Highlights from the Patents pubs.acs.org/OPRD
Highlights of the Recent U.S. Patent Literature: Focus on Biocatalysis temperatures. Nitrilases have been developed for a handful of industrial applications,3−5 notably the manufacture of nicotinic acid from 3-cyanopyridine (Lonza),6,7 (R)-mandelic acid from benzaldehyde (Mitsubishi Rayon, BASF),8 and desymmetrization of 3-hydroxylglutaronitrile en route to the atorvastatin side chain (Diversa).9,10 Many members of the same Diversa team (now a part of BASF) are inventors on the patent and patent application that describe second generation nitrilase enzymes that have wider substrate scope and improved selectivity, stability, and consistency of manufacture, as presented in Table 1. The patent and patent application describe the following advances in nitrilase substrate scope: 1. Enantioselective preparation of a range of mandelic acid analogues from aryl aldehydes; 2. Dynamic kinetic resolution of a series of substituted arylacetaldehydes to form aryllactic acids; 3. Desymmetrization of 3-hydroxyglutaronitrile to provide either product enantiomer; 4. Preparation of α-amino acids from aldehydes. Mandelic Acid Derivatives. The inventors report kinetic dynamic resolution for the conversion of the cyanohydrins of substituted benzaldehyes to the corresponding α-hydroxyacids, including 2-Cl, 2-Br, 2-Me, 3-Cl, 3-Br, and 4-F mandelic acids. In addition, the 1- and 2-naphthyl analogs, 3-pyridyl, and 3thienyl analogues were successfully prepared. Yields ranged from 70 to 92% with ee’s of 91−99%. While the absolute configurations were not assigned, they were assumed to be (R) based on analogy to mandelic acid (optical rotation and HPLC elution order). The preparation of mandelic acid is described on a 1 g scale from mandelonitrile. Conducting the 2chloromandelonitrile reaction at 45 mM in 20 mL provided a 67% yield with poor mass balance, while no product was observed at 90 mM concentration. Additional experiments indicated 2-chlorobenzaldehyde and 2-chlorobenzoic acid inhibited the enzyme, suggesting considerable development work is still required to render these enzymes synthetically useful. Aryllactic Acids. Conversion of cyanohydrins prepared from arylacetaldehydes to aryl lactic acids were also disclosed. As with the mandelic acid analogs, a dynamic kinetic resolution process was developed to provide single enantiomers in high yield from the racemic cyanohydrins. Since the cyanohydrin derived from phenylacetaldehyde has a less acidic proton than that derived from benzaldehyde, racemization is likely occurring via equilibration via the aldehyde, not by equilibration of the two cyanohydrin enantiomers ((R)-1 and (S)-1, Scheme 1). Eleven analogues were reported, including the parent phenyllactic acid, 2-Br, 2-F, 2-Me, 3-F, 3-Me, 1-naphthyl, 2- and 3thienyl, and 2- and 3-pyridyl, with yields ranging from 59 to 91% and ee’s 88−97%. Para-substituted substrates were sluggish, and no results were reported. The inventors provided no examples of (S)-nitrilases for these substrates.
ABSTRACT: Biocatalysis is playing an increasingly important role in organic synthesis and for industrial manufacture of fine chemicals and pharmaceutical APIs. This review highlights patents and patent applications describing new applications of enzymes for organic synthesiscyclopropanation, Diels−Alder reactions, reductive amination, and nitrile hydrolysis. These enzymatic processes are still early in development, but industrial application is likely just waiting for the right opportunity.
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arnessing biology for chemical synthesis is an expanding area of research that is increasingly finding its way into industrial laboratories and manufacturing applications. Several companies offer “off the shelf” enzyme kits, including ene reductases, aldolases, lipases, monooxygenases, amine transaminases, ketoreductases, and nitrilases,1 such that the practicing organic chemist can apply these enzymes as they would any other readily available chemical reagent. Modern protein engineering techniques, combined with modeling and computation, are making possible the creation of enzymes capable of catalyzing reactions of diverse non-natural substrates, and even reactions not known in nature, such as cyclopropanations, fluorinations, metathesis, and cycloadditions. In this article, we focus on patents and patent applications at the forefront of research on enzymatic catalysisDiels−Alder reactions, cyclopropanations, reductive aminations, and nitrilases. These patents and patent applications primarily describe proof-of-concept results and not fully developed enzymes nor applications that are ready for industrial implementation. Nontheless, as we have witnessed with the Merck−Codexis collaboration to design and develop a transamination for industrial manufacture of sitagliptin,2 focused development can occur quickly once the right opportunity is identified.
I. NITRILASE ENZYMES FOR PREPARATION OF α-AMINO ACIDS AND α-HYDROXY ACIDS Patent Application Pub. Number: US 2015/0125928 Publication Date: May 7, 2015 Title: Nitrilases, Nucleic Acids Encoding Them and Methods for Making and Using Them Applicant: BASF Enzymes LLC Inventors: K. Wong, J. M. Short, M. J. Burk, G. Desantis, R. Farwell, K. Chatman Patent Number: US 8.778,651 Publication Date: July 15, 2014 Title: Nitrilases, Nucleic Acids Encoding Them and Methods for Making and Using Them Applicant: Verenium Corporation Assignee: Verenium Corporation Inventors: K. Wong, J. M. Short, M. Burk, G. Desantis, R. Farwell, K. Chatman Nitrilase enzymes catalyze the hydrolysis of nitriles to carboxylic acids under near-neutral conditions and mild © XXXX American Chemical Society
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DOI: 10.1021/acs.oprd.6b00075 Org. Process Res. Dev. XXXX, XXX, XXX−XXX
Organic Process Research & Development
Highlights from the Patents
Table 1. Advantages of Newly Developed Nitrilases Described in BASF Patents nitrilases disclosed in ‘928 patent application
previously reported nitrilases limitations
