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4.3 Linear Cascade Reactions Involving ω-Transaminases  79

               Table 4.4  Selected results of the one-pot, two-step deracemization cascade providing enan-
               tiopure amines. a

               Entry       Substrate      First   -TA  Second   -TA  Conversion (%)  ee (%)


               1              NH 2         ATA-117     ATA-113        >99        >99 (S)
                                           ATA-114     ATA-117        <99        >99 (R)
                               NH
               2                  2        ATA-117     ATA-113        >99        >99 (S)
                           O
                                           ATA-114     ATA-117        >99         96 (R)
                               NH 2
               3                           ATA-117     ATA-113         62        >99 (S)
                                           ATA-114     ATA-117         88        >99 (R)

               4             NH 2          ATA-117     ATA-113         82        >99 (S)
                                           ATA-114     ATA-117         72        >99 (R)
                          Ph
                                   NH 2
               5             O             ATA-117     ATA-113         98        >99 (S)
                                           ATA-113     ATA-117         97        >99 (R)



               a The abbreviation ATA (amino transaminase) was introduced by Codexis for its commercial
               ω-transaminases.




               cascade for mexiletine catalytic amounts of pyruvate were sufficient using either a
               d-or l-selective amino acid oxidase (AAO) for recycling the pyruvate.
                A limitation of this concept is that for the second asymmetric amination no ω-TA
               of the first step should be present, which would otherwise lead to a diminished
               enantiomeric excess of the final product amine. Hence, in the initial studies,
               the ω-TA of the first step was inactivated by heat treatment. To improve the
               economy of the method, immobilization techniques were then successfully applied
               to further optimize the overall process [49]. After encapsulating the commercially
               available (R)-selective ω-TA ATA-117 (Codexis) in a sol–gel/celite matrix, the
               immobilized enzyme preparation was used in the initial KR of the racemic amine.
               The immobilization facilitated easy removal by either filtration or centrifugation
               from the reaction mixture, thereby avoiding the disadvantageous heat treatment.
               The second step, the reductive amination, was performed as described previously by
               employing a free (S)- or (R)-selective ω-TA. The further optimized sequence enabled
               the production of optically pure (S)-amines with up to >99% conversion, >99%
               ee, and a remarkable 95% isolated yield on a preparative scale (50 mg, entry 5).
               As demonstrated, deracemization of chiral amines is an elegant and efficient
               strategy to produce enantiomerically pure amines via sequential enzymatic cascade
               reactions.
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