1. 1

2. Abyssomicin C John Trant Department of Chemistry University of Ottawa, 2007

3. Abyssomicin C—From isolation to mechanism of action 3 An introduction to the tetrahydrofolate biosynthetic pathway The isolation identification of Abyssomicin C A brief retrosynthetic overview of Abyssomicin C K.C. Nicolaou’s synthesis: The application of Lewis- acid catalysed self-assembling (LACASA) Diels- Alder reaction The mechanism of action

4. Folic Acid: An Introduction 4  Vitamin B9  Needed for the catalysis of one-carbon transfer reactions including dTMP from dUMP, and in the catalysis of glycine synthesis.  Not synthesised in vertebrates, but synthesised in plants, archaea, fungi, bacteria, and some lower animals.

5. p-Aminobenzoic acid/Tetrahydrofolate pathway 5 Trimethoprim Sulfa Drugs

6. 6 Kozlowski, M.C et al. J. Am. Chem. Soc. 1995. 117 2128-2140.

7. p-Aminobenzoic acid/Tetrahydrofolate pathway 7 Abyssomicin C

8. The Abyssomicins 8 Verrucosispora AB-18-032 Riedlinger, J. et al. J. Antibiotics. 2004. 57, 271-279. Bister, D. et al. Angew. Chem. Intl. Ed. 2004. 43, 2574-2576.

9. Abyssomicin C as a Chorismate mimic 9 Copley, A.D.; Knowles, J.R. J. Am. Chem. Soc. 1987. 109, 5008-5013. Chorismate

10. Abyssomicin C 10

11. 11 A Short Retrosynthetic Overview Snider/Sorenson/Couladaros Approach to the initial disconnection Snider, B.B.; Zou, Y. Org. Lett. 2005. 7, 4939-4941. Zapf, C.W.; Harrison, B.A.; Drahl, C.; Sorenson, E.J. Angew. Chem. Int. Ed. 2005. 44, 6533-6537. Couladouros, E.A.; Bouzas, E.A.; Magos, A.D. Tet. Lett. 2005. 62, 5272-5279.

12. 12 A Short Retrosynthetic Overview Sorenson’s Retrosynthesis of 1 Snider’s Retrosynthesis of 1

13. A Short Retrosynthetic Overview 13 Rath, J.; Kinast, S.; Maier, M.E. Org. Lett. 2005. 7, 3089-3092. Zografos, A.L.; Yiotakis, A.; Georgiadis, D. Org. Lett. 2005. 7, 4512-4518. Maier’s and Georgiadis’ Retrosynthesis of Abyssomicin C

14. Nicolaou’s Retrosynthesis 14 Nicolaou, K.C.; Harrison, S.T. Angew. Chem. Int. Ed. 2006. 45, 3256-3260. Nicolaou, K.C.; Harrison, S.T. J. Am. Chem. Soc. 2007. 129, 429-440.

15. 15

16. But... 16 Ward, D. E.; Abaee, M.S. Org.Lett. 2000. 2, 3937-3940. 120º C

17. 17 Examples from literature Ward, D. E.; Abaee, M.S.Org.Lett. 2000. 2, 3937-3940.

18. Ward’s Solution 18 Ward, D.E.; Abaee, M.S. Org. Lett. 2000. 2, 3937-3940.

19. An Early Attempt at Olefination 19 Nicolaou, K.C.; Harrison, S.T. J. Am. Chem. Soc. 2007. 129, 429-440.

20. A Modified Julia-Olefination Strategy 20

21. 21 Ward, D.E.; Souweha, M. S. Org. Lett. 2005. 7, 3533-3536.

22. The Disadvantages 22 Stoichiometric amount of enantiopure BINOL and ZnMe 2 in the first step of the synthesis. Synthesis had become lengthy (7 steps, 38% yield).

23. Asymmetric Borane Reduction 23

24. 30%!!!! 24 Use of “sacrificial” alcohol resulted in no increase in yield. Lewis Acid scan produced no increase of the yield (TiCl 4, AlCl 3, Zn(OTf) 2, MgBr 2 OEt 2 /i-Pr 2 NEt). But...Remember that the enantioselective Diels-Alder Reaction was faster than the racemic version...

25. Bidentate Ligands 25 6

26. The Proposed Transition State 26

27. Julia-Type Reduction 27 [O]

28. 28

29. Synthesis of The Coupling Partner 29

30. 30 Various homodimerised and polymerised by-products

31. 31 Covers blue

32. A New Approach 32

33. 33

34. 34 5.6% overall yield, 19 steps from Weinreb Amide The NMR did not match that of the previously isolated natural compound. After 18 hours in CDCl 3 a new set of peaks appeared. The new peaks matched those of the previously isolated abyssomicin C.

35. Generation of Possible Atropisomers 35

36. 36 Nicolaou and Harrison J. Am. Chem. Soc. 2006, 129, 430-440.

37. The Differences 37 Nicolaou and Harrison J. Am. Chem. Soc. 2006, 129, 430-440.

38. Nicolaou’s Proposed Mechanism 38

39. 39 Biosynthetic Ramifications 39 Figure modified from Nicolaou et Harrison J. Am. Chem. Soc 2007. 129, 429-440.

40. Biosynthetic Ramifications 40 Abyssomicin C Figure modified from Nicolaou et Harrison J. Am. Chem. Soc. 2007. 129, 429-440. % Starting Material Remaining Time (h) Atrop-abyssomicin C

41. Minimum Inhibitory Concentrations for the analogues 41

42. Proposed Mechanism of Action 42 Figure adapted from Parsons, J. F. et. al. Biochem. 2002. 41, 2198-2208.

43. Narrowing the Search 43

44. Identifying the nucleophile 44 Keller, H. et. al. Angew. Chem. Intl. Ed. 2007. 46, 8284-8286.

45. 45

46. Minimum Inhibitory Concentrations for the analogues 46

47. In Conclusion 47 Examined the Folate Biosynthesis pathway. Examined Nicolaou’s application and modification to Ward’s LACASA approach to Diels-Alder Reactions using an allylic alcohol diene. Delved into Nicolaou’s Approach for the total synthesis of Abyssomicin C. Demonstrated how Nicolaou’s synthetic work uncovered the potent inhibitor, atrop-abyssomicin C, leading to a better understanding of the abyssomicin mechanism of action.

48. Acknowledgements 48 Roger Tam Pawel Czechura Jennifer Chaytor Elisabeth Von Moos Tahir Rana Wendy Campbell Sandra Ferreira Ruoying “Gloria” Gong Jaqueline Tokarew Ivan Petrov Dr. Michael Souweha Dr. Matthieu Leclere Dr. Robert Ben And NSERC for providing funding to make this possible

49. Couladouros’ Retrosynthesis of 2 49

50. Synthesis of Building Block 5 50 Takeda et al. J. Org. Chem. 1987, 52, 4135-4137

51. Synthesis of 4 51

52. Putting The Pieces Together 52

53. Putting The Pieces Together Take 2 53

54. 54

55. Synthesis of Vinyl Iodide 3 55

56. Isomerisation Conditions 56

57. Isolation and Identification 57 Isolated in 2004 by Süssmuth from an actinomycete of genus Verrucosipora, discovered in a sediment sample from the Japanese Sea. Investigated because of pABA inhibition identified by an agar-plate diffusion assay Structure determination by NMR and X-Ray

58. Whoopdeedoo 58 Folic acid, Vitamin B12 is an essential vitamin. Not synthesised in vertebrates Fungi, Bacteria, Plants, Achaea, and insects and anthropods all synthesise it. So it has the possibility of providing broad spectrum antibiotics. Sulfonamides and Trimethoprim are existing antibiotics that inhibit the Folate Synthesis Pathway

59. Andrus’ Lactone 59

60. Agar Plate Diffusion Assay 60 Screened 930 extracts. B. subtilis in complex medium Lives B. subtilis in simple medium Lives No Activity Dies Metabolic Activity Dies General Toxin Riedlinger, J. et al. J. Antibiotics. 2004. 57, 271-279

61. B. Subtilis in simple medium pAba+Trp +Phe+Tyr pAbaTrpPhe+Tyr B. Subtilis in simple medium pAba+Trp +Phe+Tyr pAbaTrpPhe+Tyr B. Subtilis in simple medium pAba+Trp +Phe+Tyr pAbaTrpPhe+Tyr B. Subtilis in simple medium pAba+Trp +Phe+Tyr pAbaTrpPhe+Tyr 61 ibid Verrucosispora AB-18-032

62. Borane Transition State 62

63. A Shorter Route 63

64. 64 Kinetic Studies Irreversible Inhibition: K I app =390μM; k inact =0.8 min -1 Keller, H et. al. Angew. Chem. Intl. Ed. 2007. 46, 8284-8286

65. Ward’s DA evidence 65