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

~ 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

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

~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

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

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M+Na HPLC peak F

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

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

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Only 1 compound found - details match with F a

Now examine contents of microtitre plate well containing F

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

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

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

Now examine contents of microtitre plate well containing F

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

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

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

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

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

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