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Biomolecular Catalysis of Diels-Alder Reactions

Biomolecular Catalysis of Diels-Alder Reactions. Organic Seminar March 7 th , 2002 Lisa Jungbauer. Outline. Introduction The Diels-Alder Reaction Biomolecule Catalysts of Diels-Alder Reactions Catalytic Antibodies (Abzymes) Ribozymes (Catalytic RNA)

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Biomolecular Catalysis of Diels-Alder Reactions

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  1. Biomolecular Catalysis of Diels-Alder Reactions Organic Seminar March 7th, 2002 Lisa Jungbauer

  2. Outline • Introduction • The Diels-Alder Reaction • Biomolecule Catalysts of Diels-Alder Reactions • Catalytic Antibodies (Abzymes) • Ribozymes (Catalytic RNA) • Biocatalysis of Diels-Alder Reactions in Biosynthesis and Organic Synthesis • Conclusions

  3. The Diels-Alder Reaction • 4+2 Cycloaddition • Concerted • Stereospecific Diene (e- rich) Dienophile (e- poor) Hetero-Diels Alder Retro-Diels Alder Inverse Electron Demand Diels-Alder

  4. Regiochemistry LUMO “ortho” HOMO Figure adapted from Eaton, B. E.; Tarasow, T. M. Cell. Mol. Life Sci.1999, 55, 1463-1472.

  5. Stereochemistry Figure adapted from Eaton, B. E.; Tarasow, T. M. Cell. Mol. Life Sci.1999, 55, 1463-1472.

  6. Products of the Diels-Alder Reaction TS4 DS‡ : Rotational and translational entropy DH‡: Reactivity of substrates TS3 DDG‡ TS2 TS1 DG4‡ DG3‡ DG2‡ DG1‡ SM P4 P3 P1 P2 DG‡ = DH‡ - TDS‡

  7. Catalysis G‡cat < G‡uncat Blackburn, G. M.; Datta, A.; Denham, H.; Paul Wentworth, J. Adv. Phys. Org. Chem. 1998, 31, 249-369. Bartlett, P. A.; Mader, M. M. Chem. Rev.1997, 97, 1281-1301.

  8. Catalysis of Diels-Alder Reactions • Typical Methods for catalysis are • Lewis Acids • ZnCl2, AlCl3, SnCl4, TiCl4, Et2AlCl • Medium Effects (the hydrophobic effect of aqueous solvent) and pressure also facilitate the reaction

  9. Stereochemical Outcome of Diels-Alder Reactions • Stereoselectivity is influenced by • Chiral auxiliaries • Chiral metal complexes • Lewis acid catalysts typically enhance regioselectivity and stereoselectivity • Utility of Diels-Alder reactions increases with ability to influence stereochemical outcome • Catalysts that direct stereoselectivity are valuable tools

  10. Biomolecules are Suitable Catalysts for Diels-Alder Reactions The Diels-Alder reaction…. Biocatalysts…. 1) Large activation entropy (-30 to -40 cal K-1 mol-1) 2) Potential to form stereoisomeric products 3) No enzymatic example of a Diels-Alder biocatalyst 4) Challenge for catalysis since there are no ionic intermediates and little charge separation in the transition state • Compensate for loss in entropy by • binding the substrates in an active site • 2) Inherent chirality of biomolecules • may direct stereoselectivity • 3) Expand the limits of biocatalysis • from enzymology to organic synthesis • The binding site should recognize the • transition state based on shape and • structure Hilvert, D.; Hill, K. W.; Nared, K. D.; Auditor, M.-T. M. J. Am. Chem. Soc. 1989, 111, 9261-9262. Chen, J.; Deng, Q.; Wang, R.; Houk, K. N.; Hilvert, D. ChemBioChem2000, 1, 255-261.

  11. Outline • Introduction • The Diels-Alder Reaction • Biomolecule Catalysts of the Diels-Alder Reaction • Catalytic Antibodies (Abzymes) • Antibody structure, function and production • 6 examples of Diels-Alder catalytic antibodies • Limitations • Future directions • Ribozymes (Catalytic RNA) • Biocatalysis of the Diels-Alder Reaction in Biosynthesis and Organic Synthesis • Conclusions

  12. Brief History of Catalytic Antibodies 1947:Enzyme catalysis achieved by stabilization of the transition state through binding (Linus Pauling) 1969:Catalytic antibodies were proposed (William P. Jencks) 1986:1st successful catalytic antibody (reported independently by Lerner et al. and Schultz et al.) 1989:1st Antibody catalysis of the Diels-Alder (Hilvert, then Schultz) 1995:First synthetic application: antibody catalysis used to set stereochemistry in total synthesis of -Multistratin (pheromone) 2002:16 years of development and detailed studies • Application in pharmaceuticals, total synthesis • One antibody commercially available from Sigma (aldolase antibody 38C2, Aldrich #47,995-0, $108.70/10 mg) Keinan, E.; Lerner, R. A. Isr. J.Chem. 1996, 36, 113-119. Hasserodt, J. Synlett1999, 12, 2007-2022.

  13. Many Types of Catalytic Antibodies • Difficult chemical transformations and rerouting reaction outcomes • Syn-eliminations • Exo-Diels-Alder cycloadditions • 6-endo-tet ring closures • Conversion of enol ether to cyclic ketal in water • Cationic olefin cyclization • Sigmatropic rearrangements • Metal insertion • Hydrolysis • Ester • Amide • Phosphate ester • Glycoside • Redox reactions • Aldol reactions • Michael reactions • Acyl transfer Over 100 different reactions have been accelerated by antibodies Hilvert, D. Annu. Rev. Biochem.2000, 69, 751-793. Stevenson, J. D.; Thomas, N. R. Nat. Prod. Rep.2000, 17, 535-577.

  14. Antibody Structure Legend V = variable region C = constant region H = heavy chain L = light chain CDR = complementarity determining region Davies, D. R.; Chacko, S. Acc. Chem. Res.1993, 26, 421-427. Hilvert, D.; MacBeath, G.; Shin, J. A. The Structural Basis of Antibody Catalysis; Hecht, S. M., Ed.; Oxford University Press: New York, 1998, pp 335-366.

  15. Antibody Functions • Antibodies are involved in the immune response, one of the most important biological defense mechanisms • Antibodies are rapidly produced as advanced, complex receptors to tightly bind potentially harmful foreign substances • In order to recognize an enormous range of molecules, the immune system is capable of generating an incredibly diverse library of antibodies Blackburn, G. M.; Datta, A.; Denham, H.; Paul Wentworth, J. Adv. Phys. Org. Chem.1998, 31, 249-369.

  16. Immunological Methods to Generate Catalytic Antibodies Affinities for hapten: (KD=10-4 to 10-10 M) Hapten Hapten 6-10 weeks Anti-hapten antibodies Screen for catalytic activity Hapten = The small organic molecule to be bound by the antibody (transition state analog, TSA) Burton, D. R. Acc. Chem. Res.1993, 26, 405-411. Hasserodt, J. Synlett1999, 12, 2007-2022.

  17. The Diels-Alder Reaction Transition State • Highly ordered cyclic overlap of  electrons • Product-like • Boat-like conformation • Partial formation of new sigma and  bonds

  18. Hapten Design Strategies for Diels-Alder Reactions • Transition state analog • Shape complementarity • Must avoid product inhibition • Diels-Alder adduct undergoes further transformation • Conformationally restricted analogs • Conformationally flexible analogs Hilvert, D. Annu. Rev. Biochem.2000, 69, 751-793. Hilvert, D.; Hill, K. W.; Nared, K. D.; Auditor, M.-T. M. J. Am. Chem. Soc.1989, 111, 9261-9262.

  19. Diels-Alder Catalyst Antibody 1E9 • 1st biocatalyst of the Diels-Alder reaction • Exploited the chemical and conformational differences between transition state and product Hapten mimics the endo transition state Hilvert, D.; Hill, K. W.; Nared, K. D.; Auditor, M.-T. M. J. Am. Chem. Soc.1989, 111, 9261-9262.

  20. Crystal structure of Fab fragment of 1E9 Diels-Alder Catalyst Antibody 1E9 Uncatalyzed rxn. in H2O H‡= 15.5 kcal mol-1 S‡= -21.5 cal K-1 mol-1 (e.u.) kuncat = 0.013 M-1min-1 kcat = 13 min-1 KM = 2.4 mM (diene) ; 29 mM (dienophile) kcat/kuncat = 1000 M Antibody 1E9 catalyzed rxn. H‡= 11.3 kcal mol-1 S‡= -22.1 cal K-1 mol-1 (e.u.) Hilvert, D.; Hill, K. W.; Nared, K. D.; Auditor, M.-T. M. J. Am. Chem. Soc.1989, 111, 9261-9262. Jian Xu, Q. D., Chem, J., Houk, J. N., Bartek, J., Hilvert, D., Wilson, I. A. Science1999, 286, 2345-2348.

  21. Diels-Alder Catalyst Antibody 39-A11 Locked boat-like conformation Kinetic Parameters for Antibody 39-A11 kuncat = 1.9 M-1 s-1 kcat = 0.67 sec-1 KM = 1.2 mM (diene); 0.74 mM (dienophile) kcat/kuncat= 0.35 M Braisted, A. C.; Schultz, P. G. J. Am. Chem. Soc.1990, 112, 7430-7431.

  22. Binding Site of 1E9 vs. 39-A11 1E9 39-A11 Jian Xu, Q. D., Chem, J., Houk, K. N., Bartek, J., Hilvert, D., Wilson, I. A. Science1999, 286, 2345-2348. Hilvert, D. Annu. Rev. Biochem.2000, 69, 751-793. Chen, J.; Deng, Q.; Wang, R. Houk, K. N.; Hilvert, D. ChemBioChem2000, 1, 255-261. Golinelli-Pimpaneau, B. Curr. Op. Struct. Biol.2000, 10, 697-708.

  23. Diels-Alder Antibody 22C8 Mixture of endo and exo products formed in the absence of a catalyst Gouverneur, V. E.; Houk, K. N.; Pascual-Teresa, B. d.; Beno, B.; Janda, K. D.; Lerner, R. A. Science1993, 262, 204-208.

  24. Diels Alder Antibody 22C8 G‡TS = 1.9 kcal/mol Gouverneur, V. E.; Houk, K. N.; Pascual-Teresa, B. d.; Beno, B.; Janda, K. D.; Lerner, R. A. Science1993, 262, 204-208.

  25. Diels-Alder Antibody 22C8 Gouverneur, V. E.; Houk, K. N.; Pascual-Teresa, B. d.; Beno, B.; Janda, K. D.; Lerner, R. A. Science1993, 262, 204-208.

  26. Exo-Diels-Alder Antibody 22C8 kuncat = 1.75 x 10–4 M-1 min-1 kcat = 3.17 x 10-3 min-1 KM = 0.7 mM (diene) ; 7.5 mM (dienophile) kcat/kuncat = 18 M Uncatalyzed rxn. in H2O Regioselective for ortho product 66:34 endo/exo (toluene) 85:15 endo/exo (aqueous) Both enantiomers produced Antibody 22C8 catalyzed rxn. Regioselective for ortho product 0:100 endo/exo > 97% ee Gouverneur, V. E.; Houk, K. N.; Pascual-Teresa, B. d.; Beno, B.; Janda, K. D.; Lerner, R. A. Science1993, 262, 204-208.

  27. Conformationally Unrestricted Hapten Yli-Kaukaluoma, J. T.; Ashley, J. A.; Lo, C.-H.; Tucker, L.; Wolfe, M. M.; Janda, K. D. J. Am. Chem. Soc.1995, 117, 7041-7047.

  28. Diels-Alder Antibody 13G5 Crystal Structure of Fab with a Ferrocenyl Hapten Mimic Bound in the Cavity Yli-Kaukaluoma, J. T.; Ashley, J. A.; Lo, C.-H.; Tucker, L.; Wolfe, M. M.; Janda, K. D. J. Am. Chem. Soc. 1995, 117, 7041-7047. Heine, A.; Stura, E. A.; Yli-Kauhaluoma, J. T.; Gao, C. Science1998, 279, 1934-1940.

  29. Exo-Diels-Alder Antibody Catalyst 13G5 Uncatalyzed Reaction Both diastereomers formed No enantiomeric preference Catalyzed Reaction kuncat = 1.75 x 10-4 M-1 min-1 kcat = 1.20 x 10-3 min-1 KM = 2.7 mM (diene) 10 mM (dienophile) kcat/kuncat= 6.9 M >98 % de; 95% ee Yli-Kaukaluoma, J. T.; Ashley, J. A.; Lo, C.-H.; Tucker, L.; Wolfe, M. M.; Janda, K. D. J. Am. Chem. Soc.1995, 117, 7041-7047. Heine, A.; Stura, E.A.; Yli-Kauhaluoma, J.T.; Gao, C. Science1998, 279, 1934-1940.

  30. Hetero-Diels-Alder Catalytic Antibody kuncat = 7.0 x 10-5 s-1 kcat = 1.83 x 10-1 s-1 KM = 3.94 mM (diene); kcat / kuncat = 2618 > 95% of targeted regioisomer was formed in 82% ee Meekel, A. A. P.; Resmini, M.; Pandit, U. K. Bioorg. Med. Chem.1996, 4, 1051-1057. Meekel, A. A. P.; Resmini, M.; Pandit, U. K. J. Chem. Soc., Chem. Comm.1995, 5, 571-572.

  31. Retro-Diels-Alder Catalytic Antibody 9D9 Bahr, N.; Guller, R.; Reymond, J.-L.; Lerner, R. A. J. Am. Chem. Soc.1996, 118, 3550-3555.

  32. Retro-Diels-Alder Catalytic Antibody 9D9 Uncatalyzed rxn. in H2O Spontaneous in aqueous buffer 2% per hour at 20 ºC t1/2 (substrate) = 36 hr. Antibody 9D9 catalyzed rxn. kuncat = 3 x 10-4 min-1 kcat = 0.07 min-1 KM = 0.1 mM kcat/kuncat = 233 • Unique hapten design to avoid product inhibition • Antibody as potential prodrug release system • Successful generation of antibody with hapten • in conformation equilibrium Bahr, N.; Guller, R.; Reymond, J.-L.; Lerner, R. A. J. Am. Chem. Soc.1996, 118, 3550-3555.

  33. Antibody Catalysts of the Diels-Alder Reaction Current Limitations • Moderate to low catalytic efficiency • Selected for binding energy not catalytic activity • Expensive and time consuming to produce • High substrate specificity not ideal for practical application Stevenson, J. D.; Thomas, N. R. Nat. Prod. Rep.2000, 17, 535-577. Liu, D. R.; Schultz, P. G. Angew. Chem. Int. Ed.1999, 38, 36-54. Hilvert, D. Top. Stereochem.1999, 22, 83-135. Hasserodt, J. Synlett1999, 12, 2007-2022. Hilvert, D. Annu. Rev. Biochem.2000, 69, 751-793.

  34. Antibody Catalysts of the Diels-Alder Reaction Advantages and Future Directions • Valuable ability to direct regioselectivity, diastereoselectivity and enantioselectivity of Diels-Alder reactions • Forthcoming advances in immunological technology, screening and selection to discover improved catalytic efficiency • Hapten redesign/optimization • Explore broadening of substrate specificity and complexity of substrates • Metallo-antibodies • Gain insight from comparative analysis with “Diels-alderase” enzymes Stevenson, J. D.; Thomas, N. R. Nat. Prod. Rep.2000, 17, 535-577. Liu, D. R.; Schultz, P. G. Angew. Chem. Int. Ed.1999, 38, 36-54. Hilvert, D. Top. Stereochem.1999, 22, 83-135. Hasserodt, J. Synlett1999, 12, 2007-2022. Eaton, B. E.; Tarasow, T. M. Cell. Mol. Life Sci.1999, 55, 1463-1472.

  35. Outline • Introduction • The Diels-Alder Reaction • Biomolecule Catalysts of the Diels-Alder Reaction • Catalytic Antibodies (Abzymes) • Ribozymes (Catalytic RNA) • Ribozyme Structure, Function and Production • Examples of Diels-Alder Ribozymes • Limitations and Future Directions • “Diels-Alderases” (Enzymes in nature) • Biocatalysis of the Diels-Alder Reaction and Organic Synthesis • Conclusions

  36. A Brief History of Ribozymes 1989: Sidney Altman and Thomas R. Cech won the Nobel Prize for their discovery (in 1982) of catalytic properties of RNA 1990: Tuerk & Gold and Szostak independently develop in vitro selection strategies (SELEX) 1995: First non-phosphate centered reaction catalyzed by RNA (self-alkylating RNA discovered by Wilson & Szostak) 1997: First Diels-Alder reaction catalyzed by RNA (Bruce Eaton) 2002: Exploration of the scope of ribozyme catalysis—are there limits to the types of reactions catalyzed by RNA?

  37. Ribozyme Structure Tarasow, T. M.; Tarasow, S. L.; Eaton, B. E. J. Am. Chem. Soc.2000, 122, 1015-1021. Jaschke, A.; Seeling, B.; Keiper, S.; Stuhlmann, F. Angew. Chem. Int. Ed.2000, 39, 4576-4579.

  38. Increasing Catalytic Ability of Ribozymes Nucleotide ligation Nucleotide cleavage Peptide bond formation Phosphotransfer Phosphoester hydrolysis Natural ribozymes Ribozymes generated in vitro Aminoacylation Metallation Peptide bond formation Phosphorylation Acylation Alkylation Wilson, D. S.; Szostak, J. W. Annu. Rev. Biochem.1999, 68, 611-647.

  39. Strategies to Isolate New Ribozymes • Selection against transition state analogs • Isolate RNA with affinity for immobilized TSA • Screen for catalytic activity • Direct selection • Self-modified RNA is created by reaction with a substrate • Screen for catalytic activity Jaschke, A. Biol. Chem.2001, 382, 1321-1325. Jaschke, A.; Frauendorf, C.; Hausch, F. Synlett1999, 6, 825-833. Wilson, D. S.; Szostak, J. W. Annu. Rev. Biochem.1999, 68, 611-647.

  40. Direct Selection of Ribozymes with Linker-Coupled Reactants Systematic Evolution of Ligands by eXponential Enrichment (SELEX) Jaschke, A. Curr. Opin. Struct. Biol..2001, 11, 321-1326. Jaschke, A. Catalysis of Organic Reactions by RNA-Strategies for the Selection of Catalytic RNAs; Eggleston, D. S., Prescott, C. D. and Pearson, N. D., Ed.; Academic Press: San Diego, 1998, pp 179-190.

  41. RNA Binds a Diels-Alder TSA…. 1st report of RNA binding a nonplanar/hydrophobic ligand Much lower binding affinity than antibody 1E9 • 21 nucleotide consensus sequence in all RNA that bound the ligand • Predicted to be in a bulge stem loop structure …..but no catalytic activity Morris, K. N.; Tarasow, T. M.; Julin, C. M.; Simons, S. L.; Hilvert, D.; Gold, L. Proc. Natl. Acad. Sci., USA1994, 91, 13028-13032.

  42. Diels-Alder Reaction is Catalyzed by RNA PEG 100N PEG 100N Tarasow, T. M.; Tarasow, S. L.; Eaton, B. E. Nature1997, 389, 54-57.

  43. The First RNA Catalyst of a Diels-Alder Reaction • Modified base • Presence of cupric ion 800-fold rate acceleration ((kcat/Km) / kuncat) kuncat = 5.42 x 10-3 M-1 s-1 kcat = 0.011 ± 0.002 s-1 KM = 2.3 ± 0.5 mM (dienophile) kcat / kuncat = 2 M • 10 nucleotide consensus sequence • No other sequence/structural homology Morris, K. N.; Tarasow, T. M.; Julin, C. M.; Simons, S. L.; Hilvert, D.; Gold, L. Proc. Natl. Acad. Sci., USA1994, 91, 13028-13032. Tarasow, T. M.; Tarasow, S. L.; Tu, C.; Kellogg, E.; Eaton, B. E. J. Am. Chem. Soc.1999, 121, 3614-3617. Tarasow, T. M.; Tarasow, S. L.; Eaton, B. E. J. Am. Chem. Soc. 2000, 122, 1015-1021.

  44. Diels-Alder Ribozyme Jaschke, A. Curr. Opin. Struct. Biol..2001, 11, 321-1326.

  45. True Catalysis of Diels-Alder by RNA Tethering of substrate to RNA is not necessary 1100-fold rate acceleration ((kcat/Km) / kuncat) kuncat = 3.2 M-1min-1 kcat = 21 min-1 KM = 0.37 mM (diene); 8 mM (dienophile) kcat/kuncat = 6.6 M 6 transformations per minute Uncatalyzed reaction yields racemic products Jaschke, A.; Seeling, B.; Keiper, S.; Stuhlmann, F. Angew. Chem. Int. Ed.2000, 39, 4576-4579.

  46. Predicted control of stereochemistry 95 % enantiomeric excess in both cases Jaschke, A.; Seeling, B.; Keiper, S.; Stuhlmann, F. Angew. Chem. Int. Ed.2000, 39, 4576-4579.

  47. Diels-Alder Ribozymes Current Limitations • SELEX is a very time consuming process • Substrate specificity not practical • RNA highly susceptible to nucleases Jaschke, A.; Frauendorf, C.; Hausch, F. Synlett1999, 6, 825-833. Jaschke, A. Biol. Chem.2001, 382, 1321-1325. Eaton, B. E.; Tarasow, T. M. Cell. Mol. Life Sci.1999, 55, 1463-1472.

  48. Diels-Alder Ribozymes Advantages and Future Directions • Highly stereoselective catalysts • Phenotype is directly linked to genotype • In vitro selection strategies—direct screen for function • Less expensive and smaller in size than antibodies • Easy to create novel features via incorporation of modified nucleotides Eaton, B. E.; Tarasow, T. M. Cell. Mol. Life Sci.1999, 55, 1463-1472. Jaschke, A.; Frauendorf, C.; Hausch, F. Synlett1999, 6, 825-833. Jaschke, A. Biol. Chem.2001, 382, 1321-1325. Golinelli-Pimpaneau, B. Curr. Opin. Struct. Biol.2000, 10, 697-708.

  49. Outline • Introduction • The Diels-Alder Reaction • Biomolecule Catalysts of the Diels-Alder Reaction • Catalytic Antibodies (Abzymes) • Ribozymes (Catalytic RNA) • Ribozyme Structure, Function and Production • Examples of Diels-Alder Ribozymes • Limitations and Future Directions • Biocatalysis of the Diels-Alder Reaction in Biosynthesis and Organic Synthesis • “Diels-Alderases” (natural enzymes) • Biomolecule catalysts vs. other catalysts of the Diels-Alder reaction • Conclusions

  50. Evidence for Diels-Alderases in Biosynthesis Synthesis of Solanopyrones Pohnert, G. ChemBioChem2001, 2, 873-875. Laschat, S. Angew. Chem. Int. Ed. Engl.1996, 35, 289-291. Oikawa, H.; Suzuki, Y.; Katayama, K.; Naya, A.; Sakana, C.; Ichihara, A. J. Chem. Soc., Perkin Trans. 1 1999, 1225-1232. Oikawa, H.; Kobayashi, T.; Katayama, K.; Suzuki, Y.; Ichihara, A. J. Org. Chem. 1998, 63, 8748-8756.

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