(26d) Scalable Continuous Synthesis of Enantiomerically Pure Amine APIs in an Enzyme Membrane Reactor

Authors: 
Bommarius, A. S., Georgia Institute of Technology
Franklin, R. D., Georgia Institute of Technology
Bommarius, B., Georgia Institute of Technology
Chiral amine groups were found in 11% of the 100 highest selling drugs in 2015. Notable examples include oseltamivir phosphate (sold as Tamiflu®), used for treating influenza symptoms, dexmethylphenidate (sold as Focalin®), an ADHD medication, and sitagliptin (sold as Januvia®), an anti-type II diabetes medicine. Amine dehydrogenases (AmDHs), which were first developed in 20121, catalyze the synthesis of a chiral amine through reductive amination of a prochiral ketone with ammonia as the nitrogen source and NADH as cofactor, to produce an (R)-amine, with NAD+ and water as by-products. To date, the largest scale at which AmDHs have been used was in a 100 mL batch process with an isolated yield of 213 mg of amine product. Because of the high cost of NADH, formate dehydrogenase (FDH) as a cofactor regeneration enzyme to convert NAD+ to NADH. Atom economy for this process is extremely high.

We present the first example of a continuous process for chiral amine synthesis with AmDHs, employing an enzyme membrane reactor (EMR). The EMR consists of a continuously stirred reaction volume with a semipermeable UF membrane at the outlet which traps macromolecules within the reactor, but allows the small molecule substrates and products to pass2-3. Conversion of substrates is measured by following the change in the optical rotation of the outlet stream with a polarimeter. This rapid, online measurement allows for constant control of conversion, which is crucial for process optimization.
In this presentation, we outline the initial process development for this new reactor system for the synthesis of a model chiral amine compound. This begins with the characterization of the kinetic parameters of both enzymes which will determine rates and inhibition patterns. After a stable operating point is determined, the space-time yield (STY) can be determined. As we examine the enzyme deactivation behavior over a period of multiple days, the total turnover number (TTN) for both enzymes is obtained and will be reported. Data from these experiments will be used to optimize STY and TTN. Lastly, we will analyze continuous amine production via EMR with Green Chemistry metrics.

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2. Bommarius, A. S.; Drauz, K.; Gunther, K.; Knaup, G.; Schwarm, M., L-methionine related L-amino acids by acylase cleavage of their corresponding N-acetyl-DL-derivatives. Tetrahedron-Asymmetry 1997, 8 (19), 3197-3200.

3. Kragl, U.; Kruse, W.; Hummel, W.; Wandrey, C., Enzyme engineering aspects of biocatalysis: Cofactor regeneration as example. Biotechnology and Bioengineering 1996, 52 (2), 309-319.