undertaken which revealed an impressive number of stereo-complementary ω-TAs
[1906–1909]. The most widely used enzymes are obtained from Vibrio fluvialis
[1910], Chromobacterium violaceum [1911, 1912], Pseudomonas aeruginosa
[1913], Bacillus megaterium [1914], and Alcaligenes denitrificans [1915]. Thermostable mutants were derived from an ω-TA from Arthrobacter citreus [1916].
Because the transamination is reversible, the synthesis of nonracemic amines
using ω-transaminases can be operated in two modes (Scheme 2.222):
• Enantioselective deamination starts from a racemic amine via kinetic resolution,
where one enantiomer is converted into the corresponding ketone, leaving the
desired amine enantiomer untouched, which can be recovered in 50% theoretical
yield. For thermodynamic reasons, pyruvate was employed as preferred amine
acceptor yielding D- or L-alanine as by-product, depending on the
stereopreference of the enzyme.
• Desymmetrization of prochiral ketones via asymmetric amination is preferred
for its superior efficiency. Depending on the substrate preference of the
employed transaminase, sacrificial amine donors derived from the α-aminoacid
pool (e.g. Ala, Phe, Glu, Asp) or simple amines (i-Pr-NH 2 , 2-Bu-NH 2 ) are
commonly employed. It should be kept in mind that the absolute configuration
of a chiral amine-donor has to match the stereospecificity of the ω-TA in order to
be accepted.
In transamination, equilibrium constants are close to unity at best and the amino
transfer from an α-amino acid to a ketone is strongly disfavored.
53 To even worsen
the situation, ω-TAs often show cosubstrate and/or coproduct inhibition at
elevated concentrations, which prevents to push amine formation by employing
an excess of amine donor [1917]. In contrast, pulling the equilibrium by co-product
removal is much more effective. The following strategies have been developed
(Scheme 2.222) [1918]:
• The most simple approach is to use i-Pr-NH 2 as amine donor and to remove the
coproduct acetone at elevated temperature by evaporation [1919, 1920].
• Non-volatile coproducts are usually removed by an additional enzymatic step:
For instance, decarboxylation of an α-ketoacid (e.g., pyruvate or
phenylpyruvate, formed from alanine or phenylalanine, respectively) using
pyruvate or phenylpyruvate decarboxylase, yields an aldehyde and CO 2
[1921, 1922]. Although this provides a strong driving force, the aldehyde thus
formed is usually a good substrate and gets aminated.
• Carbonyl-reduction of the keto-coproduct by a suitable dehydrogenase in presence
of NAD(P)H-recycling yields the corresponding alcohol. For instance, pyruvate
can be conveniently reduced to lactate by lactate dehydrogenase [1923].
53 The equilibrium constant between acetophenone and alanine was reported to be 8.8 Â 10
À4
,
see [1916].
2.6 Transfer Reactions
247
[1906–1909]. The most widely used enzymes are obtained from Vibrio fluvialis
[1910], Chromobacterium violaceum [1911, 1912], Pseudomonas aeruginosa
[1913], Bacillus megaterium [1914], and Alcaligenes denitrificans [1915]. Thermostable mutants were derived from an ω-TA from Arthrobacter citreus [1916].
Because the transamination is reversible, the synthesis of nonracemic amines
using ω-transaminases can be operated in two modes (Scheme 2.222):
• Enantioselective deamination starts from a racemic amine via kinetic resolution,
where one enantiomer is converted into the corresponding ketone, leaving the
desired amine enantiomer untouched, which can be recovered in 50% theoretical
yield. For thermodynamic reasons, pyruvate was employed as preferred amine
acceptor yielding D- or L-alanine as by-product, depending on the
stereopreference of the enzyme.
• Desymmetrization of prochiral ketones via asymmetric amination is preferred
for its superior efficiency. Depending on the substrate preference of the
employed transaminase, sacrificial amine donors derived from the α-aminoacid
pool (e.g. Ala, Phe, Glu, Asp) or simple amines (i-Pr-NH 2 , 2-Bu-NH 2 ) are
commonly employed. It should be kept in mind that the absolute configuration
of a chiral amine-donor has to match the stereospecificity of the ω-TA in order to
be accepted.
In transamination, equilibrium constants are close to unity at best and the amino
transfer from an α-amino acid to a ketone is strongly disfavored.
53 To even worsen
the situation, ω-TAs often show cosubstrate and/or coproduct inhibition at
elevated concentrations, which prevents to push amine formation by employing
an excess of amine donor [1917]. In contrast, pulling the equilibrium by co-product
removal is much more effective. The following strategies have been developed
(Scheme 2.222) [1918]:
• The most simple approach is to use i-Pr-NH 2 as amine donor and to remove the
coproduct acetone at elevated temperature by evaporation [1919, 1920].
• Non-volatile coproducts are usually removed by an additional enzymatic step:
For instance, decarboxylation of an α-ketoacid (e.g., pyruvate or
phenylpyruvate, formed from alanine or phenylalanine, respectively) using
pyruvate or phenylpyruvate decarboxylase, yields an aldehyde and CO 2
[1921, 1922]. Although this provides a strong driving force, the aldehyde thus
formed is usually a good substrate and gets aminated.
• Carbonyl-reduction of the keto-coproduct by a suitable dehydrogenase in presence
of NAD(P)H-recycling yields the corresponding alcohol. For instance, pyruvate
can be conveniently reduced to lactate by lactate dehydrogenase [1923].
53 The equilibrium constant between acetophenone and alanine was reported to be 8.8 Â 10
À4
,
see [1916].
2.6 Transfer Reactions
247