Research Project Description

    Stewart Research Group
    Department of Chemistry, University of Florida
    Enzymes as Practical Catalysts for Organic Synthesis

Because they consist of optically pure amino acids, enzymes can be used to introduce and manipulate stereocenters in synthetic intermediates. Our recent work has focused on asymmetric carbonyl and alkene reductions. We identify candidate enzymes by genome analysis¹ and use rapid cloning and expression methods to isolate pure proteins. After identifying those with the desired substrate- and stereoselectivities by screening, enzymes are used in key transformations in total synthesis. These efforts are supported by the NSF and several companies.

1. Stereoselective alkene reductions to prepare chiral building blocks. Asymmetric alkene reductions offer especially attractive routes to chiral building blocks since two adjacent stereocenters are established by a single reaction. Cyclohexenone reduction results

We have cloned and expressed a library of alkene reductases and examined their stereoselectivities. For example, 2- and 3-alkyl cyclohexenones were reduced with high stereoselectivity by the old yellow enzyme (OYE) from Saccharomyces pastorianus,¹ providing important cyclohexanone building blocks.² We also used the same enzyme in a concise chemo-enzymatic route to beta²-amino acids, Beta-2 amino acid synthesis

which are becoming increasingly important in medicinal chemistry.³ We are currently working to expand the substrate range of these reductions and increasing their synthetic utility by combining alkene reduction with C-C bond formation in a one-pot, multi-step cascade.

2. Accessing both product stereoisomers. One key observation from our profiling studies of alkene reductase enzymes was that all OYE family members possess very similar stereoselectivities. This allows access to one product enantiomer, but how can we obtain the other? Expanding the range of these While members of the old yellow enzyme (OYE) family all gave very similar results, We have prepared both enantiomers of the N-benzoyl-phenylisoserine Taxol side-chain from a common starting material.² Screening a collection of 18 purified bakers' yeast reductases³ yielded two that catalyzed the correct dynamic kinetic resolutions. Enantiomeric glycidic esters prepared from the resulting chlorohydrins were opened in Ritter reactions with benzonitrile to afford oxazolines that were hydrolyzed to the final targets. Perillaldehyde reduction results

nzymatic reduction of an alpha-chloro-beta-keto ester was the key step in our route to this natural product with both anticancer and antibiotic activities.⁴ Bestatin retrosynthesis

This dynamic kinetic resolution introduced both stereochemical centers in >98% diastereomeric and enantiomeric excess. The homochiral chlorohydrin was converted to a known alpha-hydroxy-beta-amino acid that had previously been converted to bestatin by Umezawa, which completed our formal total synthesis of this molecule.⁵

3. Asymmetric alkene reductions. After analyzing a variety of bacterial, plant and animal genomes, we have cloned and expressed 20 known and putative alke Enone reduction

ne reductases. These enzymes carry out asymmetric hydrogenations on electron-poor alkenes. We are currently characterizing our collection and applying it to preparing chiral intermediates.

Selected References

Formerly Saccharomyces carlsbergenesis.
Stereoselective Enone Reductions by Saccharomyces carlsbergensis Old Yellow Enzyme. M.A. Swiderska and J.D. Stewart, J. Mol. Catal. B: Enzymatic 2006, 42, 52-54.
Asymmetric Bioreductions of beta-Nitroacrylates as a Route to beta²-Amino Acids.” M.A. Swiderska and J.D. Stewart, Org. Lett. 2006, 8, 6131-6133.
Chemoenzymatic Formal Total Synthesis of (-)-Bestatin. B.D. Feske and J.D. Stewart, Tetrahedron: Asymmetry 2005, 16, 3124-3127.
Chemical Synthesis of Bestatin. H. Suda, T. Takita, T. Aoyagi and H. Umezawa, J. Antibiot. 1976, 29, 600-601.