Methyl acetoacetate

The engineered E. coli strain was previously reconstructed by our group and preserved in our laboratory.^{15,16 (link)}Methyl acetoacetate (99.9% purity, MAA) was from Sigma-Aldrich (USA). Methyl (S)-3-hydroxybutyrate (98% purity, (S)-HBME), methyl (R)-3-hydroxybutyrate (98% purity, (R)-HBME), n-decane (>99.9% purity), His, Arg, Lys, Na₂CO₃ and C₆H₁₁NaO₇ (>99.9% purity) were also obtained from Sigma-Aldrich (USA). All other reagents and solvents were purchased from commercial sources and were of analytical pure grade.

Sulfamic acid, methyl acetoacetate, ammonium acetate and aromatic aldehydes were purchased from Sigma-Aldrich Chemical Co. and used without purification. All organic solvents used for the synthesis were of analytical grade. Column chromatography was performed using silica gel 60 Å (ACROS Organics, 0.035–0.070 mesh). The reactions were monitored by thin-layer chromatography (TLC) performed on glass plates coated with silica gel (Merck 60GF245); a mixture of hexane : ethyl acetate was used as the eluent, and the products were visualized using iodine vapor. The melting points were obtained using a Fisatom 430D apparatus and were reported as uncorrected values. ¹H NMR and ¹³C NMR spectra were recorded on Bruker Ascend 400 MHz operating at 400 MHz and 100 MHz, respectively, in deuterated chloroform (CDCl₃) as the solvent. The chemical shift data were reported in units of δ (ppm) downfield from tetramethylsilane (TMS), which was used as an internal standard. Infrared spectra were obtained using potassium bromide (KBr) pellets or sodium chloride (NaCl) disks on a Shimadzu-IR Prestige-21 spectrometer.