Download Biocatalysis Center of Emphasis

Development of an Enzymatic Process for Lipitor
Dave Bauer, Mike Burns*, Simon Davidson, Alastair Denholm, Aoife Fahy,
Cathal Healy, John O’Shaughnessy, Éanna Ó Maitiú, Floriana Stomeo,
Gillian Whittaker, and John Wong
The 12th Annual
Green Chemistry & Engineering Conference
Washington, DC
June 24-26, 2008
Biocatalysis Center of Emphasis
Centered in Groton, Connecticut; colleagues in Sandwich, UK
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Biocatalysis Center of Emphasis
Multidisciplinary group consisting of:
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Biocatalysis Center of Emphasis
Multidisciplinary group consisting of:
Organic Chemists
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Biocatalysis Center of Emphasis
Multidisciplinary group consisting of:
Organic Chemists
Microbiologists
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Biocatalysis Center of Emphasis
Multidisciplinary group consisting of:
Organic Chemists
Microbiologists
Molecular Biologists
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Biocatalysis Center of Emphasis
Multidisciplinary group consisting of:
Organic Chemists
Microbiologists
Molecular Biologists
Access to Pfizer resources that include:
Chemical Engineers
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Biocatalysis Center of Emphasis
Multidisciplinary group consisting of:
Organic Chemists
Microbiologists
Molecular Biologists
Access to Pfizer resources that include:
Chemical Engineers
Statisticians
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Biocatalysis Center of Emphasis
Multidisciplinary group consisting of:
Organic Chemists
Microbiologists
Molecular Biologists
Access to Pfizer resources that include:
Chemical Engineers
Statisticians
Protein Chemists and Crystallographers
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Biocatalysis Center of Emphasis
Multidisciplinary group consisting of:
Organic Chemists
Microbiologists
Molecular Biologists
Access to Pfizer resources that include:
Chemical Engineers
Statisticians
Protein Chemists and Crystallographers
Primary function is to develop chemoenzymatic routes to small molecule
compounds.
This includes all stages of development and approved products.
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New Process for Atorvastatin
OH
O
NC
OH
O
O
NC
O
OH
O
O
NC
O
hydroxyketone
hydroxynitrile
OH
cis diol
O
N
H
N
OH
OH
O
O
OH
O
NC
O
O
TBIN
F
Atorvastatin
The reduction of hydroxyketone to cis diol is now a biocatalytic step
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Comparison of Chemical and
Biocatalytic Reactions
OH
NC
O
Chemical Route
NaBH4
Et3B
THF/MeOH/HOAc
cryogenic temp
O
O
OH
NC
Biocatalytic Route
enzyme
aqueous buffer
ambient temperature
OH
O
O
• Chemical process is slow: 80 hours for 6 x methanol distillations to
remove boronic wastes. Enzymatic reaction is faster and has a relatively
simple work-up.
• Quality: Enzymes are highly selective, giving improved cis: trans ratio.
• TEB: pyrophoric and toxic
• NaBH4: Safety hazard. H2 source. Toxic
• Multiple solvents and low temperature requirement eliminated
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Biocatalytic Routes to Chiral Alcohols
R1
Hydroxylation
O
R1
R2
R2
Red
-H
OH
R1
Red
ucti
on
R2
ion
t
u
sol
Re
OH
Red
+
R1
R2
Biocatalytic Reductions: From Lab Curiosity to “First Choice”
Oliver May, et. al., Acc. Chem. Res. (2007), 40(12) - DSM
“…ketoreductases are the preferred catalyst for ketone reduction…”
Jeffrey C. Moore, et. al., Acc. Chem. Res. (2007), 40(2) - Merck
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Reaction Mechanism for Ketone Reduction
H
O
H
O
O
NH2
S
NH2
L
N
R
NADH
N
R
NAD+
http://mgl.scripps.edu/people/goodsell/pdb/pdb13/pdb13_1.html
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Reaction Mechanism for Ketone Reduction
O
O-
O
O P O-
N
O
O
-
H
H2N
O P O
O
H
H
H
H
OH
H
OH
O H
R H
R2
R1
* R2
NAD(P)+cat
NAD(P)H
Cosubstrate(ox)
O
Cosubstrate-H(red)
KRED2
H
O P O H
O-
R1
OH
H
KRED
O
N
N
N
NH2
KRED: Ketone reductase
KRED2: Enzyme used for cofactor regeneration
NAD(P)H: Nicotinamide Adenine Dinucleotide
Cofactor in the reduced form
N
R = H (NADH)
= Phosphate (NADPH)
• The NAD(P)H provides the hydride for the reduction
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Co-factor Recycling Systems
Substrate Coupled Co-factor Regeneration
OH O
O
NC
OH OH O
NC
O
O
ketoester
cis diol
NADH
alcohol
+
dehydrogenase NAD
isopropanol
acetone
Enzyme Coupled Co-factor Regeneration
OH O
O
NC
OH OH O
alcohol
dehydrogenase
O
NC
O
ketoester
cis diol
NADPH
NADP+
glucose
gluconic acid
glucose
dehydrogenase
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Intellectual Property Landscape
OH
NC
O
Enzyme
O
O
OH
OH
NC
O
O
Reeve (Zeneca), US Patent 6,001,615
Microbial reductases obtainable from Beauveria, Candida,
Kluyveromyces, Debaromyces, and Pichia for reduction of
cyanoketoester to diol
Kizaki (Kaneka), US Patents 6,472,544 and 6,645,746
Recombinant E. coli expressing reductase from Candida magnoliae,
and glucose dehydrogenase from Bacillus megaterium for reduction of
chloroketoester
Microorganisms and enzymes from in-house screen – lots of hits.
We needed to develop our own or license a biocatalyst
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Development of Biocatalytic Route
OH O
O
NC
OH OH O
triethanolamine buffer
45C
NC
O
O
ketoester
cis diol
NADH
Ox 28 alcohol
dehydrogenase
NAD+
acetone
isopropanol
Decided not to pursue the development of proprietary biocatalyst
Evaluated biocatalysts from vendors
Licensed Ox28 alcohol dehydrogenase from IEP (Wiesbaden, Germany).
Developed reaction. Main issues were:
Work-up / Biocatalyst formulation
Reaction time – unstable hydroxyketone
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Biocatalyst Production
Wild type enzymes cloned into E.coli
IEP Ox28
lacI
Ox 28
Promoter
pOX28
4909 bp
oriV
Kan
par
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Development of the biocatalyst formulation
Whole frozen E. coli cells
Cell debris caused
problems with
phase splits during
product work-up
Lysate stabilized
with isopropanol
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Biocatalyst Production
E. coli fermentation
growth of cells In stirred tank
Centrifugation
Separation of
Unwanted biomass
to give clear supernate
Ultrafiltration
1.
2.
Concentration of Solution
Removal of water
Cell separation
Cell slurry
microfiltration or
centrifugation
dilute with water
Heat Treatment
Homogenisation
Denature unwanted
proteins
Cell Disruption
to break cell wall
Enzyme
Stabilisation
Add Isopropyl
Alcohol
Refined Enzyme
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Development of the Reaction
Work-up  New Enzyme Formulation
Reaction Time  Reaction Kinetics
OH O
O
NC
triethanolamine
buffer
O
OH OH O
NC
O
ketoester
cis diol
NADH
acetone
Ox 28 alcohol
dehydrogenase
NAD+
isopropanol
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Development of the Reaction
Conversion of hydroxyketone
% Conversion
100
80
60
40
20
0
Hours
final
DOE
Initial
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Green Chemistry
Prevent waste
Design safer chemicals and products
Design less hazardous chemical syntheses
Use renewable feedstocks
Use catalysts, not stoichiometric reagents
Avoid chemical derivatives
Maximize atom economy
Use safer solvents and reaction conditions
Increase energy efficiency
Design chemicals and products to degrade after use
Analyze in real time to prevent pollution
Minimize the potential for accidents
Paul Anastas and John Warner in Green Chemistry: Theory
and Practice (Oxford University Press: New York, 1998)
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Twelve Principles of Green Chemistry
Prevent waste
Design safer chemicals and products
Design less hazardous chemical syntheses
Use renewable feedstocks
Use catalysts, not stoichiometric reagents
Avoid chemical derivatives
Maximize atom economy
Use safer solvents and reaction conditions
Increase energy efficiency
Design chemicals and products to degrade after use
Analyze in real time to prevent pollution
Minimize the potential for accidents
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Solvent Savings
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
enzymatic
IW
D
Ac
Et
O
eO
H
M
F
TH
Ac
e
to
ne
chemical
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Solvent Savings
Chemical
6000000
5000000
4000000
Enzymatic
3000000
Chemical
Enzymatic
2000000
1000000
0
Aqueous Waste
Organic Waste
Organic solvent usage and waste reduced by 65% per annum
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Elimination of Toxic Chemicals
Triethyborane – pyrophoric and toxic
B
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Elimination of Toxic Chemicals
Triethyborane – pyrophoric and toxic
B
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Elimination of Toxic Chemicals
Sodium borohydride
Safety hazard. H2 source
H
Na
+
-
B H
H
H
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Elimination of Toxic Chemicals
Sodium borohydride
Safety hazard. H2 source.
H
Na
+
-
B H
H
H
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Elimination of Cryogenic Conditions
Cryogenic Conditions
-75°C, high energy use
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Elimination of Cryogenic Conditions
Cryogenic Conditions
-75°C, high energy use
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Green Advantages of Biocatalytic Processes
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Green Advantages of Biocatalytic Processes
Reduce solvent usage
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Green Advantages of Biocatalytic Processes
Reduce solvent usage
Biocatalyst are produced from renewable
resources.
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Green Advantages of Biocatalytic Processes
Reduce solvent usage
Biocatalyst are produced from renewable
resources.
Gentle reaction conditions (pH & temp)
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Green Advantages of Biocatalytic Processes
Reduce solvent usage
Biocatalyst are produced from renewable
resources.
Gentle reaction conditions (pH & temp)
Catalytic
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Green Advantages of Biocatalytic Processes
Reduce solvent usage
Biocatalyst are produced from renewable
resources.
Gentle reaction conditions (pH & temp)
Catalytic
Very regio-selective and functionally
selective; may alleviate need for protection
and deprotection steps.
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Green Advantages of Biocatalytic Processes
Reduce solvent usage
Biocatalyst are produced from renewable
resources.
Gentle reaction conditions (pH & temp)
Catalytic
Very regio-selective and functionally
selective; may alleviate need for protection
and deprotection steps.
Biodegradable
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Summary and Conclusions
Enzymatic Lipitor Process has Advantages
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Summary and Conclusions
Enzymatic Lipitor Process has Advantages
Greener Process
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Summary and Conclusions
Enzymatic Lipitor Process has Advantages
Greener Process
Better Selectivity
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Summary and Conclusions
Enzymatic Lipitor Process has Advantages
Greener Process
Better Selectivity
Biocatalytic Processes are Commercially
Feasible
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Summary and Conclusions
Enzymatic Lipitor Process has Advantages
Greener Process
Better Selectivity
Biocatalytic Processes are Commercially
Feasible
Pregabalin and Atorvastatin (Pfizer)
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Summary and Conclusions
Enzymatic Lipitor Process has Advantages
Greener Process
Better Selectivity
Biocatalytic Processes are Commercially
Feasible
Pregabalin and Atorvastatin (Pfizer)
The Future of Biocatalysis is now!
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Development of an Enzymatic Process for Lipitor
Dave Bauer, Mike Burns*, Simon Davidson, Alastair Denholm, Aoife Fahy,
Cathal Healy, John O’Shaughnessy, Éanna Ó Maitiú, Floriana Stomeo,
Gillian Whittaker, and John Wong
The 12th Annual
Green Chemistry & Engineering Conference
Washington, DC
June 24-26, 2008
Questions?
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Questions?
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