Directed Evolution of Stereoselective Biocatalysts

David KnappCHEM575 Literature Seminar

3-13-2008

Importance of Stereoselective Synthesis

Catalytic Approaches

Small Molecules Enzymes

•High substrate scope•Stability/Solubility•Synthetic accessibility•Decades of development

•Poor solubility/stability•Greener chemistry•Tremendous complexity•Excellent stereoselectivity

How can we get better enzymatic catalysts?

Cloning naturalenzymes De novo design

Rational modification

Directed evolution

Directed Evolution

RandomMutagenesis

Transformation &Expression

Selection /Screening

Cloned DNA

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

*

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.

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

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!

Successful examples

Baeyer Villiger Oxidation

Reetz, M. T., et. al. Angew. Chem. Int. Ed. 2004, 43, 4075-4078.

Mechanism

Chiral Products

Enzymatic Baeyer Villiger Oxidation

Reetz, M. T., et al. Angew. Chem. Int. Ed. 2004, 43, 4075-4078.

Cyclohexanone Monooxygenase (CHMO)

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.

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

+

Results of Directed Evolution

Reetz, M. T., et al. Angew. Chem. Int. Ed. 2004, 43, 4075-4078.

Improved R Variant

Substrate Scope

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

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)

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

Structural Considerations

Williams, G. J., et al. J. Am.. Chem. Soc. 2006, 128, 16238-16247.

4R-selectiveRed

4S-selectiveGreen

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

Structural Considerations

Williams, G. J., et al. J. Am.. Chem. Soc. 2006, 128, 16238-16247.

4R-selectiveE192NT167VS208V

4S-selectiveE192NT167G

Substrate analog

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.

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.

Results of Directed Evolution

Best Hits Displayed:• Increased activity• Reduced substrate inhibition

Jennewein, S., et al. Biotechnol. J. 2006, 1, 537-548.

DE of an Enantioselective Aldolase

Jennewein, S., et al. Biotechnol. J. 2006, 1, 537-548.

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

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

• Professor Silverman• Professor Burke• The Burke Group• CHEM575 Class

Acknowledgements