Protein-Engineered Biocatalysts in Industrychemlabs.princeton.edu/macmillan/wp-content/... ·...
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Protein-Engineered Biocatalysts in Industry
June 18th, 2015Group Meeting
Stefan McCarver
N
O O
NN
N
FF
F
CF3
N
O
NN
N
FF
F
CF3
NH2
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Sanchez, S.; Demain, A. L. Org. Process Res. Dev. 2011, 15 , 224–230.
■ World enzyme market in 2003
Protein-Engineered Biocatalysts in IndustryDiverse applications of enzymes
Product USD (Millions)
Detergents
Foods
Agriculture and feed
Textile processing
Pulp, paper, leather, and chemicals
789
634
376
237
222
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Koeller, K. M.; Wong, C.-H. Nature 2001, 409, 232–240.
■ Enzymes can provide many advantages for chemical synthesis
Protein-Engineered Biocatalysts in IndustryDiverse applications of enzymes
Higher enantioselectivity and regioselectivity
Can be effective in both aqueous and organic media
Typically do not require protecting groups
Operate under mild conditions with high efficiency
HN
OAc
OHN
OAc
O
MeCHO
OH
N3
HOO
OPO32–
MeOH
N3 OH
OH
OOPO32–
lipase
aldolase
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■ Naturally occuring enzymes usually do not have desired catalytic reactivity
Protein-Engineered Biocatalysts in IndustryUtilizing natural enzymes for chemical reactions
Naturally occuring enzyme Ideal biocatalyst
Activity for specific substrate(s)
Unstable under process conditions
Activity for desired substrate(s)
Tolerates process conditions
How do chemists transform natural enzymes into useful biocatalysts?
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Bloom, J. D.; Arnold, F. H. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 , 9995–10000.
■ Proteins are modified through iterative mutation and screening
Protein-Engineered Biocatalysts in Industry
Parent enzyme
Modification of enzyme catalysts
mutagenesis screeningand
selection
Evolved enzyme
■ Evolved proteins are used as parents for another iteration
■ The process is continued until desired reactivity is reached
Mutant proteins
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Primrose, S. B.; Twyman, R. In Principles of Gene Manipulation and Genomics, 7th ed.; Wiley-Blackwell, 2006; ch. 8
■ Methods for mutagenesis
Protein-Engineered Biocatalysts in IndustryGenerating protein diversity
Error-prone PCR
Gene shuffling
Site-directed mutagenesisA 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
The polymerase chain reaction reaction is naturally error prone
This can be amplified by increasing Mg2+, adding Mn2+ and by using unequal nucleotide concentrations
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
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Primrose, S. B.; Twyman, R. In Principles of Gene Manipulation and Genomics, 7th ed.; Wiley-Blackwell, 2006; ch. 8
■ Methods for mutagenesis
Protein-Engineered Biocatalysts in IndustryGenerating protein diversity
Error-prone PCR
Gene shuffling
Site-directed mutagenesisA 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
The polymerase chain reaction reaction is naturally error prone
This can be amplified by replacing some Mg2+ with Mn2+ and by using excess of certain nucleotides
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
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Primrose, S. B.; Twyman, R. In Principles of Gene Manipulation and Genomics, 7th ed.; Wiley-Blackwell, 2006; ch. 8
■ Methods for mutagenesis
Protein-Engineered Biocatalysts in IndustryGenerating protein diversity
Error-prone PCR
Gene shuffling
Site-directed mutagenesisA 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
The polymerase chain reaction reaction is naturally error prone
This can be amplified by increasing Mg2+, adding Mn2+ and by using unequal nucleotide concentrations
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
![Page 9: Protein-Engineered Biocatalysts in Industrychemlabs.princeton.edu/macmillan/wp-content/... · Primrose, S. B.; Twyman, R. In Principles of Gene Manipulation and Genomics, 7th ed.;](https://reader034.fdocuments.in/reader034/viewer/2022042306/5ed2033f11fd342e3924d2fb/html5/thumbnails/9.jpg)
Primrose, S. B.; Twyman, R. In Principles of Gene Manipulation and Genomics, 7th ed.; Wiley-Blackwell, 2006; ch. 8
■ Methods for mutagenesis
Protein-Engineered Biocatalysts in IndustryGenerating protein diversity
Error-prone PCR
Gene shuffling
Site-directed mutagenesisA 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
The polymerase chain reaction reaction is naturally error prone
This can be amplified by replacing some Mg2+ with Mn2+ and by using excess of certain nucleotides
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
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Protein-Engineered Biocatalysts in IndustryCase studies
NCO2Ca0.5
OH OHiPr
Ph
O
NHPh
F
N
O
NN
N
FF
F
CF3
NH2
Atorvastatin (Lipitor)
Me
OMe
O
HO
O
MeMe Me
Simvastatin (Zocor)
Sitagliptin (Januvia)
O
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Protein-Engineered Biocatalysts in IndustrySynthesis of atorvastatin
NCO2Ca0.5
OH OHiPr
Ph
O
NHPh
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
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Ma, S. K. et. al. Green Chem. 2012, 12 , 81–86.
■ Process route
Protein-Engineered Biocatalysts in Industry
key hydroxynitrile
Synthesis of atorvastatin
NC CO2EtOH
LDA, THF
Me OtBu
O
NCOH O
CO2tBu NCOH OH
CO2tBu
NaBH4, MeOBEt2
THF/MeOH –90 °C
2,2-DMP
Acetone, MsOH NCO O
CO2tBu
MeMeH2, Raney-Ni
MeOH, NH3
O OCO2tBu
MeMe
H2N
OiPr O
Ph
PhHN
OF
heptane, tolueneTHF, pivalic acid
NCO2tBu
O OiPr
Ph
O
NHPh
F
MeMe
deprotection
salt formation
Atorvastatincalcium
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Ma, S. K. et. al. Green Chem. 2012, 12 , 81–86.
■ Previous routes to hydroxynitrile starting material
Protein-Engineered Biocatalysts in Industry
key hydroxynitrile
Synthesis of atorvastatin
NC CO2EtOH
■ Estimated demand 100 mT per year
■ Simplified route that reduces waste is highly desirable
HO ClOH
HO CO2EtOH
Br CO2EtOH
NC CO2EtOH
from kineticresolution
3) EtOH, H+
1) NaCN2) NaOH HBr NaCN
kinetic resolution - max 50% yield
two steps involving the use of cyanide
HBr required to form bromohydrin
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Ma, S. K. et. al. Green Chem. 2012, 12 , 81–86.
■ Previous routes to hydroxynitrile starting material
Protein-Engineered Biocatalysts in Industry
key hydroxynitrile
Synthesis of atorvastatin
NC CO2EtOH
■ Estimated demand 100 mT per year
■ Simplified route that reduces waste is highly desirable
NC CO2EtOH
lactoseO O
HO HBr
EtOH
1) HO–, H2O2
2) H+ Br CO2EtOH NaCN
uses chiral pool materials
HBr required to form bromohydrin
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Ma, S. K. et. al. Green Chem. 2012, 12 , 81–86.
■ Previous routes to hydroxynitrile starting material
Protein-Engineered Biocatalysts in Industry
key hydroxynitrile
Synthesis of atorvastatin
NC CO2EtOH
■ Estimated demand 100 mT per year
■ Simplified route that reduces waste is highly desirable
NC CO2EtOH
O
O
Cl CO2EtO
Cl CO2EtOHasymmetric
hydrogenation
NaCNCl2
EtOH
high pressure H2 for asymmetric reduction
harsh cyanation conditions
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Ma, S. K. et. al. Green Chem. 2012, 12 , 81–86.
■ Previous routes to hydroxynitrile starting material
Protein-Engineered Biocatalysts in Industry
key hydroxynitrile
Synthesis of atorvastatin
NC CO2EtOH
■ 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
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Ma, S. K. et. al. Green Chem. 2012, 12 , 81–86.
■ Enzymatic route to hydroxynitrile starting material
Protein-Engineered Biocatalysts in Industry
key hydroxynitrile
Synthesis of atorvastatin
NC CO2EtOH
■ Estimated demand 100 mT per year
■ Simplified route that reduces waste is highly desirable
ClO
CO2Et ClOH
CO2Et NCOH
CO2Et
ketonereductase
glucosedehydrogenase
NADPH NADP
glucosegluconate
CO2EtO– HCl HCN
Halohydrin dehalogenase
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Kratzer, R.; Wilson, D. K.; Nidetzky, B. Life 2006, 58, 499–507.
■ Ketone reductase mechanism
Protein-Engineered Biocatalysts in IndustrySynthesis of atorvastatin
ClO
CO2Et ClOH
CO2Et
ketonereductase
glucosedehydrogenase
NADPH NADP
glucosegluconate
O
RlargeRsmall
N N H
His OH
Tyr
NH HH Lys
AspO
O–
N
H H O
NH2
OO
OHNADPH
H
■ Hydrogen bond to histidine orients substrate
■ Proton transfer occurs from tyrosine residue
■ Lysine provides electrostatic stabilization
■ NADPH positioned by hydrogen bond to asparagine
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Ma, S. K. et. al. Green Chem. 2012, 12 , 81–86.
■ Enzymatic route to hydroxynitrile starting material
Protein-Engineered Biocatalysts in Industry
key hydroxynitrile
Synthesis of atorvastatin
NC CO2EtOH
■ Estimated demand 100 mT per year
■ Simplified route that reduces waste is highly desirable
ClO
CO2Et ClOH
CO2Et NCOH
CO2Et
ketonereductase
glucosedehydrogenase
NADPH NADP
glucosegluconate
CO2EtO– HCl HCN
Halohydrin dehalogenase
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Janssen, D. B. et. al. J. Bacteriol. 2001, 183 , 5058–5066.
■ Halohydrin dehalogenase mechanism
Protein-Engineered Biocatalysts in IndustrySynthesis of atorvastatin
■ Deprotonation by tyrosine residue
■ Hydrogen bonding catalysis from serine
■ Binding pocket for departing halide
■ NADPH positioned by hydrogen bond to asparagine
NCOH
CO2Et
CN–
Halohydrin dehalogenase
ClOH
CO2Et
ClOH
CO2Et
H2N N
ArgH
HO–
Tyr
OSerH
halidebinding pocket
CO2Et
H2N N
ArgH
HO
Tyr
OSerH
halidebinding pocket
H
O
Cl–
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ClO
CO2Et ClOH
CO2Et NCOH
CO2Et
ketonereductase
glucosedehydrogenase
NADPH NADP
glucosegluconate
CO2EtO– HCl HCN
Halohydrin dehalogenase
Ma, S. K. et. al. Green Chem. 2012, 12 , 81–86.
■ Directed evolution of KRED and GDH enzymes using DNA shuffling
Protein-Engineered Biocatalysts in IndustrySynthesis of atorvastatin
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Ma, S. K. et. al. Green Chem. 2012, 12 , 81–86.
■ Directed evolution of KRED and GDH enzymes using DNA shuffling
Protein-Engineered Biocatalysts in IndustrySynthesis of atorvastatin
Initial process (wild-type enzymes) Final process (evolved enzymes)
Substrate (g/L)
Reaction time (h)
Enzyme loading (g/L)
Isolated yield (%)
Product ee (%)
80 160
24 8
9 0.9
85 95
> 99.5 > 99.9
ClO
CO2Et ClOH
CO2Et NCOH
CO2Et
ketonereductase
glucosedehydrogenase
NADPH NADP
glucosegluconate
CO2EtO– HCl HCN
Halohydrin dehalogenase
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Ma, S. K. et. al. Green Chem. 2012, 12 , 81–86.
■ Directed evolution of HDDH enzyme using DNA shuffling
Protein-Engineered Biocatalysts in IndustrySynthesis of atorvastatin
ClO
CO2Et ClOH
CO2Et NCOH
CO2Et
ketonereductase
glucosedehydrogenase
NADPH NADP
glucosegluconate
CO2EtO– HCl HCN
Halohydrin dehalogenase
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Ma, S. K. et. al. Green Chem. 2012, 12 , 81–86.
■ Directed evolution of HDDH enzyme using DNA shuffling
Protein-Engineered Biocatalysts in IndustrySynthesis of atorvastatin
Inital process (wild-type enzyme) Final process (evolved enzyme)
Substrate (g/L)
Reaction time (h)
Enzyme loading (g/L)
Isolated yield (%)
Product ee (%)
20 140
72 5
30 1.2
67 92
> 99.5 > 99.5
ClO
CO2Et ClOH
CO2Et NCOH
CO2Et
ketonereductase
glucosedehydrogenase
NADPH NADP
glucosegluconate
CO2EtO– HCl HCN
Halohydrin dehalogenase
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Protein-Engineered Biocatalysts in IndustrySynthesis of sitagliptin
■ Antidiabetic dipeptidyl peptidase-4 inhibitor
■ Developed and marketed by Merck
■ Often used as a combination therapy with other medicines
N
O
NN
N
FF
F
CF3
NH2
Sitagliptin (Januvia)
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Desai, A. A. Angew. Chem. Int. Ed. 2011, 50, 1974–1976.
■ First generation process route
Protein-Engineered Biocatalysts in IndustrySynthesis of sitagliptin
FF
F
OCO2Me
1) [(S)-binapRuCl2]HBr, H2
2) NaOH83%
FF
F
OH
1) BnONH2•HClEDC, LiOH
2) DIAD, PPh3
81%
CO2H
FF
F
N O
BnO
FF
F
NHBnO
N
O
NN
N
CF3
FF
F
NH3+
N
O
NN
N
CF3
H2PO4–1) H2, Pd/C2) H3PO4
78% (4 steps)
1) base2) EDC, NMM
HNN
NN
CF3
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Desai, A. A. Angew. Chem. Int. Ed. 2011, 50, 1974–1976.
■ Second generation process route
Protein-Engineered Biocatalysts in IndustrySynthesis of sitagliptin
FF
F
CO2H
1) [{Rh(cod)Cl}2], NH4Cl,tBu-josiphos, H2
2) heptane/iPrOH3) H3PO4
FF
F
O–
then NH4OAc
FF
F
NH2
N
O
NN
N
CF3
FF
F
NH3+
N
O
NN
N
CF3
H2PO4–
HNN
NN
CF3
tBuCOCl, iPr2NEt83%
O
O
O
O
MeMe
O
O
O
O
MeMe
iPr2HNEt+
CF3CO2H
one pot, 82% yield 81% yield> 99.9% ee
•HCl
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Savile, C. K. et. al. Science 2010, 329, 305–309.
■ Third generation process route
Protein-Engineered Biocatalysts in IndustrySynthesis of sitagliptin
transaminasepyridoxal phosphate
FF
F
O
N
O
NN
N
CF3
FF
F
NH2
N
O
NN
N
CF3
iPrNH2
■ 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
O
Me
NH2
MeATA-117
> 99.9% ee
Chem. Commun. 2009, 2127–2129.
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Savile, C. K. et. al. Science 2010, 329, 305–309.
Protein-Engineered Biocatalysts in IndustryTransaminase mechanism
NH
OH
Me
NH
H2O3PO
Lys
NH
OH
Me
NH
H2O3PO
MeMe
NH2
Lys
H
NH
OH
Me
NH
H2O3PO
MeMe
NH3
Lys
NH
OH
Me
NH
H2O3PO
MeMe
NH2
Lys
NH
OH
Me
NH2
H2O3PO
NH3
Lys
NH
OH
Me
NH
H2O3PO
NH2
Lys
PhMe
NH
OH
Me
NH
H2O3PO
PhMe
NH3
Lys
NH
OH
Me
NH
H2O3PO
PhMe
NH2
Lys
NH2
PhMe
O
PhMe
H2O
NH2
MeMe
NH2
MeMe
O
PhMe
NH2
PhMe
O
MeMe
H
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Savile, C. K. et. al. Science 2010, 329, 305–309.
■ Design of a transaminase catalyst - site saturation libraries in small and large pockets
Protein-Engineered Biocatalysts in IndustrySynthesis of sitagliptin
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Savile, C. K. et. al. Science 2010, 329, 305–309.
Protein-Engineered Biocatalysts in IndustrySynthesis of sitagliptin
Orange: Large pocket
Green: Catalytic residues
Teal: Small Pocket
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Savile, C. K. et. al. Science 2010, 329, 305–309.
■ Third generation process route
Protein-Engineered Biocatalysts in IndustrySynthesis of sitagliptin
transaminasepyridoxal phosphate
FF
F
O
N
O
NN
N
CF3
FF
F
NH2
N
O
NN
N
CF3
iPrNH2
■ 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
![Page 33: Protein-Engineered Biocatalysts in Industrychemlabs.princeton.edu/macmillan/wp-content/... · Primrose, S. B.; Twyman, R. In Principles of Gene Manipulation and Genomics, 7th ed.;](https://reader034.fdocuments.in/reader034/viewer/2022042306/5ed2033f11fd342e3924d2fb/html5/thumbnails/33.jpg)
Savile, C. K. et. al. Science 2010, 329, 305–309.
■ Process optimization
Protein-Engineered Biocatalysts in IndustrySynthesis of sitagliptin
Me
NH2
N
O
NN
N
CF3
NH2
N
O
NN
N
CF3
Activity on model substrate Activity on sitagliptin ketone
38% conversion
10 g/L enzyme
2 g/L substrate
0.5 M iPrNH2
5% DMSO / triethanolamine
10 g/L enzyme
2 g/L substrate
0.5 M iPrNH2
5% DMSO / triethanolamine
76% conversion
Optimized process
0.6 g/L enzyme
200 g/L substrate
1 M iPrNH2
50% DMSO / triethanolamine
92% yield, > 99.9% ee
F
F
F
NH2
N
O
NN
N
CF3
F
F
F
![Page 34: Protein-Engineered Biocatalysts in Industrychemlabs.princeton.edu/macmillan/wp-content/... · Primrose, S. B.; Twyman, R. In Principles of Gene Manipulation and Genomics, 7th ed.;](https://reader034.fdocuments.in/reader034/viewer/2022042306/5ed2033f11fd342e3924d2fb/html5/thumbnails/34.jpg)
Savile, C. K. et. al. Science 2010, 329, 305–309.
■ Process optimization
Protein-Engineered Biocatalysts in IndustrySynthesis of sitagliptin
Me
NH2
N
O
NN
N
CF3
NH2
N
O
NN
N
CF3
Activity on model substrate Activity on sitagliptin ketone
38% conversion
10 g/L enzyme
2 g/L substrate
0.5 M iPrNH2
5% DMSO / triethanolamine
10 g/L enzyme
2 g/L substrate
0.5 M iPrNH2
5% DMSO / triethanolamine
76% conversion
Optimized process
0.6 g/L enzyme
200 g/L substrate
1 M iPrNH2
50% DMSO / triethanolamine
92% yield, > 99.9% ee
F
F
F
NH2
N
O
NN
N
CF3
F
F
F
![Page 35: Protein-Engineered Biocatalysts in Industrychemlabs.princeton.edu/macmillan/wp-content/... · Primrose, S. B.; Twyman, R. In Principles of Gene Manipulation and Genomics, 7th ed.;](https://reader034.fdocuments.in/reader034/viewer/2022042306/5ed2033f11fd342e3924d2fb/html5/thumbnails/35.jpg)
Savile, C. K. et. al. Science 2010, 329, 305–309.
■ Process optimization
Protein-Engineered Biocatalysts in IndustrySynthesis of sitagliptin
Me
NH2
N
O
NN
N
CF3
NH2
N
O
NN
N
CF3
Activity on model substrate Activity on sitagliptin ketone
38% conversion
10 g/L enzyme
2 g/L substrate
0.5 M iPrNH2
5% DMSO / triethanolamine
10 g/L enzyme
2 g/L substrate
0.5 M iPrNH2
5% DMSO / triethanolamine
76% conversion
Optimized process
0.6 g/L enzyme
200 g/L substrate
1 M iPrNH2
50% DMSO / triethanolamine
92% yield, > 99.9% ee
F
F
F
NH2
N
O
NN
N
CF3
F
F
F
![Page 36: Protein-Engineered Biocatalysts in Industrychemlabs.princeton.edu/macmillan/wp-content/... · Primrose, S. B.; Twyman, R. In Principles of Gene Manipulation and Genomics, 7th ed.;](https://reader034.fdocuments.in/reader034/viewer/2022042306/5ed2033f11fd342e3924d2fb/html5/thumbnails/36.jpg)
Savile, C. K. et. al. Science 2010, 329, 305–309.
■ Third generation process route
Protein-Engineered Biocatalysts in IndustrySynthesis of sitagliptin
transaminasepyridoxal phosphate
FF
F
O
N
O
NN
N
CF3
FF
F
NH2
N
O
NN
N
CF3
iPrNH2
■ Increase in overall yield relative to second generation process: 13%
■ Waste reduction relative to second generation process: 19%
![Page 37: Protein-Engineered Biocatalysts in Industrychemlabs.princeton.edu/macmillan/wp-content/... · Primrose, S. B.; Twyman, R. In Principles of Gene Manipulation and Genomics, 7th ed.;](https://reader034.fdocuments.in/reader034/viewer/2022042306/5ed2033f11fd342e3924d2fb/html5/thumbnails/37.jpg)
Protein-Engineered Biocatalysts in IndustrySynthesis of simvastatin
■ Lipid lowering medication - HMG CoA reductase inhibitor
■ Developed and marketed by Merck
■ Produced by semisynthesis from lovastatin
Me
OMe
O
HO
O
MeMe Me
Simvastatin (Zocor)
O
![Page 38: Protein-Engineered Biocatalysts in Industrychemlabs.princeton.edu/macmillan/wp-content/... · Primrose, S. B.; Twyman, R. In Principles of Gene Manipulation and Genomics, 7th ed.;](https://reader034.fdocuments.in/reader034/viewer/2022042306/5ed2033f11fd342e3924d2fb/html5/thumbnails/38.jpg)
Xie, X.; Watanabe, K.; Wojcicki, W. A.; Wang, C. C. C.; Tang, Y. Chem. Biol. 2006, 13 , 1161–1169.
■ Produced by semisynthesis
Protein-Engineered Biocatalysts in IndustrySynthesis of simvastatin
xxxsteps
xxxxxx
Me
OMe
O
HO
O
MeMe Me
O
Me
OMe
O
HO
O
Me
O
Me
Lovastatin Simvastatin
Isolated from Aspergillus terreus
Hydroxymethylglutaryl CoA reductase inhibitor
Increased inhibitory properties
Lower undesirable side effects
![Page 39: Protein-Engineered Biocatalysts in Industrychemlabs.princeton.edu/macmillan/wp-content/... · Primrose, S. B.; Twyman, R. In Principles of Gene Manipulation and Genomics, 7th ed.;](https://reader034.fdocuments.in/reader034/viewer/2022042306/5ed2033f11fd342e3924d2fb/html5/thumbnails/39.jpg)
Xie, X.; Watanabe, K.; Wojcicki, W. A.; Wang, C. C. C.; Tang, Y. Chem. Biol. 2006, 13 , 1161–1169.
■ Typical semisynthetic route
Protein-Engineered Biocatalysts in IndustrySynthesis of simvastatin
xxx
xxxxxx
Me
OMe
O
HO
O
MeMe Me
O
Me
OMe
O
HO
O
Me
O
Me
xxxhydrolysis
xxxxxx
Me
OHMe
O
HO O
Me
OHMe
O
TBSO O
TBSCl
Me
OHMe
O
TBSO O
xxx
xxxxxx
Cl
O
MeMe Me
Me
OMe
O
TBSO
O
MeMe Me
O
xxx
xxxxxx
deprotect
![Page 40: Protein-Engineered Biocatalysts in Industrychemlabs.princeton.edu/macmillan/wp-content/... · Primrose, S. B.; Twyman, R. In Principles of Gene Manipulation and Genomics, 7th ed.;](https://reader034.fdocuments.in/reader034/viewer/2022042306/5ed2033f11fd342e3924d2fb/html5/thumbnails/40.jpg)
Xie, X.; Watanabe, K.; Wojcicki, W. A.; Wang, C. C. C.; Tang, Y. Chem. Biol. 2006, 13 , 1161–1169.
■ Potential synthesis utilizing biocatalysis
Protein-Engineered Biocatalysts in IndustrySynthesis of simvastatin
Me
OMe
O
HO
O
MeMe Me
O
Me
OMe
O
HO
O
Me
O
Me
xxxhydrolysis
xxxxxx
Me
OHMe
O
HO O
xxxxxx
Direct enzymatic route:
■ Higher yielding process, cost savings
■ Remove need for protecting groups
S
O
MeMe Me
CoA
![Page 41: Protein-Engineered Biocatalysts in Industrychemlabs.princeton.edu/macmillan/wp-content/... · Primrose, S. B.; Twyman, R. In Principles of Gene Manipulation and Genomics, 7th ed.;](https://reader034.fdocuments.in/reader034/viewer/2022042306/5ed2033f11fd342e3924d2fb/html5/thumbnails/41.jpg)
US Patent WO/2011/041231
■ Selective biocatalytic acylation reaction
Protein-Engineered Biocatalysts in IndustrySynthesis of simvastatin
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Xie, X.; Watanabe, K.; Wojcicki, W. A.; Wang, C. C. C.; Tang, Y. Chem. Biol. 2006, 13 , 1161–1169.
■ Selective biocatalytic acylation reaction
Protein-Engineered Biocatalysts in IndustrySynthesis of simvastatin
Me
OMe
O
HO
O
MeMe Me
O
Me
OHMe
O
HO O
Directed
evolution
xxxsaturation mutagenesis
error-prone PCRxxx
Improved thermal stability
Improved acyltransferase activity
Use of a small molecule acyl donor
S
O
MeMe Me
CO2Me
![Page 43: Protein-Engineered Biocatalysts in Industrychemlabs.princeton.edu/macmillan/wp-content/... · Primrose, S. B.; Twyman, R. In Principles of Gene Manipulation and Genomics, 7th ed.;](https://reader034.fdocuments.in/reader034/viewer/2022042306/5ed2033f11fd342e3924d2fb/html5/thumbnails/43.jpg)
US Patent WO/2011/041231
■ Directed evolution of acyltransferase
Protein-Engineered Biocatalysts in IndustrySynthesis of simvastatin
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US Patent WO/2011/041231
■ Directed evolution of acyltransferase
Protein-Engineered Biocatalysts in IndustrySynthesis of simvastatin
0"
1"
2"
3"
4"
5"
6"
0" 1" 2" 3" 4" 5" 6" 7" 8" 9" 10"
k cat
(min
-1)
Directed evolution round
Improved Catalytic Activity
![Page 45: Protein-Engineered Biocatalysts in Industrychemlabs.princeton.edu/macmillan/wp-content/... · Primrose, S. B.; Twyman, R. In Principles of Gene Manipulation and Genomics, 7th ed.;](https://reader034.fdocuments.in/reader034/viewer/2022042306/5ed2033f11fd342e3924d2fb/html5/thumbnails/45.jpg)
US Patent WO/2011/041231
■ Directed evolution of acyltransferase
Protein-Engineered Biocatalysts in IndustrySynthesis of simvastatin
0"
1"
2"
3"
4"
5"
6"
0" 1" 2" 3" 4" 5" 6" 7" 8" 9" 10"
k cat
(min
-1)
Directed evolution round
Improved Catalytic Activity
0"
50"
100"
150"
200"
250"
0" 1" 2" 3" 4" 5" 6" 7" 8" 9" 10"
Solu
bilit
y (m
g/L)
Directed evolution round
Improved Protein Solubility
![Page 46: Protein-Engineered Biocatalysts in Industrychemlabs.princeton.edu/macmillan/wp-content/... · Primrose, S. B.; Twyman, R. In Principles of Gene Manipulation and Genomics, 7th ed.;](https://reader034.fdocuments.in/reader034/viewer/2022042306/5ed2033f11fd342e3924d2fb/html5/thumbnails/46.jpg)
US Patent WO/2011/041231
■ Directed evolution of acyltransferase
Protein-Engineered Biocatalysts in IndustrySynthesis of simvastatin
0"
1"
2"
3"
4"
5"
6"
0" 1" 2" 3" 4" 5" 6" 7" 8" 9" 10"
k cat
(min
-1)
Directed evolution round
Improved Catalytic Activity
0"
50"
100"
150"
200"
250"
0" 1" 2" 3" 4" 5" 6" 7" 8" 9" 10"
Solu
bilit
y (m
g/L)
Directed evolution round
Improved Protein Solubility
35#
37#
39#
41#
43#
45#
47#
49#
51#
0# 1# 2# 3# 4# 5# 6# 7# 8# 9# 10#
T m (°
C)
Directed evolution round
Improved Protein Stability
![Page 47: Protein-Engineered Biocatalysts in Industrychemlabs.princeton.edu/macmillan/wp-content/... · Primrose, S. B.; Twyman, R. In Principles of Gene Manipulation and Genomics, 7th ed.;](https://reader034.fdocuments.in/reader034/viewer/2022042306/5ed2033f11fd342e3924d2fb/html5/thumbnails/47.jpg)
Xie, X.; Watanabe, K.; Wojcicki, W. A.; Wang, C. C. C.; Tang, Y. Chem. Biol. 2006, 13 , 1161–1169.
■ Selective biocatalytic acylation reaction
Protein-Engineered Biocatalysts in IndustrySynthesis of simvastatin
Me
OMe
O
HO
O
MeMe Me
O
xxx
Me
OHMe
O
HO O
S
O
MeMe Me
CO2Me
■ 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
![Page 48: Protein-Engineered Biocatalysts in Industrychemlabs.princeton.edu/macmillan/wp-content/... · Primrose, S. B.; Twyman, R. In Principles of Gene Manipulation and Genomics, 7th ed.;](https://reader034.fdocuments.in/reader034/viewer/2022042306/5ed2033f11fd342e3924d2fb/html5/thumbnails/48.jpg)
EPA Presidential Green Chemistry Award 2012
EPA Presidential Green Chemistry Award 2010
EPA Presidential Green Chemistry Award 2006
Protein-Engineered Biocatalysts in IndustryCase studies
NCO2Ca0.5
OH OHiPr
Ph
O
NHPh
F
N
O
NN
N
FF
F
CF3
NH2
Atorvastatin (Lipitor)
Me
OMe
O
HO
O
MeMe Me
Simvastatin (Zocor)
Sitagliptin (Januvia)
O