Methods of using engineered ketoreductase polypeptides for the stereoselective reduction of acetophenones
Inventors
Liang, Jack • Jenne, Stephane J. • Mundorff, Emily • Ching, Charlene • Gruber, John M. • Krebber, Anke • Huisman, Gjalt W.
Assignees
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Publication Number
US-8512973-B2
Publication Date
2013-08-20
Expiration Date
Abstract
The present disclosure provides engineered ketoreductase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase enzyme. Also provided are polynucleotides encoding the engineered ketoreductase enzymes, host cells capable of expressing the engineered ketoreductase enzymes, and methods of using the engineered ketoreductase enzymes to synthesize a variety of chiral compounds.
Core Innovation
The invention relates to stereoselectively reducing a 2′,6′-substituted acetophenone substrate, optionally substituted at one or more positions selected from 3′, 4′, and 5′, to the corresponding substituted (S)-1-phenethanol product. The reduction is performed by contacting the substrate with an engineered ketoreductase polypeptide under reaction conditions suitable for stereoselectively reducing or converting the substrate.
The engineered ketoreductase polypeptide comprises an amino acid sequence at least 90% identical to a reference sequence of SEQ ID NO: 2, 4, or 98. The polypeptide further comprises an alanine, proline, or cysteine residue at a position corresponding to position 190 of the reference sequence and is capable of stereoselectively reducing acetophenone to (S)-1-phenethanol.
The disclosure further describes high stereomeric excess, improved rates, faster conversion performance, and possible thermostability improvements relative to a reference engineered ketoreductase. It also describes polynucleotides encoding the engineered ketoreductases, host cells for expression, and cofactor requirements involving NAD(P)H with optional cofactor regeneration systems.
Claims Coverage
The independent claim covers stereoselective conversion of an optionally substituted 2′,6′-substituted acetophenone to the corresponding substituted (S)-1-phenethanol using an engineered ketoreductase polypeptide defined by sequence identity, a specified residue at a position corresponding to 190, and (S)-selective acetophenone reduction capability, totaling three inventive features.
Engineered ketoreductase polypeptide for stereoselective reduction
The method comprises contacting the 2′,6′-substituted acetophenone substrate, optionally substituted at one or more of 3′, 4′, and 5′, with an engineered ketoreductase polypeptide under reaction conditions suitable for stereoselectively reducing or converting the substrate to the corresponding substituted (S)-1-phenethanol product.
Sequence identity and residue requirement at position corresponding to 190
The engineered ketoreductase polypeptide comprises an amino acid sequence at least 90% identical to the reference sequence of SEQ ID NO: 2, 4, or 98 and comprises an alanine, proline, or cysteine residue at a position corresponding to position 190 of the reference sequence.
Capability to stereoselectively reduce acetophenone to (S)-1-phenethanol
The engineered ketoreductase polypeptide is capable of stereoselectively reducing acetophenone to (S)-1-phenethanol.
Across the independent claim, the inventive concept is anchored on stereoselective reduction of the specified acetophenone substrates to the corresponding substituted (S)-1-phenethanol using an engineered ketoreductase polypeptide defined by sequence identity to SEQ ID NO: 2, 4, or 98, a specified residue at the position corresponding to 190, and (S)-selective acetophenone reduction capability.
Stated Advantages
Stereoselective conversion to the corresponding substituted (S)-1-phenethanol product.
High stereomeric excess, including an explicit example of greater than 99% stereomeric excess.
Faster conversion performance, including conversion in less than 24 hours and rate improvements relative to a reference engineered enzyme.
Possible thermostability improvements are described.
Utility in synthesis of protein kinase inhibitors.
Documented Applications
Use of the reduction step as part of synthesis of protein kinase inhibitors.
Cofactor usage and regeneration systems are described in the context of performing the stereoselective reduction to substituted (S)-1-phenethanol products.
Synthesis of enantiomerically pure compounds of structural formula (VII) and intermediates reduced or converted using engineered ketoreductase polypeptides.
Preparation of intermediates for a protein tyrosine kinase inhibitor scaffold of structural formula (VIII) using engineered ketoreductase polypeptides.
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