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Rapid, Small-scale Dereplication of Bioactive Extracts John Blunt University of Canterbury New Zealand 1.

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Presentation on theme: "Rapid, Small-scale Dereplication of Bioactive Extracts John Blunt University of Canterbury New Zealand 1."— Presentation transcript:

1 Rapid, Small-scale Dereplication of Bioactive Extracts John Blunt University of Canterbury New Zealand 1

2 ~ 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 Dereplication Bioactive Discovery 2abc

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

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

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

6 6

7 7

8 M+Na HPLC peak F

9 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 1 H NMR-recognisable features 9 ab

10 10

11 solvent 3 x doublet methyls 1 H CapNMR spectrum of 15  g of F CD 3 OD, 2 min, presat 11a

12 12

13 Only 1 compound found - details match with F a

14 Now examine contents of microtitre plate well containing F

15 M-H - +ve ion MS of F not useful, but –ve ion MS suggests M-H =

16 1 H CapNMR spectrum of 6  g of F CD 3 OD, 2 min, presat Recognise 1 doublet methyl signal, and 2 aromatic singlets probably indicating 1,2,3,5-tetrasubstitution pattern solvent 1 x doublet methyl 2 x aromatic singlet H 16

17 Search in AntiMarin for 1 doublet CH 3, 1,2,3,5-substituted benzene, and Mr = 194 One hit only – data matches that for F

18 Now examine contents of microtitre plate well containing F

19 +ve ion MS of F shows M+Na = 669 Search in AntiMarin gives 19 hits 19a

20 1 H CapNMR spectrum of 13  g of F CD 3 OD, 2 min, presat Recognise 5 doublet methyl signals solvent 5 x doublet methyls 20

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

22 7 compounds from F8095 identified by HPLC-microtitre-MS-NMR/database method 15  g 6  g 16  g 8  g 12  g 22

23 time costcost 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  g HPLC-bioassay-(UV)-(MS)-NMR ~500  g crude extract database searching dereplication spectroscopic analysis 2-50  g complete structure J. Nat. Prod., 2008, 71, abcdef

24 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


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