Directed Evolution of Enantioselective Biocatalysts · Directed Evolution of Baeyer Villigerases...
Transcript of Directed Evolution of Enantioselective Biocatalysts · Directed Evolution of Baeyer Villigerases...
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Directed Evolution of Stereoselective Biocatalysts
David KnappCHEM575 Literature Seminar
3-13-2008
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Importance of Stereoselective Synthesis
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Catalytic Approaches
Small Molecules Enzymes
•High substrate scope•Stability/Solubility•Synthetic accessibility•Decades of development
•Poor solubility/stability•Greener chemistry•Tremendous complexity•Excellent stereoselectivity
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How can we get better enzymatic catalysts?
Cloning naturalenzymes De novo design
Rational modification
Directed evolution
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Directed Evolution
RandomMutagenesis
Transformation &Expression
Selection /Screening
Cloned DNA
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Mutagenesis Methods
DNA Shuffling
Error Prone PCR (epPCR)Taq
MnCl2Cloned DNA
Cadwell, R. C.; Joyce, G. F. PCR Methods Appl. 1994, 3, 136-140.Soi, C. F.; et al. Eur. J. Biochem. 2002, 269, 4495-4504.Stemmer, W. P. C. Proc. Natl. Acad. Sci. 1994, 91, 10747-10751.
Primer
Site Saturation MutagenesisRandomizedPrimers
*
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The Sorting Problem
SelectionScreeningEfficientScalableNot SimpleNot General
Brute ForceTime/Labor/Resource IntensiveSimpleGeneral
Large Libraries:Good for diversity, Bad for sorting
Taylor, S. V.; Kast, P; Hilvert, D. Angew. Chem. Int. Ed. 2001, 40, 3310-3335.
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Solutions to the Sorting ProblemSelection
Water insoluble Water soluble
Taylor, S. V.; Kast, P; Hilvert, D. Angew. Chem. Int. Ed. 2001, 40, 3310-3335.Reetz, M. T. Angew. Chem. Int. Ed. 2001, 40, 284-310.
Chorismate Mutase Libraries expressed in cells lacking Chorismate Mutase
prephenate
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Solutions to the Sorting Problem
SpectroscopyUV-visFluorescence
Chiral ChromatographyHPLCGC
Capillary ElectrophoresisInfrared Thermogenic ImagingCircular DichroismMass Spectrometry
Screening
Reetz, M. T. Angew. Chem. Int. Ed. 2001, 40, 284-310.
• Involves analysis of reaction products
• Throughput is key!
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Successful examples
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Baeyer Villiger Oxidation
Reetz, M. T., et. al. Angew. Chem. Int. Ed. 2004, 43, 4075-4078.
Mechanism
Chiral Products
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Enzymatic Baeyer Villiger Oxidation
Reetz, M. T., et al. Angew. Chem. Int. Ed. 2004, 43, 4075-4078.
Cyclohexanone Monooxygenase (CHMO)
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Directed Evolution of Baeyer Villigerases
• Mutagenesis StrategyepPCR – 10,000 in round 1
2,000 in round 2
• ScreeningChiral GC – 800 variants/day
• Libraries expressed in E. coli.
• Screen reaction run with whole cells
Reetz, M. T., et al. Angew. Chem. Int. Ed. 2004, 43, 4075-4078.
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Directed Evolution of Baeyer Villigerases
Reetz, M. T., et al. Angew. Chem. Int. Ed. 2004, 43, 4075-4078.
First round hits
O
OH
O2
CHMOmutants
OO
H OH
(R)-3OO
H OH
(S)-3
+
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Results of Directed Evolution
Reetz, M. T., et al. Angew. Chem. Int. Ed. 2004, 43, 4075-4078.
Improved R Variant
Substrate Scope
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Conclusions from Reetz Study
How is this study significant?
•Unselective enzyme made synthetically useful •Significant reversal of stereoselectivity• Simplistic mutagenic strategy• Substrate scope• Prior knowledge requirements•Superiority to other approaches
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N-acetylneuraminic lyase (NAL)
Williams, G. J., et al. J. Am.. Chem. Soc. 2006, 128, 16238-16247.
Sialic acidsWild type NAL (S:R) = 74:26
(Aldolase)
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Directed Evolution of NAL
Williams, G. J., et al. J. Am.. Chem. Soc. 2006, 128, 16238-16247.
• Mutagenesis StrategyepPCR – 2500/roundsite-saturation mutagenesissemi-rational design
• Screening
Pr2NO
OH
OH
OMe CO2
OO
OAcHN
OH
CO2
OHPr2N O
OAcHN
OH
CO2Pr2N
OH
++
lactatedehydrogenase
NADHNAD+
Me CO2
OH+
NAL
variant
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Structural Considerations
Williams, G. J., et al. J. Am.. Chem. Soc. 2006, 128, 16238-16247.
4R-selectiveRed
4S-selectiveGreen
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Directed Evolution of NAL
Williams, G. J., et al. J. Am.. Chem. Soc. 2006, 128, 16238-16247.
4S-product4R-productaldehyde
Best4S-Selective
Best4R-Selective
66% Yieldd.r. >98:2
70% Yieldd.r. >98:2
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Structural Considerations
Williams, G. J., et al. J. Am.. Chem. Soc. 2006, 128, 16238-16247.
4R-selectiveE192NT167VS208V
4S-selectiveE192NT167G
Substrate analog
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Evolution of an Enantioselective Aldolase
deoxy-D-ribose 5-phosphate aldolase (DERA)
Proposed Application
• e.r. > 99.9:0.1• Low activity• Limited substrate scope• Substrate Inhibition
Lys
Gijsen, H. J. M.; Wong, C. H. J. Am. Chem. Soc. 1994, 116, 8422‐8423.Greenberg, W. A., et al. PNAS 2004, 101, 5788‐5793.
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Directed Evolution of an EnantioselectiveAldolase
•GoalsImprove the activity of DERADecrease substrate inhibition
•Mutagenesis StrategyepPCR – 3,000 clones per roundDNA Shuffling of best hits
• ScreeningTarget reaction run in cell free extractActivity determined by GCThroughput: 300 samples/day
Jennewein, S., et al. Biotechnol. J. 2006, 1, 537-548.
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Results of Directed Evolution
Best Hits Displayed:• Increased activity• Reduced substrate inhibition
Jennewein, S., et al. Biotechnol. J. 2006, 1, 537-548.
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DE of an Enantioselective Aldolase
Jennewein, S., et al. Biotechnol. J. 2006, 1, 537-548.
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Atorvastatin (Lipitor)
N OH
OOHOHMeMe
O
NH
F
N OH
OOHOHMeMe
O
NH
F
•Statin•Inhibits HMG-CoA-Reductase•Marketed by Pfizer•2006 Sales: $12.9 billion
Hu, S.; Tao, J.; Xie, Z. PCT Int. Appl. 2006, 34pp, WO 2006134482 A1.“Pfizer wins Lipitor Patent Extension”, myiRIS, 4-3-2007.
Single-Enantiomer Synthesis
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Conclusions
Directed Evolution stands as an underutilized, yet potentially general and powerful way to access stereoselective catalysts
BenefitsExceptional catalyst stereoselectivityMethodological complementarity to transition metalsStrategic generalityGreen chemistry
LimitationsCurrent enzymatic scope/availabilityOverhead
Future Directions
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• Professor Silverman• Professor Burke• The Burke Group• CHEM575 Class
Acknowledgements