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4.3 Linear Cascade Reactions Involving ω-Transaminases 73

with the aminotransferase originating from C. violaceum [31]. The applicability
of this sequential three-step cascade was successfully demonstrated for octane
and methyl dodecanoate as substrates, yielding the corresponding amines besides
significant amounts of the corresponding acid due to overoxidation. Even though
the reactions were performed at low substrate concentrations (0.5–2.9 mM), this
artificial cascade represents a excellent starting point for further developments
toward functionalizations that are difficult to achieve by pure chemical means.
Transformation of secondary alcohols to the primary α-chiral amines is another
challenging task, as the stereochemistry of the starting material (alcohol) and the
product (amine) needs to be considered, in addition to the cofactor dependency
of the ADHs. For instance, the oxidation of a racemic alcohol requires in general
two enantiocomplementary ADHs in order to gain full conversion to the ketone.
Moreover, both enzymes have to be NAD-dependent to be compatible with the
NADH-dependent AlaDH (see above). As the formed intermediate (ketone) is
prochiral, the stereochemistry of the amine should be exclusively controlled by the
utilized ω-TA. Taking all these deliberations into account, a novel redox-neutral
cascade was designed and investigated [36]. For the oxidation of the secondary
alcohols, the (S)-selective ADH originating from Rhodococcus ruber [37] and the
(R)-selective ADH007 from Codexis were found to be appropriate. Reductive
aminations were performed with various (S)-selective ω-TAs from C. violaceum
[31], V. fluvialis [27], or Bacillus megaterium [38]. A first proof-of-concept trial led
to significant product formation employing racemic and enantiomerically pure
alcohols as starting materials (Table 4.2). For example, (S)-octane-2-ol (entry 1) was
converted with 86% conversion, affording the corresponding ketone in 32% and
the amine in 54% yield (78% ee) under optimized conditions. The diminished
optical purity in comparison to previous results using this ω-TA was attributed
to the constant backward and forward reactions in an equilibrating, and thus
racemizing, system [39]. Nevertheless, excellent conversions of 72% were found
using enantiopure (S)-4-phenylbutan-2-ol (entry 2) or (S)-1-phenylethanol (entry 3)
as substrates.
Promising results were also obtained with racemic alcohols, whereby (R)- and
(S)-selective ADHs were employed simultaneously: conversions of 85% and 78%
were detected in case of rac-4-phenylbutane-2-ol (entry 4) and achiral cyclopentanol
(entry 5), respectively. Noteworthy, in both cases also unconsumed ketone remained
up to 35%. In order to gain a better understanding of whether the transformation
reached equilibrium, and thus the overall amination stopped, further experiments
and modifications were conducted. In a first attempt, various oxidases were
integrated into the NADH recycling of the oxidation (Scheme 4.9). Among the
various oxidases tested, NOX 2 from Streptococcus mutans [40] was used to simulate
the formation of higher amounts of ketone but also amine observed with crude
enzyme preparations. In a further experiment, the removal of the formed pyruvate
to lactate instead of recycling to alanine was investigated as a possible alternative;
thus the AlaDH in Scheme 4.6 was substituted by an LDH. Also, in this case
enhanced amine formation could be detected. Since still both reactions (oxidation
and reductive amination) are interconnected via the LDH, the process is still