Enzymes nitro compound reduction

The enantiomeric reduction of 2-nitro-l-phenylprop-l-ene has been studied in a range of Gram-positive organisms including strains of Rhodococcus rhodochrous (Sakai et al. 1985). The enantiomeric purity of the product depended on the strain used, the length of cultivation, and the maintenance of a low pH that is consistent with the later results of Meah and Massey (2000). It has been shown that an NADPH-linked reduction of a,p-unsaturated nitro compounds may also be accomplished by old yellow enzyme via the flcf-nitro form (Meah and Massey 2000). This is formally analogous to the reduction and dismutation of cyclic enones by the same enzyme (Vaz et al. 1995), and the reductive fission of nitrate esters by an enzyme homologous to the old yellow enzyme from Saccharomyces cerevisiae (Snape et al. 1997). 

Reduction of 3-nitroacrylates 43 by Saccharomyces carlshergensis Old Yellow Enzyme is the key step in the synthesis of optically pure P-2-amino acids (Scheme 2.18). High yields and enantioselectivities of the initially derived nitro-compounds 44 were achieved. The latter can be further chemically elaborated to the corresponding P-amino acids 45 [20]. 

The excretion of amines is unusual in animals. Amines are highly toxic and one method employed by vertebrates to detoxify them is via monoamine oxidase, an enzyme which has been detected in H. diminuta (569). Amines can arise from the decarboxylation of the appropriate amino acid, e.g. glycine and alanine can give rise to methylamine and ethylamine, respectively. Another possible source of amines may be the reduction of azo or nitro compounds (39) and azo- and nitro-reductase activity has been reported from M. expansa (180, 181). Furthermore, the physiologically active amines octopamine, dopamine, adrenalin and serotonin (5-hydroxytryptamine) have been demonstrated in cestodes (283, 296, 435, 681, 682, 758, 859), where they probably function predominantly as neurotransmitters (see Chapter 2). 

Nitrobenzene reductase activity has been detected in the fat body, gut, and Malpighian tubules of the Madagascar cockroach, G. portentosa (Rose and Young, 1973). Anaerobic conditions are essential for activity. The enzymes in the microsomes are strongly NADH dependent, whereas those in the soluble fraction are strongly NADPH dependent. Activity is enhanced by the addition of flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) or riboflavin. It appears that the true substrate for the nitroreductase is FMN and that the reduction of the nitro compounds occurs nonenzymatically (Figure 8.15). Similar results are obtained using azofuchsin as substrate. 

Non-cytochrome P450 enzymes may also be involved in oxidative reactions. One such enzyme is alcohol dehydrogenase whose substrates include vitamin A, ethanol, and ethylene glycol. Aldehyde dehydrogenase is another enzyme. Most reduction reactions also involve microsomal enzymes, with the exception of ketone reduction. Nitro compounds are reduced to amines and volatile anesthetics undergo dehalo-genation by microsomal enzymes. Hydrolysis reactions are involved in metabolism of compounds with amide bonds or ester linkages, as in the conversion of aspirin to salicylate (Brown, 2001). 

Baker s yeast reduces conjugated nitro compounds to nitroalkanes and also the C=C unit of conjugated ketones. Other enzymatic reductions are possible. A reductase from Nicotiana tabacum reduced a conjugated ketone to the saturated ketone, with excellent enantioselectivity. Enzyme YNAR-I and NADP-H reduces conjugated nitro compounds to nitroalkanes. ... 

The types of nitrogen-containing compounds that are most frequently involved in reductive biotransformation are those containing nitro, azo, and N-oxide functional groups. Similar enzymes are involved that are generally located in the endoplasmic reticulum or cytosol of the liver or in the intestinal microflora. Complete reduction of a nitro compound to the primary amine involves a six-electron transfer and proceeds through nitroso and hydroxylamine intermediates [Eq. (16)]. 

Polymer-supported enzymes have been combined with polymer-supported reagents in the synthesis of the bryostatins. The nitrone derived from the nitro compound 43 supported on a soluble aryl poly-ether polymer undergoes an efficient 1,3-dipolar cycloaddition with butenone to give the isoxazoline 44 and hence by reduction the racemic syn compound 45 required for the synthesis.16... 

The enzymes responsible for reduction may be located in both the microsomal fraction and the soluble cell fraction. Reductases in the microflora present in the gastrointestinal tract may also have an important role in the reduction of xenobiotics. There are a number of different reductases which can catalyse the reduction of azo and nitro compounds. Thus, in the microsomal fraction, cytochromes P-450 and possibly a flavoprotein are capable of reductase activity. NADPH is required, but the reaction is inhibited by oxygen. FAD alone may also catalyse reduction by acting as an electron donor. 

Reductive biotransformations of several compounds such as polyhalogenated, keto, nitro and azo derivatives, are catalysed by a variety of enzymes which differ according to the substrates and the species. The liver cytochrome P-450-dependent drug metabolizing system is capable of reducing Af-oxide, nitro and azo bonds, whereas the cytosolic nitrobenzene reductase activity is mainly due to cytochrome P-450 reductase, which transforms nitrobenzene into its hydroxylamino derivative. NADPH cytochrome c reductase is also able to catalyse the reduction of nitro compounds. These metabolic conversions may also be brought about by gastrointestinal anaerobic bacteria. 

In addition to the oxidative systems, liver microsomes also contain enzyme systems that catalyze the reduction of azo and nitro compounds to primary amines. A number of azo compounds, such as Prontosil ahd sulfasalazine (Fig. 10.16), are converted to aromatic primary amines by azoreductase, an NADPFI-dependent enzyme system in the liver microsomes. Evidence exists for the participation of CYP450 in some reductions. Nitro compounds (e.g., chloramphenicol and nitrobenzene) are reduced to aromatic primary amines by a nitroreductase, presumably through hitrosamine and hydroxylamine intermediates. These reductases are not solely responsible for the reduction of azo ahd hitro compouhds reductioh by the bacterial flora ih the anaerobic environment of the intestine also may occur. 

The main reason for this is almost certainly the low nitro group reduction potentials of these compounds. The 4-NOj derivative (3) has a reduction potential E(l) of about -500 mV (12), which is likely to be too low for efficient enzymic nitroreduction, and that of the 3-NOj compound (E(l) = -470 mV) is similar. Future work in this area is therefore being directed towards the development of more soluble derivatives with higher nitro group reduction potentials. 

B. Reduction The enzymology of reduction is not as well as characterized as for oxidation but, for example, reductive reactions can be catalyzed by cytochrome P-450 and P-450 reductase and soluble enzymes such as DT-diaphorase (EC 1.6.99.2) Many compounds including azo-and nitro-compounds, epoxides, heterocycles and halogenated hydrocarbons Sources of reducing equivalents for the reactions include NAD PH and NADH. Chemical groups modified include nitro, nitroso, tertiary amine oxide, hydroxylamine, azo, quinone, nitroso, alkylhalide... 

In addition to direct oxygenation, e.g. by aryl hydrocarbon hydroxylase, oxidative N- or 0-dealkylation is another process catalyzed by components of the Cytochrome P-450 System (mixed-function oxidases). Reduction also occurs in this system NADPH-cytochrome P-450 reductase has an activity similar to microsomal nitroreductase, i.e. transformation of aronaatic nitro compounds into the corresponding arylamines takes place. The oxidation may be followed by other enzymic reactions, e.g. epoxides are hydrated to vicinal diols by microsomal epoxide hydratase or they are coupled with glutathione by glutathione-S-epoxide transferase. 

Other enzymes catalyze similar reactions in the further reduction of nitrite, hydroxylamine, and organic nitro compounds. 

Hepatic microsomes possess an enzyme system which catalyses the reduction of nitro compounds such as chloramphenicol and nitrobenzene to the corresponding amines. This system requires NADPH and is strongly inhibited ( 80%) by O2 in air, possibly because the hydroxylamine intermediates undergo autoxidation readily. It is also inhibited by CO suggesting that the reaction is mediated by cytochrome P-450 ... 

PETN reductase appears to be an industrially viable enzyme due to its robustness [10,44]. It catalyzes the reduction not only of aromatic nitro compounds, but also of activated alkenes such as unsaturated aldehydes and ketones [10,44]. Of particular synthetic interest is its ability to catalyze the reduction of prochiral E- and Z-a,(3-unsaturated nitroolefins leading to chiral nitro products. The mechanism is shown in Figure 5.7. 

The enzymatic reduction of the nitro group involves the stepwise addition of six reducing equivalents potentially derived from reduced pyridine nucleotides (Fig. 8). The first reaction yields a nitroso derivative which is subsequently reduced to a hydroxylamine the hydroxylamino compound is then reduced to the amine. In most systems studied to date (Cemiglia and Somerville, this volume) a single nitroreductase enzyme is responsible for all three reactions and there is little or no accumulation of the intermediates. However, reduction of nitro compounds does not seem to be the physiological function of the enzymes that have been reported to carry out these reactions. Diaphorases (23), ferredoxin-NADPH reductase (33), and a variety of other enzymes from procaryotes and eucaryotes have been shown to catalyze the fortuitous reduction of aromatic nitro groups. 

The occurrence of the nitro radical anion during transformation of nitro compounds with crude extracts or enzyme preparations of strictly anaerobic bacteria has been detected by Angermaier and Simon (2,3). The authors described the formation of the dianion radical of 4-nitrobenzoate and further reduction reactions that may occur. The nitro radical anion is subject to dismutation with the concomitant formation of the nitroso derivative and the parent nitro compound. This dismutation reaction occurs in competition with further one-electron or two-electron reduction reactions (Fig. 4). According to Angermaier and Simon (3), chemical reduction and dismutation reactions may interfere with the reduction mediated by nitro-reducing enzymes. 

There are several reports of enzymes involved in reduction of nitro compounds by strictly anaerobic organisms. According to these reports, nitro reduction can be carried out by different anaerobic systems NAD(P)H-dependent reductases, the physiological function of which is still unclear (2, 25) extracellular nitroreductases from human gastrointestinal... 

Angermaier, L., F. Hein, and H. Simon. 1981. Investigations on the reduction of aliphatic and aromatic nitro compounds by Clostridium species and enzyme systems, p. 266-275. In Bothe, H., and A. Trebst, (ed.) Biology of inorganic nitrogen and sulfur. Springer, Berlin. 

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