Directed Evolution of Stereoselective Biocatalysts David Knapp - - PowerPoint PPT Presentation

Directed Evolution of Stereoselective Biocatalysts

David Knapp CHEM575 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 natural enzymes De novo design Rational modification Directed evolution

Directed Evolution

Random Mutagenesis Transformation & Expression Selection / Screening

Cloned DNA

Mutagenesis Methods

DNA Shuffling Error Prone PCR (epPCR)

Taq MnCl2

Cloned 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 Mutagenesis

Randomized Primers

*

The Sorting Problem

Selection Screening

Efficient Scalable Not Simple Not General Brute Force Time/Labor/Resource Intensive Simple General

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 Problem

Selection

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

Spectroscopy UV-vis Fluorescence Chiral Chromatography HPLC GC Capillary Electrophoresis Infrared Thermogenic Imaging Circular Dichroism Mass 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 Strategy

epPCR – 10,000 in round 1 2,000 in round 2

• Screening

Chiral 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 CHMO mutants O O H OH (R)-3 O O 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 acids Wild 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 Strategy

epPCR – 2500/round site-saturation mutagenesis semi-rational design

• Screening

Pr2N O OH OH O Me CO2 O O O AcHN OH CO2 OH Pr2N O O AcHN OH CO2 Pr2N OH + + lactate dehydrogenase NADH NAD+ Me CO2 OH + NAL variant

Structural Considerations

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

4R-selective Red 4S-selective Green

Directed Evolution of NAL

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

4S-product 4R-product aldehyde Best 4S-Selective Best 4R-Selective 66% Yield d.r. >98:2 70% Yield d.r. >98:2

Structural Considerations

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

4R-selective E192N T167V S208V 4S-selective E192N T167G 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 Enantioselective Aldolase

• Goals

Improve the activity of DERA Decrease substrate inhibition

• Mutagenesis Strategy

epPCR – 3,000 clones per round DNA Shuffling of best hits

• Screening

Target reaction run in cell free extract Activity determined by GC Throughput: 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 O OH OH Me Me O NH F N OH O OH OH Me Me 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

Benefits

Exceptional catalyst stereoselectivity Methodological complementarity to transition metals Strategic generality Green chemistry

Limitations

Current enzymatic scope/availability Overhead

Future Directions

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

Acknowledgements