N n dimethylaniline

Hydrolysis reactions were performed using 15 µg of phage DNA diluted to 300 µL in water. An equal volume of 4 M trifluoroacetic acid (TFA) was added and the mixture heated at 100 °C in a heating block for three hours. The samples were cooled to room temperature, lyophilized, and then reconstituted in water (60 µL). The samples were subjected to high-pH anion exchange chromatography with pulsed amperometric detection (HPAED-PAD) analysis in duplicate by injecting a 10-µL sample volume. Standards were also analyzed for identification and quantitation (the signal from 1.0 nmol of glucose gave a peak area of 20.0 and therefore a response of 0.05 nmol/area). Dionex analysis was carried out on an ICS5000 instrument using a Carbopac™ PA10 (4 × 250 mM) (Sunnyvale, CA, USA) analytical column. Samples were eluted using an isocratic 18 mM NaOH solution.
Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis was performed on a Bruker UltrafleXtreme MALDI-TOF/TOF mass spectrometer (Sunnyvale, CA, USA)in positive reflector mode. Thus, 100 mg/mL of 2,5-dihydroxybenzoic acid (Sigma-Aldrich, St. Louis, MO, USA) matrix was prepared in 1:1 H₂O/acetonitrile solution, with additional 20 μL of N,N-dimethylaniline (Sigma-Aldrich).

In order to monitor the volatile intermediates and by-products (mainly, the most concerning aromatic amines), the samples were injected in a Shimadzu GC-2010 gas chromatograph provided with flame ionization detector (FID) and split injection mode; 5.0 mL of sample were analyzed in this case. Three successive extractions were conducted with 3.0 mL portions of ethyl acetate (Panreac, 99.5%); the sample was concentrated in a Heidolph rotaevaporator at 40°C up to 2.0 mL, and then measured. Separations were made under a temperature program by using an PTA-5 Fused Silica capillary column (30 m × 0.53 mm × 1.5 μm from SUPELCO). The GC conditions of separation were: Detector and injector temperature: 220°C, carrier gas He, make up gas N₂ (40 mL/min), oven temperature program: 130–150°C under a ramp of 1.0°C/min, then 150–180°C under a ramp of 10°C/min, and finally 1 min at 180°C. Following standards were used to calculate response factors: Phenylamine (Mallinckrodt, 99.99%), N-methylaniline (Sigma-Aldrich, ≥99%), N,N-dimethylaniline (Sigma-Aldrich, 99.57%), N,N-dimethyl-p-phenylenediamine (Alfa Aesar, 96%), 3-Dimethylaminophenol (Alfa Aesar, 97%).

All reagents were purchased at the highest commercially available purity and were used without further purification processes. Bovine serum amine oxidase (amine: O₂ oxidoreductase, deaminating, E.C. 1.4.3.6) was purified according to [7 (link)]. Tannic acid, spermine, 4-amino-antipyrine, horseradish peroxidase type II (HRP, 179 U/mg), N,N-dimethylaniline were obtained from Sigma-Aldrich/Merck (St. Louis, MO, USA). Water for sample preparation and analysis was obtained from a Genie Direct-Pure (RephiLe Bioscience Ltd., Shanghai, China) ultrapure water device with a resistivity of at least 18 MΩ cm⁻¹. A series of Nd-Fe-B magnets (N35, 263–287 kJ/m3 BH, 1170–1210 mT flux density by Power magnet—Germany) was used to magnetically recover the particles.

The pCW-3A4-BMR plasmid containing the gene of human P450 3A4 fused to the reductase of cytochrome P450 BM3 from B. megaterium (BMR) linked via a Pro-Ser-Arg linker was available in our laboratory (Dodhia et al., 2006 (link)). GenElute plasmid miniprep kit was obtained from Sigma (Italy). PCR purification kit and gel extraction kit were purchased from Macherey-Nagel (Germany).
The primers for PCR and oligonucleotides for the linker region between P450 3A4 and BMR reductase were prepared by MWG (Germany). DNA Ladder, restriction endonucleases enzymes Avr II and Hind III, Vent polymerase, T4 DNA ligase enzyme and buffers used in sub-cloning were from New England Biolabs (UK) and Promega (Italy). Horse heart cytochrome c, erythromycin, testosterone, 6β-hydroxytestosterone, horseradish peroxidase type X, superoxide dismutase from bovine erythrocytes, catalase, N,N-dimethylaniline, and 4-amino-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one were purchased from Sigma (Italy). NADPH (tetrasodium salt) was acquired from Calbiochem (Germany). Human CPR was purchased from Life Technologies. All other chemicals, biochemicals used in this study were purchased from Sigma (Italy) at the highest available grade and used as recommended by the manufacturer.

Anhydrous dichloromethane (DCM, spectroscopic grade ≥ 99.8%), aniline (99.5%), N-methylaniline (98%), N,N-dimethylaniline (99%), benzylamine (99%), and toluene (anhydrous, 99.8%) were purchased from Sigma-Aldrich and used without further purification.