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 Dereplication is differentiating extracts that contain novel metabolites from those with known natural products 2a b c

Scale of Dereplication Exercise 4 mL agar slope Petri dish 100 mg sponge 0.5 – 2 mg extract Bioassay and HPLC/UV/MS/NMR evaluation 3a

Eluant collected in microtitre plate 1 mL/min, 250 µ L/well Daughter plate submitted to bioassay to locate bioactive components ~0.6 mg crude extract separated (Master plate submitted for automated on analytical C18 column ES-MS analysis of each well) DAD & ELSD detection 4

Separation of 600 µ g fungal extract (F8095) using acetonitrile/methanol gradient Jackson Lin Sun 5

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F8095.3G94M SLJ0273 223 (2.516) Cm (198:223) 1: TOF MS ES+ 407.5368 3.17e3 100 HPLC peak F8095-1 M+Na 791.9871 % 793.0001 408.5374 281.3798 793.9875 408.7168 402.5803 282.3919 814.0095 770.0074 253.4369 409.5392 0 m/z 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 8

AntiMarin – combination of compound data from MarinLit (~20,000 marine natural products) and AntiBase (~33,000 microbial natural products) All compounds coded for 1 H NMR-recognisable features Search against ~44,000 unique compounds in AntiMarin for those with M+Na = 407 9 a b

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3 x doublet methyls 1 H CapNMR spectrum of solvent 15 µ g of F8095-1 CD 3 OD, 2 min, presat 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm 11a

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Only 1 compound found - details match with F8095-1 13 a

Now examine contents of microtitre plate well containing F8095-2 14

F8095-4G191M SLJ0291 120 (2.405) Cm (118:120) 1: TOF MS ES- 192.8424 106 100 386.6739 386.6923 % M-H - 386.7290 193.6537 387.6937 306.7098 238.8127 387.8132 308.7327 238.7910 306.6853 308.7737 238.7622 15 239.7081 309.7589 261.7245 580.5323 328.6413 422.6362 588.2189 0 m/z 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 +ve ion MS of F8095-2 not useful, but –ve ion MS suggests M-H = 193

1 H CapNMR spectrum of 6 µ g of F8095-2 1 x doublet methyl CD 3 OD, 2 min, presat 2 x aromatic singlet H solvent 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 ppm Recognise 1 doublet methyl signal, and 2 aromatic singlets probably indicating 1,2,3,5-tetrasubstitution pattern 16

Search in AntiMarin for 1 doublet CH 3 , 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 F8095-7 18

F8095.9G100M SLJ0279 225 (2.545) Cm (210:225) 1: TOF MS ES+ 862 669.8434 100 669.8797 669.9039 M+Na Search in AntiMarin gives 19 hits % 670.8597 647.8329 253.4294 671.9132 685.8550 219.3950 157.2350 337.5758 391.6849 864.0764 217.3993 686.8834 279.4392 465.7622 0 m/z 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 +ve ion MS of F8095-7 shows M+Na = 669 19a

5 x doublet methyls 1 H CapNMR spectrum of 13 µ g of F8095-7 CD 3 OD, 2 min, presat solvent 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm Recognise 5 doublet methyl signals 20

Search in AntiMarin for 5 doublet CH 3 s, and M+Na = 669 One hit only – data matches that for F8095-7 21

OH O H O O O H O H O HO O O H O O OH O O O H O O O 15 µ g O OH O 16 µ g 6 µ g O H O O O O H H O O H O H O H OH O H O O O O O O O H O O O O O HO O O O H O O O HO HO O O O H O O O O O O O O O O H O O OH 8 µ g HO O H H O H O O H O O OH 12 µ g 12 µ g O O O O HO O O O O O H HO 12 µ g 7 compounds from F8095 identified by HPLC-microtitre-MS-NMR/database method 22

The pathway to bioactive compound identification c o J. Nat. Prod., 2008, 71 , 1595-99. s t complete structure/dereplication spectroscopic analysis pure compound ~1 mg complete structure bioassay-guided fractionation spectroscopic analysis 2-50 µ g scale-up & extraction database searching dereplication HPLC-bioassay-(UV)-(MS)-NMR ~500 µ g crude extract HPLC-Bioassay-UV-MS ~5-50 µ g bioassay 23a c d e f b extract sample time

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 24