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Enzymes in Organic Media

Enzymes in Organic Media. Tahir Rana University of Ottawa September 25th 2008. 1. 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. 2.

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Enzymes in Organic Media

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

  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 2

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

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

  5. Structure And Function Catalytic Scheme 5

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

  7. Applications of Enzymes Examples: Asymmetric Aldol DERA – Deoxyribose Aldolase 7 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.

  8. Applications of Enzymes Industrial Examples 8 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.

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

  10. Applications of Enzymes Amide Hydrolysis 10

  11. Limitations of Aqueous Enzymology • Solubility of non-polar substrates • Polymerization of phenols 11 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. Limitations Thermal Inactivation in Aqueous Media • Irreversible: • Molecular Aggregation • Deamidation • Reversible: • Changes in higher order structure 12 Klibanov, A.; Ahern, T. Methods of Biochemical Analysis, 1988, 33, 91-128.

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

  14. Overcoming Limitations The Solution – Organic Solvents • Increased solubility of non-polar substrates 14 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.

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

  16. Overcoming Limitations Opportunity for Synthesis 16

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

  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 18

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

  43. Total Synthesis of Fredericamycin A Synthesis of DEF Ring System- 2 R=CO2Me R=CO2Me 43 Kita, Y. et al. European J. of Chem. 2005. 11, 6286-6297.

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

  45. Total Synthesis of Fredericamycin A Synthesis of DEF Ring System - 2 R=CO2Me 45 Kita, Y. et al. European J. of Chem. 2005. 11, 6286-6297.

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

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

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

  49. Total Synthesis of Fredericamycin A 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 % 49

  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 50

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