Download Protein-Engineered Biocatalysts in Industry

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