Slide 1

Biomolecular Catalysis of Diels-Alder Reactions Organic Seminar March 7 th, 2002 Lisa Jungbauer Slide 2 2 Outline I. Introduction The Diels-Alder Reaction II. Biomolecule Catalysts of Diels-Alder Reactions Catalytic Antibodies (Abzymes) Ribozymes (Catalytic RNA) III. Biocatalysis of Diels-Alder Reactions in Biosynthesis and Organic Synthesis IV. Conclusions Slide 3 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 Slide 4 4 Regiochemistry Figure adapted from Eaton, B. E.; Tarasow, T. M. Cell. Mol. Life Sci. 1999, 55, 1463-1472. LUMO HOMO ortho Slide 5 5 Stereochemistry Figure adapted from Eaton, B. E.; Tarasow, T. M. Cell. Mol. Life Sci. 1999, 55, 1463-1472. Slide 6 6 Products of the Diels-Alder Reaction S : Rotational and translational entropy H : Reactivity of substrates TS1 TS2 TS3 TS4 P4 P3 P2 P1 G SM G1G1 G3G3 G4G4 G = H - T S G2G2 Slide 7 7 Catalysis 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. G cat < G uncat Slide 8 8 Catalysis of Diels-Alder Reactions Typical Methods for catalysis are Lewis Acids ZnCl 2, AlCl 3, SnCl 4, TiCl 4, Et 2 AlCl Medium Effects (the hydrophobic effect of aqueous solvent) and pressure also facilitate the reaction Slide 9 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 Slide 10 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 1)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 4)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. ChemBioChem 2000, 1, 255-261. Slide 11 11 Outline I. Introduction The Diels-Alder Reaction II.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) III. Biocatalysis of the Diels-Alder Reaction in Biosynthesis and Organic Synthesis IV. Conclusions Slide 12 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: 1 st successful catalytic antibody (reported independently by Lerner et al. and Schultz et al.) 1989: 1 st 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. Synlett 1999, 12, 2007-2022. Slide 13 13 Many Types of Catalytic Antibodies Sigmatropic rearrangements Metal insertion Hydrolysis Ester Amide Phosphate ester Glycoside Redox reactions Aldol reactions Michael reactions Acyl transfer Hilvert, D. Annu. Rev. Biochem. 2000, 69, 751-793. Stevenson, J. D.; Thomas, N. R. Nat. Prod. Rep. 2000, 17, 535-577. Over 100 different reactions have been accelerated by 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 Slide 14 14 Antibody Structure 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. Legend V = variable region C = constant region H = heavy chain L = light chain CDR = complementarity determining region Slide 15 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. Slide 16 16 Immunological Methods to Generate Catalytic Antibodies Burton, D. R. Acc. Chem. Res. 1993, 26, 405-411. Hasserodt, J. Synlett 1999, 12, 2007-2022. Hapten 6-10 weeks Hapten Anti-hapten antibodies Screen for catalytic activity Hapten = The small organic molecule to be bound by the antibody (transition state analog, TSA) Affinities for hapten: (K D =10 -4 to 10 -10 M) Slide 17 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 Slide 18 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. Slide 19 19 Diels-Alder Catalyst Antibody 1E9 1 st biocatalyst of the Diels-Alder reaction Exploited the chemical and conformational differences between transition state and product Hilvert, D.; Hill, K. W.; Nared, K. D.; Auditor, M.-T. M. J. Am. Chem. Soc. 1989, 111, 9261-9262. Hapten mimics the endo transition state Slide 20 20 Diels-Alder Catalyst Antibody 1E9 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. Science 1999, 286, 2345-2348. Crystal structure of Fab fragment of 1E9 k uncat = 0.013 M -1 min -1 k cat = 13 min -1 K M = 2.4 mM (diene) ; 29 mM (dienophile) k cat /k uncat = 1000 M Uncatalyzed rxn. in H 2 O H = 15.5 kcal mol -1 S = -21.5 cal K -1 mol -1 (e.u.) Antibody 1E9 catalyzed rxn. H = 11.3 kcal mol -1 S = -22.1 cal K -1 mol -1 (e.u.) Slide 21 21 Diels-Alder Catalyst Antibody 39-A11 Locked boat-like conformation Braisted, A. C.; Schultz, P. G. J. Am. Chem. Soc. 1990, 112, 7430-7431. k uncat = 1.9 M -1 s -1 k cat = 0.67 sec -1 K M = 1.2 mM (diene); 0.74 mM (dienophile) k cat /k uncat = 0.35 M Kinetic Parameters for Antibody 39-A11 Slide 22 22 Binding Site of 1E9 vs. 39-A11 Jian Xu, Q. D., Chem, J., Houk, K. N., Bartek, J., Hilvert, D., Wilson, I. A. Science 1999, 286, 2345-2348. Hilvert, D. Annu. Rev. Biochem. 2000, 69, 751-793. Chen, J.; Deng, Q.; Wang, R. Houk, K. N.; Hilvert, D. ChemBioChem 2000, 1, 255-261. Golinelli-Pimpaneau, B. Curr. Op. Struct. Biol. 2000, 10, 697-708. 1E9 39-A11 1E939- A11 Buried surface of hapten upon binding (%) 83.665.5 Cavities between hapten and antibody ( 3 ) 0117 Van der Waals contacts 12185 Slide 23 23 Diels-Alder Antibody 22C8 Gouverneur, V. E.; Houk, K. N.; Pascual-Teresa, B. d.; Beno, B.; Janda, K. D.; Lerner, R. A. Science 1993, 262, 204-208. Mixture of endo and exo products formed in the absence of a catalyst Slide 24 24 Diels Alder Antibody 22C8 Gouverneur, V. E.; Houk, K. N.; Pascual-Teresa, B. d.; Beno, B.; Janda, K. D.; Lerner, R. A. Science 1993, 262, 204-208. G TS = 1.9 kcal/mol Slide 25 25 Diels-Alder Antibody 22C8 Gouverneur, V. E.; Houk, K. N.; Pascual-Teresa, B. d.; Beno, B.; Janda, K. D.; Lerner, R. A. Science 1993, 262, 204-208. Slide 26 26 Exo-Diels-Alder Antibody 22C8 k uncat = 1.75 x 10 4 M -1 min -1 k cat = 3.17 x 10 -3 min -1 K M = 0.7 mM (diene) ; 7.5 mM (dienophile) k cat /k uncat = 18 M Uncatalyzed rxn. in H 2 O 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. Science 1993, 262, 204-208. Slide 27 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. Slide 28 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. Science 1998, 279, 1934-1940. Slide 29 29 Exo-Diels-Alder Antibody Catalyst 13G5 k uncat = 1.75 x 10 -4 M -1 min -1 k cat = 1.20 x 10 -3 min -1 K M = 2.7 mM (diene) 10 mM (dienophile) k cat /k uncat = 6.9 M Uncatalyzed Reaction Catalyzed Reaction >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. Science 1998, 279, 1934-1940. Both diastereomers formed No enantiomeric preference Slide 30 30 Hetero-Diels-Alder Catalytic Antibody 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. k uncat = 7.0 x 10 -5 s -1 k cat = 1.83 x 10 -1 s -1 K M = 3.94 mM (diene); k cat / k uncat = 2618 > 95% of targeted regioisomer was formed in 82% ee Slide 31 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. Slide 32 32 Retro-Diels-Alder Catalytic Antibody 9D9 k uncat = 3 x 10 -4 min -1 k cat = 0.07 min -1 K M = 0.1 mM k cat /k uncat = 233 Uncatalyzed rxn. in H 2 O Spontaneous in aqueous buffer 2% per hour at 20 C t 1/2 (substrate) = 36 hr. Antibody 9D9 catalyzed rxn. Bahr, N.; Guller, R.; Reymond, J.-L.; Lerner, R. A. J. Am. Chem. Soc. 1996, 118, 3550-3555. Unique hapten design to avoid product inhibition Antibody as potential prodrug release system Successful generation of antibody with hapten in conformation equilibrium Slide 33 33 Antibody Catalysts of the Diels-Alder Reaction 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. Synlett 1999, 12, 2007-2022. Hilvert, D. Annu. Rev. Biochem. 2000, 69, 751-793. Current Limitations Slide 34 34 Antibody Catalysts of the Diels-Alder Reaction 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. Synlett 1999, 12, 2007-2022. Eaton, B. E.; Tarasow, T. M. Cell. Mol. Life Sci. 1999, 55, 1463-1472. 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 Slide 35 35 Outline I. Introduction The Diels-Alder Reaction II.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) III. Biocatalysis of the Diels-Alder Reaction and Organic Synthesis IV. Conclusions Slide 36 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 catalysisare there limits to the types of reactions catalyzed by RNA? Slide 37 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. Slide 38 38 Increasing Catalytic Ability of Ribozymes Nucleotide ligation Nucleotide cleavage Peptide bond formation Phosphotransfer Phosphoester hydrolysis Aminoacylation Metallation Peptide bond formation Phosphorylation Acylation Alkylation Ribozymes generated in vitroNatural ribozymes Wilson, D. S.; Szostak, J. W. Annu. Rev. Biochem. 1999, 68, 611-647. Slide 39 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. Synlett 1999, 6, 825-833. Wilson, D. S.; Szostak, J. W. Annu. Rev. Biochem. 1999, 68, 611-647. Slide 40 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. Slide 41 41 RNA Binds a Diels-Alder TSA. Morris, K. N.; Tarasow, T. M.; Julin, C. M.; Simons, S. L.; Hilvert, D.; Gold, L. Proc. Natl. Acad. Sci., USA 1994, 91, 13028-13032. ..but no catalytic activity 1 st 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 Slide 42 42 Diels-Alder Reaction is Catalyzed by RNA Tarasow, T. M.; Tarasow, S. L.; Eaton, B. E. Nature 1997, 389, 54-57. PEG 100N PEG Slide 43 43 The First RNA Catalyst of a Diels-Alder Reaction Modified base Presence of cupric ion Morris, K. N.; Tarasow, T. M.; Julin, C. M.; Simons, S. L.; Hilvert, D.; Gold, L. Proc. Natl. Acad. Sci., USA 1994, 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. 800-fold rate acceleration ((k cat /K m ) / k uncat ) k uncat = 5.42 x 10 -3 M -1 s -1 k cat = 0.011 0.002 s -1 K M = 2.3 0.5 mM (dienophile) k cat / k uncat = 2 M 10 nucleotide consensus sequence No other sequence/structural homology Slide 44 44 Diels-Alder Ribozyme Jaschke, A. Curr. Opin. Struct. Biol.. 2001, 11, 321-1326. Slide 45 45 True Catalysis of Diels-Alder by RNA Tethering of substrate to RNA is not necessary 1100-fold rate acceleration ((k cat /K m ) / k uncat ) k uncat = 3.2 M -1 min -1 k cat = 21 min -1 K M = 0.37 mM (diene); 8 mM (dienophile) k cat /k uncat = 6.6 M 6 transformations per minute Jaschke, A.; Seeling, B.; Keiper, S.; Stuhlmann, F. Angew. Chem. Int. Ed. 2000, 39, 4576-4579. Uncatalyzed reaction yields racemic products Slide 46 46 Predicted control of stereochemistry Jaschke, A.; Seeling, B.; Keiper, S.; Stuhlmann, F. Angew. Chem. Int. Ed. 2000, 39, 4576-4579. 95 % enantiomeric excess in both cases Slide 47 47 Diels-Alder Ribozymes SELEX is a very time consuming process Substrate specificity not practical RNA highly susceptible to nucleases Current Limitations Jaschke, A.; Frauendorf, C.; Hausch, F. Synlett 1999, 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. Slide 48 48 Diels-Alder Ribozymes Highly stereoselective catalysts Phenotype is directly linked to genotype In vitro selection strategiesdirect 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. Synlett 1999, 6, 825-833. Jaschke, A. Biol. Chem. 2001, 382, 1321-1325. Golinelli-Pimpaneau, B. Curr. Opin. Struct. Biol. 2000, 10, 697-708. Advantages and Future Directions Slide 49 49 Outline I.Introduction The Diels-Alder Reaction II.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 III.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 IV.Conclusions Slide 50 50 Evidence for Diels-Alderases in Biosynthesis Pohnert, G. ChemBioChem 2001, 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. Synthesis of Solanopyrones Slide 51 51 Evidence for Diels-Alderases in Biosynthesis Watanabe, K.; Mie, T.; Ichihara, A.; Oikawa, H.; Honma, M. J. Biol. Chem. 2000, 275, 38393-38401. Pohnert, G. ChemBioChem 2001, 2, 873-875. Synthesis of benzoate (macrophomic acid) Diels-Alder Cyclization Slide 52 52 Proof of the Existence of aDiels-Alderase Lovastatin Nonaketide Synthase (LNKS) catalyzes an intramolecular Diels-Alder reaction of a substrate analog Hutchinson, C. R.; Kennedy, J.; Park, C.; Kendrew, S.; Auclair, K.; Vederas, J. Antonie van Leeuwenhoek 2000, 78, 287-294. Pohnert, G. ChemBioChem 2001, 2, 873-875. never observed only formed in presence of LNKS Formed in 1:1 mixture in the absence of LNKS Slide 53 53 Diels-Alder Biocatalysts in Total Synthesis? No examples in synthesis An important pursuit for valuable catalysts of Diels-Alder Stereoselectivity, redirect reaction route Many complex natural products Eaton, B. E.; Tarasow, T. M. Cell. Mol. Life Sci. 1999, 55, 1463-1472. Slide 54 54 Other Diels-Alder Catalysts Chiral Lewis Acids Molecularly Imprinted Polymers (MIPs) Metallo-Porphyrin Systems Zn 2+ Liu, X.-C.; Mosbach, K. Macromol. Rapid Commun. 1997, 18, 609-615. Walter, C. J.; Sanders, J. K. M. Angew. Chem. Int. Ed. Engl. 1995, 34, 217-219. Nakash, M.; Clyde-Watson, Z.; Feeder, N.; Davies, J. E.; Teat, S. J.; Sanders, J. K. M. J. Am. Chem. Soc. 2000, 122, 5286-5293. Otto, S.; Bertoncin, F.; Engberts, J. B. F. N. J. Am. Chem. Soc. 1996, 118, 7702-7707. Kundig, E. P.; Saudan, C. M.; Alezra, V.; Viton, F.; Bernardinelli, G. Angew. Chem. Int. Ed. 2001, 40, 44814485. Slide 55 55 CatalystReactionKineticsSelectivity Antibody 22C8 k cat = 3.17 x 10 -3 min -1 k cat /k uncat = 18 M Exo product > 97% ee Ribozyme (untethered) k cat = 21 min -1 k cat /k uncat = 6.6 M Exo product > 95% ee H2OH2O k 2 = 4.02 x 10 -3 M -1 s -1 84 endo :16 exo Lewis Acid (10 mM Cu(NO 3 ) 2 ) k 2 = 1.11 M -1 s -1 93 endo : 7 exo Porphyrin System k cat = 4.0 M -1 s -1 k cat /k uncat = 1030 Exo product (no turnover) Chiral metal compound 71 exo : 29 endo 85% ee MIP k cat /k uncat = 270 k cat = 3.82 x 10 -2 min -1 (no turnover) endo CH 2 Cl 2 H2OH2O H2OH2O H2OH2O H2OH2O CH 3 CN Slide 56 56 Future Directions of Biomolecular Catalysis of Diels-Alder Reactions Focus on biomolecules as catalysts used to direct the regiochemistry, stereochemistry and enantioselectivity of Diels- Alder reactions Learn from enzyme Diels-Alderases Expand substrate complexity Broaden substrate range Improve catalytic efficiency Eaton, B. E.; Tarasow, T. M. Cell. Mol. Life Sci. 1999, 55, 1463-1472. Hilvert, D. Annu. Rev. Biochem. 2000, 69, 751-793. Slide 57 57 Conclusions Remarkable selectivity provided by biomolecules should drive the pursuit of optimized catalytic efficiency Biomolecular Diels-Alder catalysts demonstrate low catalytic efficiency not necessarily due to their inherent catalytic inabilities but to suboptimal selection and screening Diels-Alder catalytic antibodies and ribozymes possess excellent features that may be exploited for the creation of tailored biocatalysts to be used in synthesis and pharmaceuticals Eaton, B. E.; Tarasow, T. M. Cell. Mol. Life Sci. 1999, 55, 1463-1472. Jaschke, A.; Frauendorf, C.; Hausch, F. Synlett 1999, 6, 825-833. Slide 58 58 Acknowledgements Dr. Silvia Cavagnero Matt Hinderaker Brenda Foster Charles Chow Jason Pontrello Margaret Biddle Whitney Erwin Courtney Bakke Val Keller Sarah Maifeld Susie Martins Clement Chow Konstantin Levitsky Ken Nikolas Sena RajagopalanEric Fulmer Jason Ellefson Mike Birkeland Thank you!