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% à40% 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