1. 1 1 Enzymes in Organic Media Tahir Rana University of Ottawa September 25th 2008

2. 2 2 Outline  Structure and Function  Applications of Enzymes  Limitations of Enzymes in Aqueous Media  Concerns  Applications of Enzymes in Organic Media  Total Synthesis of Fredericamycin A

3. 3 3 What are Enzymes ?  Enzymes are proteins  Enzymes catalyze reactions Structure And Function

4. 4 4 Enzyme Structure  Primary Structure – order of amino acids  Secondary Structure - α-helix, β-sheet  Tertiary Structure - arrangement in 3D  Quaternary Structure- interaction of subunits Sakuraba, H. et al. J. Biol. Chem. 2003, 361, 278, 10799-10806. Structure And Function

5. 5 5 Catalytic Scheme Structure And Function

6. 6 6 Factors Involved in Enzymatic Catalysis  Increase in local concentration  Positioning and enhancement of active site functional groups  Specificity  Introduction of strain into substrate Structure And Function

7. 7 7 Examples: Asymmetric Aldol Wong, C,H.; Gilsen, H. J. Am. Chem Soc. 1994, 164, 8422-8423. Wong, C.H. Liu, J. J. Angewantde Chemie. 2001, 114, 1462-1465. Applications of Enzymes DERA – Deoxyribose Aldolase

8. 8 8 Industrial Examples Anderson, B. et al. J. Am. Chem. Soc. 1995, 117, 12358-12359. Liese, A.; Seelbach, K.; Wandrey, C. Industrial Biotransformations. Wiley-VCH, 2005, 117-121. Applications of Enzymes

9. 9 9 Industrial Examples Ricks, E.; Estrada-Valdes, M; Iacobucci, G. Biotech. Prog. 1992. 8, 197-203. Liese, A.; Seelbach, K.; Wandrey, C. Industrial Biotransformations, Wiley-VCH. 2005. Applications of Enzymes

10. 10 Amide Hydrolysis Applications of Enzymes

11. 11 Limitations of Aqueous Enzymology  Solubility of non-polar substrates  Polymerization of phenols Bruno, F.; Ayyagari, S.; Akkara, J. Trends in Biotechnology. 1999, 17, 67-73. Reihmann, M.; Ritter, H. Syn. Of Pol. Using Peroxidases. Adv. Poly. Sci. Springer-Verlag. 2006, 194, 1-49.

12. 12 Thermal Inactivation in Aqueous Media  Reversible:  Changes in higher order structure  Irreversible:  Molecular Aggregation  Deamidation Klibanov, A.; Ahern, T. Methods of Biochemical Analysis, 1988, 33, 91-128. Limitations

13. 13 Domination of Hydrolysis  Water is in excess  Cannot use other nucleophiles Limitations

14. 14 The Solution – Organic Solvents  Increased solubility of non-polar substrates Bruno, F.; Ayyagari, S.; Akkara, J. Trends in Biotechnology. 1999, 17, 67-73. Reihmann, M.; Ritter, H. Syn. Of Pol. Using Peroxidases. Adv. Poly. Sci. Springer-Verlag. 2006, 194, 1-49. Overcoming Limitations

15. 15 Suppression of Thermal Inactivation in Organic Sol. % Activity of Lipase at 100 °C Klibanov, A.; Zaks, A. Science. 1984, 224, 1249-1251. Overcoming Limitations

16. 16 Opportunity for Synthesis Overcoming Limitations

17. 17 Recap - Advantages of Organic Solvents  Increased solubility of non-polar substrates  Suppression of Thermal Inactivation  Opportunity for synthesis Overcoming Limitations

18. 18 Outline  Structure and Function  Applications of Enzymes  Limitations of Aqueous Enzymology  Concerns Regarding Enzymes in Organic Solvents  Applications of Enzymes in Organic Media  Total Synthesis of Fredericamycin A

19. 19 Concerns  Structural Integrity  Mechanistic Integrity  Diminished Activity

20. 20 Structural Integrity % Alpha Helix Content of Subtilisin Klibanov, A.; Griebenow, K. J. Am. Chem. Soc. 1996, 118, 11965-119700. Concerns Addressed

21. 21 Structure of Subtilisin in Water and Acetonitrile C  Backbone Trace Active Site (Asp-32,His-64,Ser-221) Heavy lines = MeCN Light lines = water Klibanov, A. et al. Proc. Nat. Acad. Sciences. 1993, 90, 8653-8657. Concerns Addressed

22. 22 Mechanism of Transesterification Chaterjee, S.; Russell, A. Enzyme Microb. Technol. 1993, 15, 1022-1029. Concerns Addressed

23. 23 Mechanism of Transesterification Chaterjee, S.; Russell, A. Enzyme Microb. Technol. 1993, 15, 1022-1029. Concerns Addressed

24. 24 Mechanistic Integrity Ping Pong Mechanism Transesterification in Organic SolventsEster Hydrolysis in Water Conclusion: Mechanism is the same (1)Chaterjee, S.; Russell, A. Enzyme Microb. Technol. 1993, 15, 1022-1029. (2)Klibanov, A. Trends Biochem. Sci. 1989, 14, 141-144. Concerns Addressed

25. 25 Diminished Activity  Enzymes have reduced activity in dry organic solvents Due to lack of:  a) conformational mobility  b) transition state stabilization  c) entropy Klibanov, A. Trends In Biotech. 1997. 15, 97-101. Concerns Addressed

26. 26 Effect of Water on Activity  Activity can be recovered Klibanov, A. J. Biol. Chem. 1987. 263, 8017-8021. Enzyme Activity as a Function of Water Content Concerns Addressed

27. 27 Concerns Addressed  Structurally intact  Act by the same mechanism  Activity can be recovered

28. 28 Applications (1)Wong, C-H.; Koeller, K. Nature. 2001. 409, 232-241 (2)Klibanov, A.; Kirchner, G.; Scollar, P. J. Am. Chem. Soc. 1985. 107, 2072-2076. Problem: Max Conversion = 50 % Applications in Org. Media

29. 29 Resolution: Meso Diols Kim, M.J.; Lee, S. Synlett. 1993. 767-768. Applications in Org. Media

30. 30 60 % Overall Yield Kim, M.J.; Lee, S. Synlett. 1993. 767-768. Applications in Org. Media

31. 31 Applications: Desymmetrization  Loss of one or more symmetry elements  Potential for 100 % conversion Gotor, V. et al. Organic Letters. 2007. 9, 4203-4206. Applications in Org. Media

32. 32 Applications: Total Synthesis of Epoxyquinols A and B Applications in Org. Media

33. 33 Retrosynthesis Mehta, G.; Islam, K. Tett. Lett. 2004. 45, 3611-3615. Applications in Org. Media

34. 34 Desymmetrization Step Applications in Org. Media Mehta, G.; Islam, K. Tett. Lett. 2004. 45, 3611-3615.

35. 35 Outline  Structure and Function  Applications of Enzymes  Limitations of Aqueous Enzymology  Concerns  Applications of Enzymes in Organic Media  Total Synthesis of Fredericamycin A

36. 36 Total Synthesis of Fredericamycin A  Isolated from Streptomyceus griseus  Antitumor activity  7 Total Syntheses; 5 Racemic, 2 Asymmetric

37. 37 Retrosynthesis of 1 st Asymmetric Synthesis Kita. Y. et al. J. Am. Chem. Soc. 2001. 123, 3214-3222. Total Synthesis of Fredericamycin A

38. 38 Installation of Spiro Center Fredericamycin A 33 Steps 0.075 % Overall Yield FED Kita. Y. et al. J. Am. Chem. Soc. 2001. 123, 3214-3222. Total Synthesis of Fredericamycin A

39. 39 Retrosynthesis of 2 nd Asymmetric Synthesis Kita, Y. et al. European J. of Chem. 2005. 11, 6286-6297. FED C B A Total Synthesis of Fredericamycin A

40. 40 Synthesis of DEF Ring System Total Synthesis of Fredericamycin A Clive, D. J. of Heterocyclic Chemistry. 1987, 9, 804-807.

41. 41 Synthesis of DEF Ring System Total Synthesis of Fredericamycin A Kita, Y. et al. European J. of Chem. 2005. 11, 6286-6297.

42. 42 Synthesis of DEF Ring System  Difficult separation of acyl hydrazone  4.5 % overall yield (from pyridone)  Approach abandoned Total Synthesis of Fredericamycin A Kita, Y. et al. European J. of Chem. 2005. 11, 6286-6297.

43. 43 Synthesis of DEF Ring System- 2 R=CO 2 Me Total Synthesis of Fredericamycin A R=CO 2 Me Kita, Y. et al. European J. of Chem. 2005. 11, 6286-6297.

44. 44 Solution ?  Use the synthetic ability of enzymes in organic solvents Total Synthesis of Fredericamycin A

45. 45 Synthesis of DEF Ring System - 2 R=CO 2 Me Total Synthesis of Fredericamycin A Kita, Y. et al. European J. of Chem. 2005. 11, 6286-6297.

46. 46 Total Synthesis – Fredericamycin A  30 % Yield (from pyridone) Total Synthesis of Fredericamycin A Kita, Y. et al. European J. of Chem. 2005. 11, 6286-6297.

47. 47 Total Synthesis – Fredericamycin A B C DEF A Total Synthesis of Fredericamycin A Kita, Y. et al. European J. of Chem. 2005. 11, 6286-6297.

48. 48 Total Synthesis – Fredericamycin A 28 Linear Steps 0.75 % Overall Yield Total Synthesis of Fredericamycin A Kita, Y. et al. European J. of Chem. 2005. 11, 6286-6297.

49. 49 Comparison of Syntheses  Lewis Acid: 4 steps to establish chirality at spiro center  Enzymatic: 1 step to establish chirality at spiro center  Enzymatic: 28 yield steps, 0.75 % yield, Lewis Acid: 33 steps, 0.075 % Total Synthesis of Fredericamycin A

50. 50 Summary  Enzymes are valuable tools for organic synthesis  Enzymes can be used in organic solvents  There are clear advantages to using enzymes in organic media  Application to the total synthesis of Fredericamycin A

51. 51 Acknowledgements Dr. Robert Ben Taz Cheema Pawel Czechura Liz von Moos John Trant Jennifer Chaytor Sandra Ferreira Wendy Campbell Ruoying Gong Roger Tam Jackie Tokarew Taline Boghossian Dr. Michael Souweha Dr. Mathieu Leclere

52. 52 Enzyme Preparations  Enzymes are insoluble in organic solvents  Enzyme powders  Suspension of enzymes in bulk solvent or on solid supports  Covalent modifications, e.g. PEG; surfactants

53. 53 pH Memory Affect  Rate of transesterification with pH adjusted subtilisin 75x that of bottled enzyme  Enzymatic activity in organic solvent depends upon pH of the last aqueous solution enzyme was exposed to.

54. 54 Structural Integrity of Enzymes Explanation ?  Enzymes possess reduced mobility in pure organic media Evidence:  Structurally rigid e.g. decrease in motion of lipase Tyr 123 acetonitrile than in water Conclusion:  Denaturation is thermodynamically favourable, yet conformational flexibility is lacking Ref. Burke + Klibanov

55. 55 Total Synthesis of Fredericamycin A  5 Racemic 1. Kelly 1986 2. Clive 1992 3. Rao 1993 4. Julia 1993 5. Boger 1995  2 Asymmetric 1. Kita 2001 2. Kita 2005

56. 56 Reversal of Chemoselectivity Ref. Ebert Conditions Ratio of Products A B Benzene, t Amyl Alcohol 71 Pyridine 81 tAmyl Alcohol w/lipase 101 Pyridine w/ lipase 110 Benzene w/ lipase 31 Benzene (2 % pyridine) w/ lipase 31

57. 57 Reversal of Regioselectivity ConditionsRatio of Products AB Acetonitrile w/ KCN21 Toluene w/ lipase and n BuOH 21 Acetonitrile w/ lipase and n BuOH 12 Ref.

58. 58 Reversal of Regioselectivity