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 •Poor solubility/stability •Stability/Solubility •Greener chemistry •Synthetic accessibility •Tremendous complexity •Decades of development •Excellent stereoselectivity

Cloning natural De novo design enzymes How can we get better enzymatic catalysts? Rational Directed modification evolution

Transformation & Mutagenesis Expression Random Directed Evolution Cloned DNA Selection / Screening

Mutagenesis Methods Error Prone PCR (epPCR) Taq Primer MnCl 2 Cloned DNA Site Saturation Mutagenesis Randomized Primers * DNA Shuffling 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.

The Sorting Problem Large Libraries: Good for diversity, Bad for sorting Screening Selection Brute Force Efficient Time/Labor/Resource Intensive Scalable Simple Not Simple General Not General Taylor, S. V.; Kast, P; Hilvert, D. Angew. Chem. Int. Ed. 2001 , 40 , 3310-3335.

Solutions to the Sorting Problem Selection prephenate Chorismate Mutase Libraries expressed in cells lacking Chorismate Mutase 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.

Solutions to the Sorting Problem Screening • Involves analysis of reaction products • Throughput is key! Spectroscopy UV-vis Fluorescence Chiral Chromatography HPLC GC Capillary Electrophoresis Infrared Thermogenic Imaging Circular Dichroism Mass Spectrometry Reetz, M. T. Angew. Chem. Int. Ed. 2001 , 40 , 284-310.

Successful examples

Baeyer Villiger Oxidation Mechanism Chiral Products Reetz, M. T., et. al. Angew. Chem. Int. Ed. 2004 , 43 , 4075-4078.

Enzymatic Baeyer Villiger Oxidation Cyclohexanone Monooxygenase (CHMO) Reetz, M. T., et al. Angew. Chem. Int. Ed. 2004 , 43 , 4075-4078.

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 O H H O 2 OH OH + CHMO O O O O mutants (S)- 3 (R)- 3 OH First round hits Reetz, M. T., et al. Angew. Chem. Int. Ed. 2004 , 43 , 4075-4078.

Results of Directed Evolution Improved R Variant Substrate Scope Reetz, M. T., et al. Angew. Chem. Int. Ed. 2004 , 43 , 4075-4078.

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) (Aldolase) Sialic acids Wild type NAL (S:R) = 74:26 Williams, G. J., et al. J. Am.. Chem. Soc. 2006 , 128 , 16238-16247.

Directed Evolution of NAL • Mutagenesis Strategy epPCR – 2500/round site-saturation mutagenesis semi-rational design • Screening OH OH OH O OH O NAL Pr 2 N Pr 2 N Pr 2 N + O CO 2 CO 2 O AcHN AcHN + variant Me CO 2 O O OH O OH OH NADH NAD + + Me CO 2 lactate dehydrogenase Williams, G. J., et al. J. Am.. Chem. Soc. 2006 , 128 , 16238-16247.

Structural Considerations 4 S -selective Green 4 R -selective Red Williams, G. J., et al. J. Am.. Chem. Soc. 2006 , 128 , 16238-16247.

Directed Evolution of NAL Best Best 4S -Selective 4R -Selective 66% Yield 70% Yield d.r. >98:2 d.r. >98:2 4S-product 4R-product aldehyde Williams, G. J., et al. J. Am.. Chem. Soc. 2006 , 128 , 16238-16247.

Structural Considerations 4 R -selective E192N T167V S208V 4 S -selective E192N T167G Substrate analog Williams, G. J., et al. J. Am.. Chem. Soc. 2006 , 128 , 16238-16247.

Evolution of an Enantioselective Aldolase deoxy-D-ribose 5-phosphate aldolase (DERA) Proposed Application Lys • e.r. > 99.9:0.1 • Low activity • Limited substrate scope • Substrate Inhibition 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) Me Me Me Me OH OH OH OH •Statin O O NH NH •Inhibits HMG-CoA-Reductase N N OH OH O O •Marketed by Pfizer •2006 Sales: $12.9 billion F F Single-Enantiomer Synthesis Hu, S.; Tao, J.; Xie, Z. PCT Int. Appl. 2006 , 34pp, WO 2006134482 A1. “Pfizer wins Lipitor Patent Extension”, myiRIS, 4-3-2007.

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

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