Rapid Dereplication using Capillary NMR and a Database of Structures

1. Rapid Dereplication using Capillary NMR and a Database of Structures John Blunt University of Canterbury, NZ (A presentation given at the Gordon Research Conference on Marine Natural Products in Ventura, CA, February 2006. Several of the concepts contained herein had been presented earlier at other conferences and institutions during 2003-2005.)

2. 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 Goettingen) Preparation of samples for demonstration of techniques: Gill Ellis, Gerhard Lang, Jackson Sun Lin, Maya Mitova Richard Phipps & Sonia van der Sar (UC)

3. Dereplication Determining which of the bioactive components in an extract are known compounds, or are likely to be new and therefore worthy of further attention.

4. Microtitre HPLC Analysis Dionex HPLC Foxy Jr DAD ELSD • Active extracts analysed by HPLC • HPLC eluant collected into microtitre plate • Daughter plates assayed for activity • Obtain UV and Mass spectral data from the bioactive region(s) of the microtitre plate

5. C18 HPLC of extract from marine-derived Cephalosporium sp. DAD detection Bioactivity profile Provides retention time, UV and molecular weight of the active compound

6. With possible taxonomy, UV and molecular weight of the active compound(s) known, databases can be consulted to establish if the bioactive is unique or known. • MarinLit- for 16,303compounds originating from marine organisms. (Blunt/Munro; UniversityofCanterbury) • AntiBase- for 29,253compounds of microbial origin. (Laatsch; University of Goettingen/Wiley) • AntiMarin-a combination of the two databases (43,324 compounds). (Blunt/Munro/Laatsch)

7. AntiMarin Search for MW=518 and UV 410-419 and 290-299

8. Only1 hit

9. Problem – not much UV data in AntiBase • Solution– use 1H NMR data • All structures in AntiMarin are coded with the numbers of each structural feature that could be deduced from 1H NMR spectra • eg #s of CH3 of different types (s,d,t)

11. Problem: Preparing an HPLC/microtitre plate from 200 – 500 mg extract will provide 2 – 40 mg compound/peak spread over 1, 2 or 3 wells. How to obtain meaningful 1Hspectra? Solution: Use a Protasis CapNMR probe. Sample can be taken up in 6 mL solvent, introduced into probe, and spectral acquisition commenced within 2 minutes.

12. Protasis CapNMR Probe

13. HPLC with ELSD detection of 600 mg extract of unidentified endophytic fungus E9,E10,E11 G4 250 mL/well

14. Recognisable features: 5 CH3 groups, of which 1 methoxyl, 3 singlet CH3-C, 1 doublet CH3-C; 1 aldehyde 500 MHz 8 mg in 6 mL CD3OD PRESAT1.5 min

15. AntiMarin search for all compounds containing 1 aldehyde and 5 methyl groups, of which 1 is methoxyl, 3 are singlet CH3-C, and 1 is doublet CH3-C

16. only 1 hit

17. E9,E10,E11 G4 250 mL/well

18. 500 MHz 5 mg in 6 mL CD3OD 1.0 min Spectrum very similar to that of auranticin B except no aldehyde peak. MW 2 amu more than for auranticin B. New peak at 4.8 ppm.

19. 11 hits with search in AntiMarin for 5 CH3, 3 singlet CH3-C, 1 doublet CH3-C, 1 methoxyl and 1 sp3-methylene. Only 1 hit if MW = 440 included in search.

20. Bioactivity profile HPLC with ELSD detection of 400 mg extract of unidentified endophytic fungus F2,F3 E9,E10 F12

21. Recognisable features: 4 CH3 groups, of which 1 methoxyl and 3 singlet CH3-C; 1 aldehyde 500 MHz 17 mg in 6 mL CD3OD 16 sec

22. 1 hit only if MW = 346 included in search

23. HPLC with ELSD detection of 400 mg extract of unidentified endophytic fungus F2,F3 E9,E10 F12

24. Recognisable features: 3 CH3 groups, of which 1 methoxyl and 2 singlet CH3-C; 1 aldehyde. MW = 332 500 MHz 2 mg in 6 mL CD3OD PRESAT 2 min

25. only 1 hit

26. HPLC with ELSD detection of 400 mg extract of unidentified endophytic fungus F2,F3 E9,E10 F12

27. 500 MHz 14 mg in 6 mL CD3OD PRESAT 1 min Spectrum similar to that of phomosine A, but no aldehyde, and MW 2 amu more than for phomosine A. (New compound – CH2 presumably at 4.9 ppm, lost in solvent presat)

28. F2,F3,F4 HPLC with ELSD detection of 500 mg extract of unidentified endophytic fungus H11

29. 500 MHz 30 mg in 6 mL CD3OD 2 min MW 684 – unknown peptide

30. TOCSY 40 min

31. Bioactive region HPLC with UV detection of 250 mg extract of endophytic fungus E1,E2,E3 ELSD detection

32. 2 x 1,2,3-trisubstituted benzenes COSY 8 min 1H 500 MHz NMR spectrum, 30 s ~20 mg from wells E1-E3 in 6 mL CD3OD, 24 sec

33. AntiMarin search with MW = 320, 0 CH3, 2 x 1,2,3-trisubstituted benzenes

34. Only 1 hit (Not this compound, but gives clue as to structural type)

35. Other search strategies 2 x 1,2,3-trisubstituted benzenes 198 hits plus 0 x CH3 89 plus 0 x sp3-methylene 35 plus 1 x sp3-methine 3 (none of these fit data, but all contain 1,8-dioxonaphthalene fragment)

36. gHSQC 45 min gHMBC 11 hr

37. Sonia van der Sar, John W Blunt & Murray H G Munro Org Lett 2006 in press

38. ELSD detection D4,D5 well D5, ~3 mg, 8 min

39. COSY 25 min

40. Summary Rapid dereplication of bioactives : • 250-500 mg bioactive extract separated by • analytical HPLC into microtitre plate wells • In-well bioassay to locate active components • UV and MS data obtained from active wells • 1H NMR spectra obtained from active • components (> 2 mg) (CapNMR) • Recognisable structural features • searched for in AntiMarin or MarinLit • If compound unknown, collect 2D NMR data • for structure determination

41. Note–all masses of samples given in this presentation are the masses of compound in the CapNMR microcell – these are ~65% of the amounts originally in the microtitre plate well(s) from which they were taken. The masses have been estimated from a calibration of the CHD2 solvent peak integral in a solution of a known compound of known concentration.