Acremostrictin, a Highly Oxygenated Metabolite from the marine-derived fungus Acremonium strictum. Elin Julianti, PhD. Sekolah Farmasi ITB

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1 Acremostrictin, a ighly xygenated Metabolite from the marine-derived fungus Acremonium strictum Elin Julianti, PhD. Sekolah Farmasi ITB Introduction The oceans cover 70% of our planet s surface and as origin of the most primitive form of life Marine microorganisms Bacteria, actinomycetes and fungi Structurally unique and biologically active metabolites bligatory and facultative marine fungi The term marine-derived fungi 1

2 Distribution of Marine-Derived Fungi Fish Coral 1% 2% Grass/plant 1% Sediment Mollusc 3% 5% Tunicate 5% ther 5% Total Compounds Wood 13% Unknown 8% Alga 24% Sponge 33% Coral 2% Grass/plant 3% Unknown 5% Sediment 4% Mollusc 6% Tunicate 7% Fish 2% ther 6% Wood 10% ew Compounds Sponge 28% Alga 27% The distribution of all compounds reported from marine-derived fungi as a function of the fungal sources The distribution of new compounds reported from marine-derived fungi as a function of the fungal sources Bugni, T. S.; Ireland, C. M. atural product reports 2004, 21, ovel Bioactive Secondary Metabolites from Marine-Derived Fungi Chepalosporin C, Antimicrobial agents Source: Acremonium chrysogenum The first natural product of marine-derived fungi (1946) Z-, sclerotide A; E-, sclerotide B Antifungal activity against Candida albicans ( MIC = 7.0 and 3.5 M respectively) Source : Aspergillus sclerotorium Plinabulin (PI-2358) Phase II clinical trials for cancer Source: Aspergilus sp. CC 139 2

3 Methodology Upstream Process Sources Isolation of fungi Pure culture Bioactivity screening Small scale fermentation for screening purpose Chemical screening Acremonium strictum (MB05005) (IC ug/ml, inhibited diverse bacterial strains)) Large scale cultivation arvest Downstream process 3

4 Small scale fermentation 4

5 large scale fermentation large scale fermentation using fermentor 5

6 Downstream Process Extraction Fractionation Isolation Structure Elucidation Bioassay replc, 394 mg DS 10x250 mm, 55Aq/Me, 2 ml/min #3, 250 l, range = times injection

7 Isolation Scheme Acremonium strictum (MB05005) 120 flasks ml marine broth-glycerol media 28 ºC, 150 rpm, 3 weeks Mycelium Broth EtAc 700 mg Reversed-phase PLC (55% aq. Me) Reversed-phase PLC (85% aq. Me) Rp mg Rp Rp Rp Rp Rp Rp mg Acremostrictin Structure Elucidation of Compound 1 C 3 RFABMS m/z [M+] + (Calcd for C , ). C 3 C 3 C=C- C/C 2 1 -MR (500 Mz, Pyridine-d 5 ) spectrum of 1 #C = 13 C=C C/C 2 C 3 C= C= C=C 3 C- 13 C- MR (125 Mz, Pyridine-d 5 ) spectrum of 1 7

8 Structure Elucidation of Compound 1 RFABMS m/z [M+] + (Calcd for C , ), UI = 6. Key gmbc (red arrows) and 1-1 CSY (bold lines) correlations of 1 C 2 C 2 C 2 Five possible structures of 1 Structure Elucidation of Compound 1 Crystal structure of 1 by RTEP diagram S*, 6S*, 7S*, 8R*, and 9S* A packing pattern with -bonds of crystal structure of 1 Julianti, E.; h,.; Jang, K. ; Lee, J. K.; Lee, S. K; h, D.-C.; h, K.-B.; Shin, J. J. at. Prod. 2011, 74,

9 Bioassay: Antibacterial Activity Micrococcus luteus (IF 12708), MIC : 50 g/ml Salmonella typhimurium (ATCC 14028), MIC : 50 g/ml Proteus vulgaris (ATCC 3851), MIC : 12.5 g/ml Antioxidant Activity % of Control Concentration (mm) Antioxidant potential of 1 on DPP free radical : scavenging activity IC 50 of 2.1 mm Protective effects of 1 on 2 2 -induced cell death of acat human keratinocytes Phthalides and Diketopiperazines from the marine-derived fungus Acremonium strictum 9

10 Isolation Scheme Acremonium strictum (MB05005) 120 flasks ml marine broth-glycerol media 28 ºC, 150 rpm, 3 weeks Mycelium Broth EtAc 700 mg Reversed-phase PLC (55% aq. Me) Reversed-phase PLC (80% aq. Me) Rp mg Rp Rp Rp Rp mg Isolated Compounds 7-hydroxyphthalide (2), 7-methoxyphthalide (3), 4-methoxyphthalide (4), cyclo-(l-2-oxo-3shydroxy-tryptophyl-l-phenyl-alanyl), cyclo-(l-tryptophyl-l-leucyl) (6), cyclo-(l-tryptophyl-lphenylalanyl) and cyclo-(l-2,3s-dihydroxy-tryptophyl-l-phenylalanyl) (8). ew compounds : 5 and 7. 10

11 Structure Elucidation of Compound 5 Ar- -C- C- C- C- 1 -MR (500 Mz, Me-d 4 ) spectrum of 5 RFABmass : m/z [M+] + (Calcd for C , ) Ar-C C= C- C- C-C 13 C-MR (125 Mz, Me-d 4 ) spectrum of 5 Structure Elucidation of Compound 5 Key gmbc (red arrows) and 1-1 CSY (bold lines) correlations of 5 11

12 Structure Elucidation of Compound 5 Determination of Absolute Stereochemistry unit B unit A 5 1. Determination of relative stereochemistry by ESY Experiment. bserved E correlation between : -11/-14 = R/R or S/S 2. ydrolysis for determining the absolute configuration stereochemistry of phenylalanine residue (unit A) by Marfey s analysis method Structure Elucidation of Compound 5 Results of Marfey s Analysis L-Phe-L-FDAA RT =33.4 [M+] + = 418 [2M+a] + = 857 M = 417 D-Phe-L-FDAA RT =39.5 [M+] + = 418 [2M+a] + = 857 M = 417 ydrolysate of 5 RT =33.5 [M+] + = 418 [2M+a] + = 857 M = 417 Absolute configuration of Phenylalanine of 5 = L configuration 12

13 3. Determining the absolute stereochemistry of 3- of unit B. unit B = modified tryptophan (oxidation product) = dioxindolalanine C 2 C 2 C 2 2 L-Tryptophan (7) 3 (8a) (3S) (8b) (3R) 2 CD spectrum of Compound 5 in Me C3(S) = negative cotton effect : nm = positive cotton effect : C3(S) = negative cotton effect : nm = positive cotton effect : C3(R) = antipodal curve Takayama,.; Shimizu, T.; Sada,.; arada, Y.; Kitajima, M.; Aimi,. Tetrahedron 1999, 55, Bioassay : Cytotoxicity Inhibition Enzyme Activity Compound A549 cell-line LC 50 ( g/ml) a + /K + ATPase IC 50 ( g/ml) ICL IC 50 ( g/ml) Sortase A IC 50 ( g/ml) Actin depolymerization IC 50 (ug/ml) Doxorubicin 1.83 uabain P pmb > nd > >100 > >100 4 > >100 >100 > > >100 >100 6 >100 >100 >100 >100 > >100 >100 >100 >100 8 >100 >100 >100 >100 >100 13

14 Acremolin, a new 1-azirine Metabolite from the marine-derived fungus Acremonium strictum Julianti, E.; h,.; Lee,. S.; h, D.-C.; h, K.-B.; Shin, J. Tetrahedron Lett. 2012, 53, Isolation Scheme Acremonium strictum (MB05005) 10 Fernbach Flasks L YPM media 28 ºC, 150 rpm, 3 weeks Mycelium Broth Rp 1-17 Rp mg EtAc 620 mg Reversed-phase PLC (55% aq. Me) Rp ormal-phase PLC (98% EtAc/Me) mg Acremolin 14

15 Structure Elucidation of Compound 9 RFABmass : m/z [M+] + (Calcd. for C , ) -C 3 C- 2-C 3 =C- 1 -MR (400 Mz, DMS-d 6 ) spectrum of compound 9 2-C 3 #C = 11 =C C=C C- 13 C-MR (100 Mz, DMS-d 6 ) spectrum of compound 9 Structure Elucidation of Compound (8.10) (3.57) (7.37) (2.88) 22.0 (1.24) 22.0 (1.24) 9 Key gmbc (red arrows) and 1-1 CSY (bold lines) correlations of 9 15

16 Bioassay : Cytotoxicity LC 50 of g/ml against A549 cell line (doxorubicin exhibited an LC 50 of 1.83 g/ml as a positive control). Thank you 16