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Protein-Engineered Biocatalysts in Industry F F F O F O N N F NH 2 N O N N N F CF3 N N CF3 Stefan McCarver Group Meeting June 18 th, 2015 Protein-Engineered Biocatalysts in Industry Diverse applications of enzymes ■ World enzyme market in 2003 Product USD (Millions) Detergents 789 Foods 634 Agriculture and feed 376 Textile processing 237 Pulp, paper, leather, and chemicals 222 Sanchez, S.; Demain, A. L. Org. Process Res. Dev. 2011, 15 , 224–230. Protein-Engineered Biocatalysts in Industry Diverse applications of enzymes ■ Enzymes can provide many advantages for chemical synthesis O O HN HN lipase OAc OAc Higher enantioselectivity and regioselectivity Can be effective in both aqueous and organic media Typically do not require protecting groups N3 CHO Me N3 OH aldolase Operate under mild conditions with high efficiency O OPO32– Me OH O HO OH OH OPO32– Koeller, K. M.; Wong, C.-H. Nature 2001, 409, 232–240. Protein-Engineered Biocatalysts in Industry Utilizing natural enzymes for chemical reactions ■ Naturally occuring enzymes usually do not have desired catalytic reactivity Naturally occuring enzyme Ideal biocatalyst Activity for specific substrate(s) Activity for desired substrate(s) Unstable under process conditions Tolerates process conditions How do chemists transform natural enzymes into useful biocatalysts? Protein-Engineered Biocatalysts in Industry Modification of enzyme catalysts ■ Proteins are modified through iterative mutation and screening screening mutagenesis and selection Parent enzyme Mutant proteins ■ Evolved proteins are used as parents for another iteration ■ The process is continued until desired reactivity is reached Evolved enzyme Bloom, J. D.; Arnold, F. H. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 , 9995–10000. Protein-Engineered Biocatalysts in Industry Generating protein diversity ■ Methods for mutagenesis Site-directed mutagenesis A number of methods are known for specifically substituting individual amino acids in a protein Requires a lot of structural information to be useful, often a crystal structure and computational modeling Error-prone PCR The polymerase chain reaction reaction is naturally error prone This can be amplified by increasing Mg 2+, adding Mn 2+ and by using unequal nucleotide concentrations Gene shuffling DNA sequences from several similar proteins are fragmented and randomly recombined A larger extent of fragmentation results in a greater number of single site mutations Primrose, S. B.; Twyman, R. In Principles of Gene Manipulation and Genomics, 7 th ed.; Wiley-Blackwell, 2006; ch. 8 Protein-Engineered Biocatalysts in Industry Generating protein diversity ■ Methods for mutagenesis Site-directed mutagenesis A number of methods are known for specifically substituting individual amino acids in a protein Requires a lot of structural information to be useful, often a crystal structure and computational modeling Error-prone PCR The polymerase chain reaction reaction is naturally error prone This can be amplified by replacing some Mg 2+ with Mn 2+ and by using excess of certain nucleotides Gene shuffling DNA sequences from several similar proteins are fragmented and randomly recombined A larger extent of fragmentation results in a greater number of single site fragmentations Primrose, S. B.; Twyman, R. In Principles of Gene Manipulation and Genomics, 7 th ed.; Wiley-Blackwell, 2006; ch. 8 Protein-Engineered Biocatalysts in Industry Generating protein diversity ■ Methods for mutagenesis Site-directed mutagenesis A number of methods are known for specifically substituting individual amino acids in a protein Requires a lot of structural information to be useful, often a crystal structure and computational modeling Error-prone PCR The polymerase chain reaction reaction is naturally error prone This can be amplified by increasing Mg 2+, adding Mn 2+ and by using unequal nucleotide concentrations Gene shuffling DNA sequences from several similar proteins are fragmented and randomly recombined A larger extent of fragmentation results in a greater number of single site mutations Primrose, S. B.; Twyman, R. In Principles of Gene Manipulation and Genomics, 7 th ed.; Wiley-Blackwell, 2006; ch. 8 Protein-Engineered Biocatalysts in Industry Generating protein diversity ■ Methods for mutagenesis Site-directed mutagenesis A number of methods are known for specifically substituting individual amino acids in a protein Requires a lot of structural information to be useful, often a crystal structure and computational modeling Error-prone PCR The polymerase chain reaction reaction is naturally error prone This can be amplified by replacing some Mg 2+ with Mn 2+ and by using excess of certain nucleotides Gene shuffling DNA sequences from several similar proteins are fragmented and randomly recombined A larger extent of fragmentation results in a greater number of single site fragmentations Primrose, S. B.; Twyman, R. In Principles of Gene Manipulation and Genomics, 7 th ed.; Wiley-Blackwell, 2006; ch. 8 Protein-Engineered Biocatalysts in Industry Case studies HO OH iPr Ph O OH NH CO2Ca0.5 N O O O Me Ph O Me Me Me F Me Atorvastatin (Lipitor) Simvastatin (Zocor) F F NH 2 O N N F N N CF3 Sitagliptin (Januvia) Protein-Engineered Biocatalysts in Industry Synthesis of atorvastatin OH iPr Ph OH NH CO2Ca0.5 N O Ph F Atorvastatin (Lipitor) ■ Lowers blood cholesterol by inhibiting the HMG-CoA reductase enzyme ■ Discovered at Parke-Davis, later acquired by Pfizer ■ Best-selling drug of all time, with over $125 billion in total sales Protein-Engineered Biocatalysts in Industry Synthesis of atorvastatin ■ Process route O OH NC Me CO2Et OH OtBu NC LDA, THF NaBH 4, MeOBEt 2 O CO2tBu THF/MeOH –90 °C OH OH NC CO2tBu key hydroxynitrile Me 2,2-DMP O Acetone, MsOH iPr O Me Me H 2, Raney-Ni NC MeOH, NH 3 CO2tBu Me H 2N O CO2tBu Me O O iPr PhHN Ph O O O Me Ph heptane, toluene THF, pivalic acid NH CO2tBu N F O O deprotection Atorvastatin salt formation calcium Ph F Ma, S. K. et. al. Green Chem. 2012, 12 , 81–86. Protein-Engineered Biocatalysts in Industry Synthesis of atorvastatin ■ Previous routes to hydroxynitrile starting material OH NC ■ Estimated demand 100 mT per year CO2Et ■ Simplified route that reduces waste is highly desirable key hydroxynitrile 1) NaCN 2) NaOH OH HO Cl 3) EtOH, H + HBr OH HO CO2Et NaCN OH Br CO2Et OH NC CO2Et from kinetic resolution kinetic resolution - max 50% yield two steps involving the use of cyanide HBr required to form bromohydrin Ma, S. K. et. al. Green Chem. 2012, 12 , 81–86. Protein-Engineered Biocatalysts in Industry Synthesis of atorvastatin ■ Previous routes to hydroxynitrile starting material OH NC ■ Estimated demand 100 mT per year CO2Et ■ Simplified route that reduces waste is highly desirable key hydroxynitrile 1) HO –, H 2O2 HO HBr lactose 2) H + O O EtOH NaCN OH Br CO2Et OH NC CO2Et uses chiral pool materials HBr required to form bromohydrin Ma, S. K. et. al. Green Chem. 2012, 12 , 81–86. Protein-Engineered Biocatalysts in Industry Synthesis of atorvastatin ■ Previous routes to hydroxynitrile starting material OH NC CO2Et key hydroxynitrile O O Cl2 EtOH ■ Estimated demand 100 mT per year ■ Simplified route that reduces waste is highly desirable asymmetric O Cl CO2Et hydrogenation NaCN OH Cl CO2Et OH NC CO2Et high pressure H 2 for asymmetric reduction harsh cyanation conditions Ma, S. K. et. al. Green Chem. 2012, 12 , 81–86. Protein-Engineered Biocatalysts in Industry Synthesis of atorvastatin ■ Previous routes to hydroxynitrile starting material OH NC CO2Et key hydroxynitrile ■ Estimated demand 100 mT per year ■ Simplified route that reduces waste is highly desirable Issues to be addressed ■ Requirement of chiral pool materials or high pressure hydrogenation to access alcohol ■ Cyanation requires harsh conditions, challenging to separate product from byproducts Ma, S. K. et. al. Green Chem. 2012, 12 , 81–86. Protein-Engineered Biocatalysts in Industry Synthesis of atorvastatin ■ Enzymatic route to hydroxynitrile starting material OH NC ■ Estimated demand 100 mT per year CO2Et ■ Simplified route that reduces waste is highly desirable key hydroxynitrile ketone reductase O Cl CO2Et Cl CO2Et O HCN CO2Et OH NC CO2Et Halohydrin dehalogenase NADPH NADP gluconate – HCl OH glucose glucose dehydrogenase Ma, S. K. et. al. Green Chem. 2012, 12 , 81–86. Protein-Engineered Biocatalysts in Industry Synthesis of atorvastatin ■ Ketone reductase mechanism ketone reductase O Cl Tyr CO2Et OH Cl CO2Et NADPH NADP gluconate O His H H N N H N H glucose glucose Lys dehydrogenase H O R small R large O Asp H H O– O ■ Proton transfer occurs from tyrosine residue NH 2 N ■ Hydrogen bond to histidine orients substrate O O ■ Lysine provides electrostatic stabilization H ■ NADPH positioned by hydrogen bond to asparagine OH NADPH Kratzer, R.; Wilson, D. K.; Nidetzky, B. Life 2006, 58, 499–507. Protein-Engineered Biocatalysts in Industry Synthesis of atorvastatin ■ Enzymatic route to hydroxynitrile starting material OH NC ■ Estimated demand 100 mT per year CO2Et ■ Simplified route that reduces waste is highly desirable key hydroxynitrile ketone reductase O Cl CO2Et Cl CO2Et O HCN CO2Et OH NC CO2Et Halohydrin dehalogenase NADPH NADP gluconate – HCl OH glucose glucose dehydrogenase Ma, S. K. et. al. Green Chem. 2012, 12 , 81–86. Protein-Engineered Biocatalysts in Industry Synthesis of atorvastatin ■ Halohydrin dehalogenase mechanism Arg H 2N H N Tyr H Cl O– O halide binding pocket CO2Et OH NC CO2Et Halohydrin dehalogenase H Cl CN – OH H O Ser CO2Et ■ Deprotonation by tyrosine residue Arg H 2N H N Tyr ■ Hydrogen bonding catalysis from serine ■ Binding pocket for departing halide H H O ■ NADPH positioned by hydrogen bond to asparagine O H Cl – halide binding pocket O Ser CO2Et Janssen, D. B. et. al. J. Bacteriol. 2001, 183 , 5058–5066. Protein-Engineered Biocatalysts in Industry Synthesis of atorvastatin ■ Directed evolution of KRED and GDH enzymes using DNA shuffling ketone reductase O Cl CO2Et Cl CO2Et O HCN CO2Et OH NC CO2Et Halohydrin dehalogenase NADPH NADP gluconate – HCl OH glucose glucose dehydrogenase Ma, S. K. et. al. Green Chem. 2012, 12 , 81–86. Protein-Engineered Biocatalysts in Industry Synthesis of atorvastatin ■ Directed evolution of KRED and GDH enzymes using DNA shuffling ketone reductase O Cl – HCl OH CO2Et Cl CO2Et O HCN CO2Et OH NC CO2Et Halohydrin dehalogenase NADPH NADP gluconate glucose glucose dehydrogenase Initial process (wild-type enzymes) Final process (evolved enzymes) Substrate (g/L) 80 160 Reaction time (h) 24 8 Enzyme loading (g/L) 9 0.9 Isolated yield (%) 85 95 > 99.5 > 99.9 Product ee (%) Ma, S. K. et. al. Green Chem. 2012, 12 , 81–86. Protein-Engineered Biocatalysts in Industry Synthesis of atorvastatin ■ Directed evolution of HDDH enzyme using DNA shuffling ketone reductase O Cl CO2Et Cl CO2Et O HCN CO2Et OH NC CO2Et Halohydrin dehalogenase NADPH NADP gluconate – HCl OH glucose glucose dehydrogenase Ma, S. K. et. al. Green Chem. 2012, 12 , 81–86. Protein-Engineered Biocatalysts in Industry Synthesis of atorvastatin ■ Directed evolution of HDDH enzyme using DNA shuffling ketone reductase O Cl – HCl OH CO2Et Cl CO2Et O HCN CO2Et OH NC CO2Et Halohydrin dehalogenase NADPH NADP gluconate glucose glucose dehydrogenase Inital process (wild-type enzyme) Final process (evolved enzyme) Substrate (g/L) 20 140 Reaction time (h) 72 5 Enzyme loading (g/L) 30 1.2 Isolated yield (%) 67 92 > 99.5 > 99.5 Product ee (%) Ma, S. K. et. al. Green Chem. 2012, 12 , 81–86. Protein-Engineered Biocatalysts in Industry Synthesis of sitagliptin F F NH 2 O N N F N N CF3 Sitagliptin (Januvia) ■ Antidiabetic dipeptidyl peptidase-4 inhibitor ■ Developed and marketed by Merck ■ Often used as a combination therapy with other medicines Protein-Engineered Biocatalysts in Industry Synthesis of sitagliptin ■ First generation process route F F 1) [(S)-binapRuCl2] F F HBr, H 2 O CO2Me EDC, LiOH OH CO2H 2) NaOH 83% F F 2) EDC, NMM N O N CF3 F F N N N CF3 78% (4 steps) H 2PO 4– NH 3+ O 2) H 3PO 4 N N HN NH 1) H 2, Pd/C O F F BnO N 81% F BnO F 2) DIAD, PPh 3 F 1) base F 1) BnONH 2•HCl N N F N N CF3 Desai, A. A. Angew. Chem. Int. Ed. 2011, 50, 1974–1976. Protein-Engineered Biocatalysts in Industry Synthesis of sitagliptin ■ Second generation process route O O F O F CO2H O F Me Me iPr 2HNEt + O– F •HCl F O O N CF3 O 83% N O tBuCOCl, iPr 2NEt F N HN CF3CO2H Me Me then NH 4OAc F F F NH 2 1) [{Rh(cod)Cl} 2], NH 4Cl, O F N N 2) heptane/iPrOH CF3 one pot, 82% yield NH 3+ O tBu-josiphos, H 2 N N F H 2PO 4– N N F N 3) H 3PO 4 N CF3 81% yield > 99.9% ee Desai, A. A. Angew. Chem. Int. Ed. 2011, 50, 1974–1976. Protein-Engineered Biocatalysts in Industry Synthesis of sitagliptin ■ Third generation process route F F transaminase F O O NH 2 pyridoxal phosphate N N F F N N O N N iPrNH 2 F N N CF3 ■ Transition metal catalyzed hydrogenation required carbon treatment to remove Rh as well as ee upgrade through recrystallization ■ Biocatalysis presents an opportunity to attain excellent yield and near perfect selectivity under mild conditions CF3 NH 2 O ATA-117 Me Me > 99.9% ee Chem. Commun. 2009, 2127–2129. Savile, C. K. et. al. Science 2010, 329, 305–309. Protein-Engineered Biocatalysts in Industry Transaminase mechanism Lys Me Me Me NH NH NH 2 OH H 2O3PO Me Me Me Ph N H Ph Me NH H NH 2 OH H 2O3PO N H Me Me OH H 2O3PO Lys Me NH 3 N H Me Me Me Lys NH 2 NH N H Me Me Me Me Ph Ph Me N H NH H 2O Lys NH 2 OH N H Me Lys Me Ph Me H 2O3PO NH 2 OH H 2O3PO NH 2 O NH 3 Me Me O H OH N H Me NH Ph NH H 2O3PO Lys Me NH 2 NH 2 OH H 2O3PO Me Lys Ph NH 2 O OH H 2O3PO N H NH 3 Lys Me Savile, C. K. et. al. Science 2010, 329, 305–309. Protein-Engineered Biocatalysts in Industry Synthesis of sitagliptin ■ Design of a transaminase catalyst - site saturation libraries in small and large pockets Savile, C. K. et. al. Science 2010, 329, 305–309. Protein-Engineered Biocatalysts in Industry Synthesis of sitagliptin Orange: Large pocket Green: Catalytic residues Teal: Small Pocket Savile, C. K. et. al. Science 2010, 329, 305–309. Protein-Engineered Biocatalysts in Industry Synthesis of sitagliptin ■ Third generation process route F F transaminase F O O NH 2 pyridoxal phosphate N N F F N N iPrNH 2 O N N F CF3 N N CF3 ■ Site saturation and combinatorial libraries of binding pocket screened for activity ■ Most active mutant subjected to directed evolution for activity and stability under process conditions Savile, C. K. et. al. Science 2010, 329, 305–309. Protein-Engineered Biocatalysts in Industry Synthesis of sitagliptin ■ Process optimization NH 2 NH 2 Me O NH 2 O N N N N N N F N N O N N F N CF3 CF3 F CF3 N F F F Activity on model substrate Activity on sitagliptin ketone Optimized process 38% conversion 76% conversion 92% yield, > 99.9% ee 10 g/L enzyme 10 g/L enzyme 0.6 g/L enzyme 2 g/L substrate 2 g/L substrate 200 g/L substrate 0.5 M iPrNH 2 0.5 M iPrNH 2 1 M iPrNH 2 5% DMSO / triethanolamine 5% DMSO / triethanolamine 50% DMSO / triethanolamine Savile, C. K. et. al. Science 2010, 329, 305–309. Protein-Engineered Biocatalysts in Industry Synthesis of sitagliptin ■ Process optimization NH 2 NH 2 Me O NH 2 O N N N N N N F N N O N N F N CF3 CF3 F CF3 N F F F Activity on model substrate Activity on sitagliptin ketone Optimized process 38% conversion 76% conversion 92% yield, > 99.9% ee 10 g/L enzyme 10 g/L enzyme 0.6 g/L enzyme 2 g/L substrate 2 g/L substrate 200 g/L substrate 0.5 M iPrNH 2 0.5 M iPrNH 2 1 M iPrNH 2 5% DMSO / triethanolamine 5% DMSO / triethanolamine 50% DMSO / triethanolamine Savile, C. K. et. al. Science 2010, 329, 305–309. Protein-Engineered Biocatalysts in Industry Synthesis of sitagliptin ■ Process optimization NH 2 NH 2 Me O NH 2 O N N N N N N F N N O N N F N CF3 CF3 F CF3 N F F F Activity on model substrate Activity on sitagliptin ketone Optimized process 38% conversion 76% conversion 92% yield, > 99.9% ee 10 g/L enzyme 10 g/L enzyme 0.6 g/L enzyme 2 g/L substrate 2 g/L substrate 200 g/L substrate 0.5 M iPrNH 2 0.5 M iPrNH 2 1 M iPrNH 2 5% DMSO / triethanolamine 5% DMSO / triethanolamine 50% DMSO / triethanolamine Savile, C. K. et. al. Science 2010, 329, 305–309. Protein-Engineered Biocatalysts in Industry Synthesis of sitagliptin ■ Third generation process route F F transaminase F O O NH 2 pyridoxal phosphate N N F F N N O N N iPrNH 2 F CF3 N N CF3 ■ Increase in overall yield relative to second generation process: 13% ■ Waste reduction relative to second generation process: 19% Savile, C. K. et. al. Science 2010, 329, 305–309. Protein-Engineered Biocatalysts in Industry Synthesis of simvastatin HO O O Me O O Me Me Me Me Simvastatin (Zocor) ■ Lipid lowering medication - HMG CoA reductase inhibitor ■ Developed and marketed by Merck ■ Produced by semisynthesis from lovastatin Protein-Engineered Biocatalysts in Industry Synthesis of simvastatin ■ Produced by semisynthesis HO O O Me O HO xxx O Me Me xxx O O steps Me O O Me Me Me xxx Me Me Lovastatin Simvastatin Isolated from Aspergillus terreus Increased inhibitory properties Hydroxymethylglutaryl CoA reductase inhibitor Lower undesirable side effects Xie, X.; Watanabe, K.; Wojcicki, W. A.; Wang, C. C. C.; Tang, Y. Chem. Biol. 2006, 13 , 1161–1169. Protein-Engineered Biocatalysts in Industry Synthesis of simvastatin ■ Typical semisynthetic route HO O O xxx O O Me HO O hydrolysis O Me OH Me xxx O OH xxx Me Me O O O Me xxx Me OH Me xxx Me TBSO Cl Me Me O O O Me Me HO xxx Me xxx Me O O O deprotect O xxx Me xxx xxx Me TBSO O TBSCl xxx Me TBSO O Me Me Me xxx Me Me Xie, X.; Watanabe, K.; Wojcicki, W. A.; Wang, C. C. C.; Tang, Y. Chem. Biol. 2006, 13 , 1161–1169. Protein-Engineered Biocatalysts in Industry Synthesis of simvastatin ■ Potential synthesis utilizing biocatalysis HO O O Me O O xxx O hydrolysis O Me HO Me OH xxx Me xxx Me Me HO O Direct enzymatic route: Me S Me ■ Higher yielding process, cost savings ■ Remove need for protecting groups CoA O O O Me xxx Me O Me Me Me xxx Me Xie, X.; Watanabe, K.; Wojcicki, W. A.; Wang, C. C. C.; Tang, Y. Chem. Biol. 2006, 13 , 1161–1169. Protein-Engineered Biocatalysts in Industry Synthesis of simvastatin ■ Selective biocatalytic acylation reaction US Patent WO/2011/041231 Protein-Engineered Biocatalysts in Industry Synthesis of simvastatin ■ Selective biocatalytic acylation reaction HO O HO O Me O Me Me Me S Me O Me Me xxx Directed O O OH saturation mutagenesis O Me Me CO2Me Me Improved thermal stability Improved acyltransferase activity evolution error-prone PCR xxx Use of a small molecule acyl donor Xie, X.; Watanabe, K.; Wojcicki, W. A.; Wang, C. C. C.; Tang, Y. Chem. Biol. 2006, 13 , 1161–1169. Protein-Engineered Biocatalysts in Industry Synthesis of simvastatin ■ Directed evolution of acyltransferase US Patent WO/2011/041231 Protein-Engineered Biocatalysts in Industry Synthesis of simvastatin ■ Directed evolution of acyltransferase Improved Catalytic Activity 6" 5" kcat (min-1) 4" 3" 2" 1" 0" 0" 1" 2" 3" 4" 5" 6" 7" 8" 9" 10" Directed evolution round US Patent WO/2011/041231 Protein-Engineered Biocatalysts in Industry Synthesis of simvastatin ■ Directed evolution of acyltransferase Improved Improved Protein Catalytic Solubility Activity 6" 250" -1) Solubility kcat (min (mg/L) 5" 200" 4" 150" 3" 100" 2" 50" 1" 0" 0" 0" 0" 1" 1" 2" 2" 3"3" 4"4" 5"5" 6"6" 7"7" 8" 8" 9" 9" 10" Directed Directedevolution evolutionround round US Patent WO/2011/041231 Protein-Engineered Biocatalysts in Industry Synthesis of simvastatin ■ Directed evolution of acyltransferase Improved Improved ImprovedProtein Catalytic ProteinSolubility Stability Activity 6" 250" 51# 49# 5" 200" -1) Solubility kcat Tm(min (°C) (mg/L) 47# 4" 45# 150" 3" 43# 100" 41# 2" 39# 50" 1" 37# 0" 35#0" 0"0#0" 1"1#1" 2"2#2" 3"3#3" 4"4#4" 5"5#5" 6" 6# 6" 7" 7# 7" 8" 8# 8" 9" 9# 9" 10" 10# Directed Directed Directedevolution evolution evolutionround round round US Patent WO/2011/041231 Protein-Engineered Biocatalysts in Industry Synthesis of simvastatin ■ Selective biocatalytic acylation reaction HO O HO xxx O Me O Me Me Me O O OH Me Me O Me S O Me Me CO2Me Me ■ Reduced use of hazardous substances and solvents in synthesis ■ Catalyst from renewable feedstocks, byproducts all recycled ■ Produced in 97% yield compared to < 70% for previous processes Xie, X.; Watanabe, K.; Wojcicki, W. A.; Wang, C. C. C.; Tang, Y. Chem. Biol. 2006, 13 , 1161–1169. Protein-Engineered Biocatalysts in Industry Case studies HO OH iPr Ph O OH NH CO2Ca0.5 N O O O Me Ph O Me Me Me F Me Atorvastatin (Lipitor) Simvastatin (Zocor) EPA Presidential Green Chemistry Award 2006 EPA Presidential Green Chemistry Award 2012 F F NH 2 O N N F N N CF3 Sitagliptin (Januvia) EPA Presidential Green Chemistry Award 2010