Secondary Metabolite from Marine Sponge-derived

Fungus Aspergillus nomius

By Muh Ade Artasasta

Introduction

Marine Sponge

Source of Diversity
Secondary Metabolites
Introduction

Needed in large scale of biomass

Slow-growing Microbes Derived from Sponge

Shelter for other marine biota

Introduction

Neopetrosia chaliniformis
Aspergillus nomius

Endemic sponge found along

Indonesia coastline
(Thiele dkk., 1989)
Introduction
33
H 25

Tacsonomy
2 0 37
1 6 2 5 O
O H 42
29 1 2
6 1 5 H 20 N
27
2 4 22 12
3 1 1 7 2 3 O 23
O H
Kingdom : fungi
17
5 9 36
2 2 34
O 6 13 14

Division : Ascomycota
3 0 31
1 4 29 28
2 40 18 OH
4
3 2 5 9 39 26

Class : Eurotiomycetes
1 8 38
N 3 0
O
2 6 16
3 0 H 1 2 4
1 0 41

Order : Eurotiales 7 19 19
N 30
O 35 H 1

Family : Trichocomaceae
21 21 10
8 7
O 27 24
O

Genus : Aspergillus
11 13 8

11
O 15
32

Species : A. nomius
28
33

14-hydroxypaspaline 14(N,N-dimethyl-L-valyloxy)paspaline
27
20

Aspergillus nomius 28 19 9
12
18 13 18
14 2
21

13 7 0
17
12 3 2 1 11
OH 23 14
22 26
19
1 23 4
20
Aspernomine has cytotoxic 24
11
8 17 16
28 8 3
21
OH
22
activity against A549, MCF7, 29 5 0 7
27
and HT29 with IC50 of 3.09, 10 16
29 6

4.93, and 3.08 μg/mL, 6 10

30
O 15 N
9 24 H 26

respectively
15
N H 31
5
25 H
Aspernomine
4
Nomine 25

(Gloer and Rinderknecht, 1989; Staub et al., 1993).

 Isolation Method
Crude EtoAc Extract of
A. nomius (10.1334 g)

VLC (n-hexane;EtoAC;DCM;MeOH)

F. I (0.1553 g) F. II (0.8569 g) F. III (1.9442g) F. IV (6.1576 g) F. V (0.3172 g)

VLC (n-hexane;EtoAC;DCM;MeOH)

F3V1 F3V2 F3V3 F3V4 F3V5 F3V6 F3V7 F3V8 F3V9 F3V10 F3V11 F3V12

1. AR-01 12 mg
 Isolation Method
F3V7 (78.1 mg)

Sephadex column

F3V7S1 15.8 mg F3V7S2 11 mg F3V7S3 21.1 mg F3V7S4 40 mg F3V7S5 3.2 mg

Semiprep

2. ANF3V7S4P5 (AR-02) 5.23 mg

3. ANF3V7S4P4 (AR-03) 8.25 mg
 Isolation Method
F3V8 (447.8 mg)

Sephadex column

F3V8S1 12.4 mg F3V8S3 48 mg F3V8S6 62.3 mg F3V8S7 55.7 mg F3V8S8 7.2 mg

Semiprep
Semiprep
4. ANF3V8S3P3 (AR-04) 7.72 mg
5. ANF3V8S3P4 (AR-05) 3.13 mg

6 ANF3V9S4P3 (AR-06) 12 mg
 Isolation Method
F3V9 (151.85mg)

Sephadex column

F3V9S1 53.7 mg F3V9S2 19 mg F3V9S3 41.1 mg F3V9S4 45.7 mg F3V9S6 17.1 mg

Semiprep

7. ANF3V9S4P3 (AR-07) 3.67 mg

 Isolation Method
FIV (6.15 g)

VLC (n-hexane;EtoAC;DCM;MeoH)

F4V1 6.9 mg F4V2 82 mg F4V5 9.1 mg F4V7 116.3 mg F4V9 660.5 mg

F4V9Si1 6.9 mg F4V9Si2 6.9 mg F4V9Si3 6.9 mg

Semiprep

8. ANF4V9Si2P2 (AR-08) 52 mg
Compound 1. AR-01 (12 mg)

Unclear

Peak #1 100% at 2.71 min

600 AR181106 #2 AR1 UV_VIS_1 70,0
mAU WVL:235 nm % Library Hit: Eudesmic acid 976.26
1 - 2,710

214.3
400

268.8
200

-10,0 nm
-150 min
200 250 300 350 400 450 500 550 595
0,0 10,0 20,0 30,0 40,0 50,0 60,0
Compound 2 AR-02 (5.23 mg)

Unclear?

Peak #1 100% at 23.15 min

600 AR181119 #4 ANF3V7S4-P5 UV_VIS_1
70,0
mAU WVL:235 nm % Library Hit: Sterigmatocystin 956.36
1 - 23,150

241.5
400

200
313.9

min -10,0 nm
-150
0,0 10,0 20,0 30,0 40,0 50,0 60,0 200 250 300 350 400 450 500 550 595
Compound 3 AR-03 (8.25 mg)

Mightbe
344?

900 AR181119 #3 ANF3V7S4-P4 UV_VIS_1

70,0
Peak #1 100% at 22.36 min
mAU WVL:235 nm % Library Hit: Spongiacidin E 954.52
1 - 22,360

365.1
363.0
368.3

500

-200 min -10,0 nm

0,0 10,0 20,0 30,0 40,0 50,0 60,0 200 250 300 350 400 450 500 550 595
Compound 4 AR-04 (7.71 mg)

Compound 4 AR-04 (7.71 mg)

m/z =370

Peak #1 100% at 26.72 min

1.400 AR181129 #4 ANF3V8S3P3 UV_VIS_1 70,0
mAU WVL:235 nm % Library Hit: Norlichexanthone 973.95
1 - 26,723

237.4

203.1

500 313.9

min -10,0 nm
-200
0,0 10,0 20,0 30,0 40,0 50,0 60,0 200 250 300 350 400 450 500 550 595
NMR Table of AR-04

No 1H (δ, ί, m, j) 13C COSY HMBC

1 6.43 (1H, s) 91.0 - 107.4 163.4
2 - 165.0 - - O O O 3 ' O
3 - 108.3 - -
5 4 4 '
4 - 163.9 - - 3
2 '
9 1 3
5 - 161.4 - - 6 1 1
O
6 6.92 (1H, d, 8.23) 107.2 7.61 108.8, 112.8 1 '
1 0 2
7
7 7.61 (1H, d, 8.34) 135.4 6.91, 7.00 106.2 1 2
O
8 7.00 (1H, d, 8.28) 109.8 7.61 106.1, 112.8, 157.1 8 O
1

9 - 177.3 - -
10 - 156.7 - -
11 - 112.8 - -
12 - 165.0 - -
13
4-OCH3
-
3.91 (3H, s)
108.4
56.5
-
-
-
163.0
O O O O
5-OCH3 3.95 (3H, s) 56.4 - 160.2
1’ 6.51 (1H, d, 5.94) 114.9 4.19 42.3, 164.1
2’ 4.19 (1H, t, 6.72) 43.6 2.38, 6.51 36.8, 106.2, 164.1
O
3’ 2.38 (2H, m) 38.0 4.19, 5.23 42.3, 106.2, 113.7
4’ 5.24 (1H, d, 4.68) 107.7 2.38 36.8, 106.2, 164.1
O O
4’-OCH3 3.18 (3H, s) 55.0 - 106.2
Compound 5 AR-05 (3.13 mg)

Waiting for NOESY

result

m/z =370

Peak #1 100% at 28.09 min

250 AR181129 #5 ANF3V8S3P4 UV_VIS_1 70,0
mAU WVL:235 nm % Library Hit: Norlichexanthone 968.43
1 - 28,090

237.4
125
202.9

312.0
0

min -10,0 nm
-150
0,0 10,0 20,0 30,0 40,0 50,0 60,0 200 250 300 350 400 450 500 550 595
Compound 6 AR-06 (12 mg)

1.200 AR190114 #4 ANF3V8S6P3 UV_VIS_1

mAU WVL:235 nm

1 - 23,060
500

-200 min
0,0 10,0 20,0 30,0 40,0 50,0 60,0

Peak #1 100% at 23.06 min

70,0
% Library Hit: Kealiinine A 959.89

241.9

313.9

-10,0 nm
200 250 300 350 400 450 500 550 595
Compound 7 AR-06 (3.67 mg)

1.200 AR190108 #4 ANF3V9S4P UV_VIS_1

mAU WVL:235 nm

1 - 25,173
500

-200 min
0,0 10,0 20,0 30,0 40,0 50,0 60,0

Peak #1 100% at 25.17 min

70,0
% Library Hit: Norlichexanthone 989.68

242.2

205.0 317.8

-10,0 nm
200 250 300 350 400 450 500 550 595
Compound 8 AR-08 (52 mg)

3.000 AR190130 #3 ANF4V9S4Si2P2 UV_VIS_1

mAU WVL:235 nm

1 - 26,340
2.000

1.000

-200 min
0,0 10,0 20,0 30,0 40,0 50,0 60,0

Peak #1 100% at 26.34 min

70,0
% Library Hit: Cyclo(L-Tyr-L-Pro) 984.38

230.7
215.4

275.3

-10,0 nm
200 250 300 350 400 450 500 550 595
Summary
 Compound 1 (AR-01) need further analysis, such us HMBC?
 Compound 2 and 3 (AR-02 and AR-03) need resubmit to MS?
 Compound 4 (AR-04) need HRMS analysis
 Compound 6 (AR-06) need MS analysis
 Compound 7 (AR-07) need MS analysis
 Compound 8 (AR-08) need further analysis such us MS, and 2D?