200 >200 >200 >200 >200 >200 200 200 M. bovis BCG 25 50 12.5 12.5 12.5 12.5 25 50 3.1 3.1 MIC µg/mL
- 14. Isolation of anti-mycobacterial peptides from marine-derived fungi. Isolation of a glycolipid from a marine sponge.
- 15. 90%MeOH (1.2g) Hexane (0.47g) Butanol (3.7g) H2O (10.2g) 090-99 Sponge (50g) MeOH Extract (16g) H2O phase (14g) EtOAc extract (1.5g) 50 50 100 100 >200 >200 >200 >200 >200 200 >200 >200 >200 >200 >200 >200 NH2 Silica Gel Open Column …B1 B5 (128mg) B8… Butanol (0.7g/3.7g) …C1 C16…C9 (8.8mg) 12.5 12.5 50 50 12.5 12.5 25 25 Compound 6 Hypoxic Aerobic Hypoxic Aerobic M.smeg BCG MIC µg/mL - Extracted with MeOH thrice HPLC C18 45% aq. MeCN/0.1%TFA EtOAc/H2O Partition Butanol/H2O Partition Hexane/90%MeOH Partition
- 16. HR-ESI-MS m/z 1097.6982 [M+H]+ Molecular formula: C54H100N2O20 Erylusamine Analogue Fragment 1 N H N O O Fragment 3Fragment 2 Erylusamine B N. Sata,et al., Tetrahedron, 1994, 50, 1105-1110.
- 17. 481353325157 Fragmentation pattern of erylusamine B 966 833 701 569 551 481Fragmentation pattern of compound 6 Not Detected 569 n+m = 8
- 18. Vicinal coupling determined by 1DTOCSY analysis of anomeric protons RingsA, C è Xylopyranose Rings B, D è Arabinopyranose
- 19. M. smegmatis MIC of 6 (µg/mL) Strains Aerobic Hypoxic 25 25 M. bovis BCG 12.5 12.5 1 2 Reduction of Ketone à Fragmentation pattern change? 1 Position of Ketone group 2 Diol Stereochemistry Relative Stereochemistry CD Spectroscopy Absolute Stereochemistry 22 23 n+m = 8
- 20. Isolation of one new peptaibol along together with four known peptaibols from marine-derived fungi, showing previously unreported anti-bacterial activity against Mycobacteria bovis BCG both aerobic and non-replicating dormant strains. Isolation and partial structure elucidation of erylusamine analogue, showing anti-mycobacterial activity. 1 2
- 21. FDAA reagent AminoAcid Derivatized amino acid Hydrolysate or Amino acid standard FDAA reagent (1 eq.) NaHCO3, 70˚C, 1hr Derivatized amino acid(s) HCl neutralization Dilution HPLC analysis Reaction Protocol