Rapid, Small-scale Dereplication of Bioactive Extracts John Blunt - - PowerPoint PPT Presentation
Rapid, Small-scale Dereplication of Bioactive Extracts John Blunt University of Canterbury New Zealand 1 Bioactive Discovery ~ 145,000 known natural products Probability for rediscovery very high Efficient dereplication processes required
John Blunt University of Canterbury New Zealand
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~ 145,000 known natural products Probability for rediscovery very high Efficient dereplication processes required is differentiating extracts that contain novel metabolites from those with known natural products
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0.5 – 2 mg extract
4 mL agar slope Petri dish
Bioassay and HPLC/UV/MS/NMR evaluation
100 mg sponge
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~0.6 mg crude extract separated
DAD & ELSD detection Eluant collected in microtitre plate 1 mL/min, 250 µL/well Daughter plate submitted to bioassay to locate bioactive components (Master plate submitted for automated ES-MS analysis of each well) 4
Separation of 600 µg fungal extract (F8095) using acetonitrile/methanol gradient Jackson Lin Sun
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F8095.3G94M
m/z 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 % 100 SLJ0273 223 (2.516) Cm (198:223) 1: TOF MS ES+ 3.17e3
407.5368 281.3798 253.4369 402.5803 282.3919 791.9871 408.5374 408.7168 770.0074 409.5392 793.0001 793.9875 814.0095
M+Na HPLC peak F8095-1 8
Search against ~44,000 unique compounds in AntiMarin for those with M+Na = 407
AntiMarin – combination of compound data from MarinLit (~20,000 marine natural products) and AntiBase (~33,000 microbial natural products) All compounds coded for 1H NMR-recognisable features
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ppm 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
solvent 3 x doublet methyls
1H CapNMR spectrum of
15 µg of F8095-1 CD3OD, 2 min, presat 11a
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Only 1 compound found - details match with F8095-1
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Now examine contents of microtitre plate well containing F8095-2
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F8095-4G191M
m/z 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 % 100 SLJ0291 120 (2.405) Cm (118:120) 1: TOF MS ES- 106
192.8424 386.6739 193.6537 306.7098 238.8127 238.7910 238.7622 306.6853 239.7081 261.7245 308.7327 308.7737 309.7589 328.6413 386.6923 386.7290 387.6937 387.8132 580.5323 422.6362 588.2189
M-H-
+ve ion MS of F8095-2 not useful, but –ve ion MS suggests M-H = 193
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1H CapNMR spectrum of
6 µg of F8095-2 CD3OD, 2 min, presat
Recognise 1 doublet methyl signal, and 2 aromatic singlets probably indicating 1,2,3,5-tetrasubstitution pattern
ppm 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
solvent 1 x doublet methyl 2 x aromatic singlet H 16
Search in AntiMarin for 1 doublet CH3, 1,2,3,5-substituted benzene, and Mr = 194 One hit only – data matches that for F8095-2 17
Now examine contents of microtitre plate well containing F8095-718
F8095.9G100M
m/z 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 % 100 SLJ0279 225 (2.545) Cm (210:225) 1: TOF MS ES+ 862
669.8434 647.8329 253.4294 219.3950 157.2350 217.3993 337.5758 279.4392 391.6849 465.7622 669.8797 669.9039 670.8597 671.9132 685.8550 864.0764 686.8834
M+Na
+ve ion MS of F8095-7 shows M+Na = 669 Search in AntiMarin gives 19 hits
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1H CapNMR spectrum of
13 µg of F8095-7 CD3OD, 2 min, presat
Recognise 5 doublet methyl signals
ppm 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
solvent 5 x doublet methyls 20
Search in AntiMarin for 5 doublet CH3s, and M+Na = 669 One hit only – data matches that for F8095-7
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7 compounds from F8095 identified by HPLC-microtitre-MS-NMR/database method 15 µg 6 µg 16 µg 8 µg 12 µg 12 µg 12 µg
O O O O O H O O H O H OH O O OH H O O O O O O O O O O O H HO O H OH O H H O O H O O O O O O O O O O O H HO HO O H O O O O O O O O O O O H HO HO OH O H O O O O O O O O O O H O OH O H HO O H O O O O O O O O O O H HO O H O H O H H O OH
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time c
t
The pathway to bioactive compound identification
complete structure/dereplication sample extract bioassay scale-up & extraction bioassay-guided fractionation pure compound ~1 mg spectroscopic analysis HPLC-Bioassay-UV-MS ~5-50 µg HPLC-bioassay-(UV)-(MS)-NMR ~500 µg crude extract database searching dereplication spectroscopic analysis 2-50 µg complete structure
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Acknowledgements
Development of the concept and techniques for the use of HPLC-microtitre plate-capillary NMR: John Blunt & Murray Munro (UC) Kirk Gustafson (MTDP, NCI, Frederick MD) Development of the concept of and construction of databases for use in dereplication: John Blunt & Murray Munro (UC) Hartmut Laatsch (University of Göttingen) Preparation of samples for demonstration of techniques: Gill Ellis, Sultan Sadia, Jackson Lin Sun
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