Peroxidase Catalytic Nitration Of Aromatic Compounds

Nitroaromatic compounds are important intermediates in organic synthesis. The classical method used to synthesize them is chemical nitration, that involves the use of nitric acid diluted in water or acetic acid which is harmful to the environment. With the development of green chemistry, environmental friendly enzyme catalysis is increasingly employed in chemical process. However the solubility of organic substrates is a difficult problem for bioconversion. Low substrate concentration would influence enzyme catalytic efficiency, and high concentration of substrate may inhibit enzyme activity, even make the enzyme inactivation. To solve the above problems, we carried out the non-aqueous medium researching the enzyme catalytic nitration of phenol. This reaction medium solved the problem of organic substrate insolubility and the formation of by-products. The water-organic two-phase enzymatic nitration system was established.We first investigated the effects of reaction conditions on the enzyme catalytic nitration, the optimum reaction conditions were temperature 25°C and neutral pH7; when phenol and H2O2 had the same concentration, the desired nitrated products were the highest, high concentration of H2O2 would inhibit the activity of enzyme; the amount of sodium nitrite was used in at least 10-fold molar excess over phenol, the polymerization of phenol was not serious; simultaneously, the application of the water-organic two-phase system could also alleviate the inhibitory effect of larger amount of substrates to some extent. A typical reaction time-course for product accumulation in the nitration of phenol showed that the reaction reached completion nearly within 1h. The termination of the reaction was due to the inactivation of enzyme and the consumption H2O2. 12 mM phenol was added to the reaction system at one time, then supplying the same amount of HRP and H2O2 as the first time every hour, after a continuous reaction of 15 h, the total production of o-nitrophenol and p-nitrophenol could be more than 70%.On the basis of above reaction conditions, we investigated the broad spectrum of aromatic compounds that could be nitrated by enzyme catalysis and found that aromatics containing substituents of-NH2 or-OH were easy to be catalyzed. Furthermore, the analysis of the products distribution characteristics of enzymatic nitration demonstrated that the ortho and para positions of substituents in the benzene ring were the favored nitration sites. This phenomenon was in consistence with the distribution characteristics of charges on carbon atoms in benzene ring calculated by the density functional method. So the enzyme catalytic nitration is similar to the selectivity of chemical nitration.In order to understand the mechanism of peroxidase catalytic nitration, we also studied the kinetics of enzymatic nitration of phenol. According to peroxidase catalytic mechanism, double substrates ping-pong kinetic model was established. By investigating the relationship between substrate concentrations and nitration reaction rate, we found the existence of H2O2 inhibition. We selected suitable substrate concentration and determined that the no inhibition reaction process was in line with double substrates ping-pong mechanism. The kinetic parameters were Km H2O2 = 1.09 mM, Km PhOH = 9.45 mM, Vmax = 0.196 mM·min-1, and the kinetic equation was obtained. Finally we used the nonlinear fitting method to compare the experimental data with the model calculation values and found that the fitting result was good, fitting degree R2 = 0.98732.