今日推送的文章是发表在ChemBioChem上的“Synthesis of Pharmaceutically Relevant Arylamines Enabled by a Nitroreductase from Bacillus tequilensis”，通讯作者为格罗宁根大学的Gerrit J. Poelarends。
Aromatic amines are an important component in the manufacture of valuable drugs, pigments, and dyes. However, their current industrial production involves the use of chemical catalytic procedures that can have a significant impact on the environment. Therefore, flavin-dependent nitroreductase (NR) has received increasing attention as a sustainable catalyst for more environmentally friendly aromatic amine synthesis. In this study, the authors evaluated a novel NR from Bacillus tequilensis (named BtNR) for the synthesis of drug-relevant aromatic amines, including valuable synthetics for the manufacture of blockbuster drugs such as vemodegib, sonogib, linezolid and sildenafil.
Results & Discussion
The three flavonases are well-characterized NRs and amine synthesis has been reported to be initiated from nitroaromatic compounds, namely NfnB from Mycobacterium smegmatis, SNR from Streptomyces mirabilis, and NfnB from sphingosine sp. In addition, a putative NR from Bacillus tequilensis was chosen, named BtNR. The nitroreductase activity of the purified enzyme was assessed using four model substrates (1b, 1c, 1e, and 1g) and converted to the corresponding amine product by GC-MS analysis (Table 1). Nitroreductase BtNR was selected for further substrate range analysis and reaction optimization due to its satisfactory conversion to the desired amine product and its ability to outperform the other three candidate enzymes. The substrate range of BtNR was analyzed using a panel of thirteen nitro compounds, including four nitroaromatic hydrocarbons that produce amine precursors of blockbuster drugs vimodegib, sonegib, linezolid and sildenafil, as well as biaryl compounds of pharmaceutical and industrial value.
Figure 1
The enzyme is active against large volumes of nitrobenzene, nitropyridine, and nitropyrazole and has moderate to excellent amine product conversion (table 2). Notably, no regioselectivity was observed for the dinitrosubstrates 1 g and 1 J as the diamine compounds 2 g and 2 j were mainly formed from a small amount of partially reduced by-products. Under anaerobic conditions, the conversion of amines was significantly increased at 1 g (from 56% to 92%), 1 k (from 60% to 98%), and 1 l (from 2% to 18%). However, no significant differences were found for substrate 1 h (from 64% to 57%) and 1 J (from 84% to 81%). These results suggest that the presence of molecular oxygen does hinder the conversion to amines. The observation that BtNR-catalyzed aerobic biotransformation resulted in reduced nitro reduction in some substrates but not in others, suggesting that the negative effects of oxygen may be related to substrates and intermediates.
The stability of BtNR under various co-solvents and different temperatures was tested. We chose nitro compound 1a as the substrate for this analysis because of its poor solubility in aqueous buffers, so it is important to optimize its conversion to amine 2a (Figure 2A). Notably, the conversion of the final amine was almost tripled when using 20% DMSO and methanol at percentages of 10% and 20% (Figure 2A). It was found that the optimal co-solvent was DMSO at a concentration of 20%, but the enzyme also performed well in aqueous buffers containing methanol and isopropanol. The yield of the amine product increased to 21% at 37 °C, which may be due to increased substrate solubility, suggesting that low solubility in water may be a limiting factor in the conversion of compound 1a (Figure 2A).
Figure 2
Biotransformation was performed using nitro substrates 1a, 1b, 1c, 1k, 1l and 1 m, and anaerobic conditions were applied to minimize the formation of by-products. Increase the substrate concentration of the half-preparative scale reaction to 8 mM volume using 20 μM BtNR, 100 mM glucose, 1 mM NAD+, and 4 μM GDH containing 10% DMSO (10 mL total) in pH 7.0 0.1 M sodium phosphate buffer. The reaction was performed at room temperature (substrate 1a at 37°C) for 48 h. The enzymatic prepared products were purified using flash chromatography and identified as corresponding amines by 1H-NMR and HRMS analysis. The final separation yield of the amine product was determined by 1 H NMR to be 6% of product 2a, 45% of product 2b, 56% of product 2c, 44% of product 2k, 18% of product 2l and 2 m of product 13. Yields can be further improved by optimizing extraction and purification procedures.