June 1998
SYNLETT
631
Kynureninase in Organic Synthesis : Preparation of γ-Hydroxy-α-L-Amino Acids
*
Tsuyoshi Miura, Noriko Masuo, Yuki Fusamae, Tetsuya Kajimoto, Yoshiteru Ida
School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
Received 24 March 1998
Abstract: Kynureninase, which is known to catalyze the transaldol
reaction between benzaldehyde and kynurenine, accepted many kinds of
other aromatic aldehydes and propargyl aldehydes as the substrates to
afford novel γ-hydroxy-α-L-amino acids. The L-configuration of the α-
carbons was confirmed by an enzymatic method using both D- and L-
amino acid oxidases. The stereochemistry of the newly formed chiral
center (γ-position) in major isomers was determined to be R-
configuration by the observed NOEs in the NMR spectroscopy of
lactones derived from the γ-hydroxy-α-L-amino acids.
containing pyridoxal-5'-phosphate (65 mmol), and this reaction mixture
was gently shaken at 30 °C for 8 hours. Dimethylsulfoxide (DMSO) can
be used as a cosolvent up to 10% of volumetric concentration unless
aldehydes dissolve well in the buffer solution at the reaction temperature
(30 °C). Phenylacetaldehyde, phenylpropylaldehyde, N-phthalimido-
acetaldehyde, benzyloxyacetaldehyde, 1,1-dimethylethyl-2,2-dimethyl-
5
4(R)-formyloxazolidine-3-carboxylate (Garner's aldehyde) were not
consumed in the reaction and more than 90 % of these aldehydes were
3
recovered from the reaction medium. The neighboring sp -carbons to
the aldehyde group seemed to interrupt the enzyme-substrate (ES)
complex formation due to the van der Waals repulsion which can not be
relieved by rotation even in the case of methylene carbon. Neither
oxygen nor nitrogen functional groups in the aldehydes affected the
progress of the reaction. Therefore, several aldehydes bearing sp -
carbon at the α-position were next tested as the substrates. Many
aromatic aldehydes were converted to the corresponding γ-hydroxy-α-
amino acids in good yields (Table 1) although the appropriate products
were not obtained from p-dimethylaminobenzaldehyde, 1-methylindole-
Recently, we reported a facile method for the preparation of β-hydroxy-
α-L-amino acids by using L-threonine aldolase (LTA) catalyzed
1
reaction, and succeeded in short step synthesis of some bioactive
2b
2
2
compounds by using this enzymatic reaction as a key step. Enzymatic
3
aldol reaction including LTA catalyzed reaction is a practical method
for achieving asymmetric carbon-carbon bond formation on a large
scale, since these reactions require neither absolute non-aqueous
atmosphere nor extremely low temperature.
3-carboxaldehyde,
and
cinnamic
aldehyde.
Furthermore,
phenylpropargylaldehyde (3a) which carrys sp-carbon at the α-position
was smoothly transformed into γ-phenylpropargyl-γ-hydroxy-α-amino
acid (3b) in spite of the low chemical yield (20%). The substrate
specificity of kynureninase implies that the catalytic site of
kynureninase should be narrow and sterically hindered since only the
flat and/or straight molecules can be incorporated as the substrates.
γ-Hydroxy-α-L-amino acids as well as β-hydroxy-α-amino acids are
useful building blocks for the synthesis of many kinds of bioactive
compounds. The anion which possesses a negative charge on the β-
carbon of L-alanine is an essential species for the aldol reaction to
prepare the γ-hydroxy-α-L-amino acids. Fortunately, kynureninase (L-
kynurenine hydrolase, E.C. 3.7.1.3) was shown to generate the anion in
its natural reaction from kynurenine (1) to anthranilic acid (2) and
alanine. The reaction mixture of benzaldehyde and kynurenine (1) in the
presence of kynureninase afforded γ-phenyl-γ-hydroxy-α-L-amino-
Finally, the stereochemistry of α- and γ-carbons and the ratios of the
isomeric products were determined. All of the products were obtained as
diastereomixtures; however, the absolute configuration at the α-
positions was confirmed to retain L-configuration by means of
experiments in which prepared γ-hydroxy-α-amino acids were subjected
4
butyric acid with anthranilic acid. It seems that the β-anion of alanine
generated during the enzymatic bond cleavage between β- and γ-carbons
of kynurenine (1) attacked the carbonyl group of benzaldehyde before
being trapped with a proton from the aqueous medium. Kynureninase is
one of the best-researched enzymes in terms of biochemical study,
however, the substrate specificity of the kynureninase catalyzed carbon-
carbon bond formation has not been studied at all. In this article, we
intend to report the substrate specificity of this aldol reaction (precisely
transaldol reaction) catalyzed by kynureninase from Pseudomonas
fluorescens (ATCC 11250). Moreover, the stereochemistry of the
products is discussed in order to utilize this reaction widely and
practically in organic synthesis.
6
to reactions with D- and L-amino acid oxidase. These compounds were
substrates for L-amino acid oxidase but monitoring with TLC showed
them to be inactive toward D-amino acid oxidase. The ratios of the
diastereomers were evaluated by deriving lactones from the γ-hydroxy-
α-amino acids. For instance, the amino group of γ-furyl-γ-hydroxy-α-
amino acid (4) was protected with tert-butyloxycarbonyl group to afford
N-Boc amino acid (5), from which cesium salt was prepared by adding
Cs CO in aqueous methanol. Addition of methyl iodide into the DMF
2
3
7
8
solution of 5 afforded γ-lactones (6a, 6b ), which can be separated by
Kynureninase (ca. 7 units) prepared according to a method in the
4
literature was added into a mixture of an aldehyde (2.0 mmol) and
kynurenine (1, 0.25 mmol) in Tris. buffer(pH 8, 65 mM, 40 mL)
Miura, Tsuyoshi
