Jeffrey C. Moore

Biocatalytic Asymmetric Synthesis of Chiral Amines from Ketones Applied to Sitagliptin Manufacture

Biocatalytic Boost Enzymes tend to direct reactions toward specific products much more selectively than synthetic catalysts. Unfortunately, this selectivity has evolved for cellular purposes and may not promote the sorts of reactions chemists are seeking to enhance (see the Perspective by Lutz). Siegel et al. (p. 309) now describe the design of enzymes that catalyze the bimolecular Diels-Alder reaction, a carbon-carbon bond formation reaction that is central to organic synthesis but unknown in natural metabolism. The enzymes display high stereoselectivity and substrate specificity, and an x-ray structure of the most active enzyme confirms that the structure matches the design. Savile et al. (p. 305, published online 17 June) applied a directed evolution approach to modify an existing transaminase enzyme so that it recognized a complex ketone in place of its smaller native substrate, and could tolerate the high temperature and organic cosolvent necessary to dissolve this ketone. This biocatalytic reaction improved the production efficiency of a drug that treats diabetes. An engineered enzyme offers substantial efficiency advantages in the production-scale synthesis of a drug. Pharmaceutical synthesis can benefit greatly from the selectivity gains associated with enzymatic catalysis. Here, we report an efficient biocatalytic process to replace a recently implemented rhodium-catalyzed asymmetric enamine hydrogenation for the large-scale manufacture of the antidiabetic compound sitagliptin. Starting from an enzyme that had the catalytic machinery to perform the desired chemistry but lacked any activity toward the prositagliptin ketone, we applied a substrate walking, modeling, and mutation approach to create a transaminase with marginal activity for the synthesis of the chiral amine; this variant was then further engineered via directed evolution for practical application in a manufacturing setting. The resultant biocatalysts showed broad applicability toward the synthesis of chiral amines that previously were accessible only via resolution. This work underscores the maturation of biocatalysis to enable efficient, economical, and environmentally benign processes for the manufacture of pharmaceuticals.

Development of an Amine Dehydrogenase for Synthesis of Chiral Amines

A leucine dehydrogenase has been successfully altered through several rounds of protein engineering to an enantioselective amine dehydrogenase. Instead of the wild-type α-keto acid, the new amine dehydrogenase now accepts the analogous ketone, methyl isobutyl ketone (MIBK), which corresponds to exchange of the carboxy group by a methyl group to produce chiral (R)-1,3-dimethylbutylamine.

Asymmetric bioreductions: application to the synthesis of pharmaceuticals

Selected examples of asymmetric bioreductions of pharmaceutically relevant prochiral ketones are reviewed. These data show that microbial screens lead to the identification of appropriate biocatalysts, and that the use of miniaturized and semi-automated technology can greatly reduce both labor and lead times. The same data also highlight the need to evaluate a . relatively large andror diverse microbial population highlighting biodiversity . We also found that in many instances the luxury of producing either enantiomers with high optical purity, enantiocomplementarity, can be achieved when employing different microbial strains. Process development studies reviewed here demonstrate that it is possible in some cases to understand and control the production of an unwanted enantiomer or by-product. Finally, a specific example, the asymmetric bioreduction of a ketone by Candida sorbophila, shows that process development studies which optimized, the bioreduction . environmental conditions pH, temperature . . . , the addition of ketone, and the implementation of a nutrient feeding strategy in conjunction with the use of a defined cultivation medium were key in achieving increased bioreduction rates and product titers. When scaled-up in pilot plant bioreactors, the bioreduction process supported the production of several kilograms of . . . R -alcohol enantiomeric excess e.e. ) 98% , with an isolated product yield of about 80%. q 2001 Elsevier Science B.V. All rights reserved.

Overcoming “speed limits” in high throughput chromatographic analysis

The combination of high speed autosampler technology and ultrafast chromatographic separations enables faster high throughput analysis. With an injection cycle time of 10.6 s, MISER (Multiple Injection in a Single Experimental Run) HPLC-MS analysis of a 96 well microplate can be completed in only 17min. As chromatographic separations in the sub 5s range become increasingly common, even faster autosamplers will be needed to realize further speed improvements in high throughput LC-MS analysis. Indeed with proper hardware sampling approaches, chromatographic analysis of microplates could approach speeds of spectrophotometric plate readers while maintaining the advantage of multicomponent detection and monitoring.

Enabling Biocatalysis by High-Throughput Protein Engineering Using Droplet Microfluidics Coupled to Mass Spectrometry

Directed Evolution is a key technology driving the utility of biocatalysis in pharmaceutical synthesis. Conventional approaches to Directed Evolution are conducted using bacterial cells expressing enzymes in microplates, with catalyzed reactions measured by HPLC, high-performance liquid chromatography-mass spectrometry (HPLC-MS), or optical detectors, which require either long cycle times or tailor-made substrates. To better fit modern, fast-paced process chemistry development where solutions are rapidly needed for new substrates, droplet microfluidics interfaced with electrospray ionization (ESI)-MS provides a label-free high-throughput screening platform. To apply this method to industrial enzyme screening and to explore potential approaches that may further improve the overall throughput, we optimized the existing droplet–MS methods. Carryover between droplets, traditionally a significant issue, was reduced to undetectable level by replacing the stainless steel ESI needle with a Teflon needle within a capillary electrophoresis (CE)–MS source. Throughput was improved to 3 Hz with a wide range of droplet sizes (10–50 nL) by tuning the sheath flow within the CE–MS source. The optimized method was demonstrated by screening reactions using two different transaminase libraries. Good correlations (r2 ∼ 0.95) were found between the droplet–MS and LC–MS methods, with 100% match on hit variants. We further explored the capability of the system by performing in vitro transcription–translation inside the droplets and directly analyzing the intact reaction mixture droplets by MS. The synthesized protein attained comparable activity to the protein standard, and the complex samples appeared well tolerated by the MS. The success of the above applications indicates that the MS analysis of the microfluidic droplets is an available option for considerably accelerating the screening of enzyme evolution libraries.

9.13 Industrially Relevant Enzymatic Reductions

Biocatalysis has benefited tremendously from the development of genetic tools through the last two decades. New activities are now discovered computationally, tuned to the needs of industrial manufacturing, and produced cheaply through fermentation. With bioreductions, the enzymatic reduction of ketones to alcohols is the most mature; ketoreductases with high selectivities and broad ranges of activities are well known. In a narrower substrate range the amino acid dehydrogenases, which reduce a-keto acids to amino acids, are also industrially relevant in making natural and unnatural amino acids with high enantiospecificity. Transaminases convert ketones to amines. Recent developments have broadened and improved their activity to commercial relevance, and these biocatalysts are well on their way to becoming an important fixture in the synthetic landscape. Finally enoate reductases, whose enzymes that reduce alkenes in conjugation with carbonyls, are still in their infancy. These enzymes have demonstrated a broad range of synthetically interesting reductions, but to date without the activity sufficient for use industrially. The success of genetic tools to improve reductases suggests that this limitation can be readily overcome. Together these enzymatic reductions provide highly ‘green‘ catalysts to reduce CO, CN, and CC bonds in and environmentally friendly, commercially productive, and economically competitive manner.

CCDC 1417569: Experimental Crystal Structure Determination

Related Article: John Y. L. Chung, Benjamin Marcune, Hallena R. Strotman, Rositza I. Petrova, Jeffrey C. Moore, Peter G. Dormer|2015|Org.Process Res.Dev.|19|1418|doi:10.1021/acs.oprd.5b00259