Promiscuous activity of C-acyltransferase from Pseudomonas protegens: synthesis of acetanilides in aqueous buffer

A C-acyltransferase was found to show promiscuous activity catalyzing C–N bond formation in aqueous buffer instead of C–C bond formation.

and EtOAc was used as solvent. 1 H-and 13 C-NMR spectra were recorded at 20 °C on a 300 MHz Bruker NMR or 500 MHz Bruker NMR. The conversions were measured at 25 °C by HPLC using a Shimadzu-Prominence liquid chromatograph, equipped with a SPD-M20A diode array detector and an achiral C18 column (Phenomenex Luna C18 (2) 100A (0.46 cm × 25 cm, 5 µm particle size). The following gradient elution with

Screening Procedure
Amine acceptor 1a-k (0.01 mmol, 10 mM final concentration) was suspended in potassium phosphate buffer (100 mM, pH 7.5). Then, cell-free extract of recombinant ATase (0.066 U) was added to the reaction mixture. The bioacylation was started by addition of the donor at the following final concentrations: DAPG (15 mM), IPEA (100 mM), PA (15 mM). DAPG was dissolved in DMSO (100 μL) to improve its solubility in buffer (10 vol % final concentration). The reaction mixture was shaken for 18 h at 35 ˚C and 750 rpm in an orbital shaker. Reactions were quenched by addition of acetonitrile (1 mL). The precipitated protein was removed by centrifugation (20 min, 14,000 rpm) and the supernatant was subjected to HPLC for determination of conversions. As a negative control, reactions without enzyme were performed.

Semi-preparative-scale Friedel-Crafts bioacetylation of amines
Amine (10 mM final concentration) was dissolved in potassium phosphate buffer (100 mM, pH 7.5) in a shaking flask. Cell-free extract containing the PpATaseCH (2.5 mL, 1.65 U) was added to the reaction mixture and the bioacetylation was started by adding PA (54.9 μL, 15 mM final concentration). The bioacetylation (25 mL total volume) was run at 35 °C and 140 rpm for 24 h. The resulting suspension was extracted with EtOAc (2 × 50 mL). The organic layers were pooled in a separation funnel, washed with brine (2 × 40 mL), dried over anhydrous Na2SO4 and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography using hexane: EtOAc as an eluent. Compounds were characterized by 1 H-NMR, 13 C-NMR and GC-MS.

Activity Assay
ATase-batch activities were measured on a Thermo Scientific Genesys 10 UV Scanning UV/Vis spectrophotometer according to a modified procedure from literature. 3 When following the disproportionation of MAPG into DAPG and PG spectrophotometrically, an increase of absorption is recorded due to the formation of DAPG (ε = 20 mM -1 cm -1 , λ = 370 nm). One unit of activity was defined as the µmol of product formed by an enzyme in 1 min per 1 milligram of protein under the following conditions: potassium phosphate buffer (960 µL, 100 mM, pH 7.5) and MAPG (1.2 µmol, 30 µL of a 40 mM stock solution prepared in S4 DMSO) were added to a cuvette and preheated to 35 °C. The reaction (1 mL total volume, 3 vol% DMSO) was started by the addition of the enzyme-containing cell-free extract (10 µL ≡ 1.43 mg wet cells). The reaction was followed for 1 minute. All reactions were performed as a duplicate. The protein concentration (Bradford) was measured [(ε = 0.083 mL mg -1 cm -1 , λ = 595 nm)] and specific activities were determined as units per mg protein.
Scheme S1. Reaction scheme of assay.

Purification of PpATaseCH
Purification of the PpATaseCH was achieved by size-exclusion chromatography with a Superdex 200 16/600 HiLoad-column. The column was initially washed with water, followed by conditioning (potassium phosphate buffer, 50 mM, pH 7.5, 100 mM NaCl). The cell-free extract (4 mL ≡ 0.57 g wet cells) was filtered (0.45 μm) prior to loading onto the column. The PpATaseCH eluted after ~66 min with a flow-rate of 0.75 mL min -1 and the size of the protein (~98 kDa) was determined by comparison to a GelFiltration standard (BioRad). The purity of the PpATaseCH-fractions was estimated by SDS-PAGE ( Figure S1). All enzyme-containing fractions (10 × 500 µL) were combined and concentrated to approximately 2.5 mL with a Vivaspin column (MWCO 30,000). NaCl was removed by filtration through a PD-10desalting column (final buffer = potassium phosphate buffer, 50 mM, pH 7.5) and the enzyme solution was concentrated again. Initial rates and protein concentrations were measured to determine the batch activity. In total, 13.1 mg of purified enzyme (0.1 U mg -1 ) was obtained from 0.57 g wet cells.

Chemical Synthesis -General Procedure for Synthesis of Reference Compounds
In a round-bottomed flask, a mixture of a suitable amine (1 mmol) and vinyl acetate (1 mL, 9.2 mmol) was stirred at room temperature for 24 h. The progression of the reaction was monitored by TLC. Crude reaction mixture was concentrated in vacuo and purified by column chromatography on silica gel (hexane/ethyl acetate) to afford corresponding acetamides. 2