10.1002/cctc.201801413
ChemCatChem
COMMUNICATION
Cascade reactions with METase–eBCAT or METase–DATA
For this, a solution containing 50 mM L-Met, 50 mM L-Glu or D-Ala in
phosphate buffer (50 mM pH 7 with 1 mM PLP) was prepared. This
solution was adjusted to pH 7 with 10 N NaOH. To this reaction solution
lyophilized enzymatic extracts were added (1 mg/mL METase and 1
mg/mL eBCAT or DATA). Samples were taken after 0, 5, 10, 15, 20, 30,
60, 90, 120, 1440 and 2880 min. All analyzes were performed by HPLC
OPA-MCE derivatization.
Acknowledgements
We thank the Brazilian foundation of Coordination for the
Improvement of Higher Education Personnel (CAPES) for the
research scholarship.
Keywords: asymmetric synthesis, cascade reaction, L-
methionine γ-lyase; amino acid aminotransferase, L- and D-
homoalanine
Preparative-scale synthesis of L- and D-homoalanine
For the cascade reaction with METase and eBCAT or METase and
DATA on a larger scale, 0.3 g (2 mmol) of L-Met and 0.296 g (2 mmol) of
L-Glu (in the case of eBCAT) and 0.1782 g (2 mmol) of D-Ala (in the case
of DATA) were used. A final concentration of 1 mg/mL of each enzyme
was used. After the reaction, the product was derivatized with di-tert-butyl
dicarbonate (Boc). For the derivatization step, 1.2 eq. of Boc and 2 eq. of
K2CO3 were added for each eq. of amino acids in the reaction. The
reaction mixture was solubilized in THF:H2O (1:1) and monitored for 24 h.
After the end of the reaction, the supernatant was evaporated and the
remaining solid was resuspended in ethanol and subjected to a silica
column using a butanol:H2O:acetic acid (3:1:1) mixture as the mobile
phase. The fractions were monitored by TLC (ninhydrin staining),
relevant fractions were pooled and the solvent was evaporated. This
gave 0.1347 g (32.5% conversion) in the case of eBCAT reactions and
0.3577 g (87.5% conversion) in the case of DATA. Product identity was
confirmed by NMR spectroscopy.
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containing 40 mM o-phthalaldehyde (OPA) and 1% (v/v) 2-
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Chiral HPLC analyses
For the determination of enantiomeric excess the D,L-homoalanine was
derivatized using the methodology described by Nimura and coworkers.31
For analysis a derivatization solution containing 20 µL 2,3,4,6-tetra-O-
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