Carbon double bonds, cytochrome

Insertion of oxygen atom from Cpd I into the carbon-carbon double bond with formation of epoxide (Scheme Ic) reveals features characteristic for a concerted process, although formation of radical intermediates is possible in many cases. A unified description of this alternative is also provided by the two-state mechanism of catalysis by Cpd I (see the section on Hydroxylation of hydrocarbons). Essentially, the concerted oxygen insertion represents a low-spin reaction surface, whereas the distinct radical intermediate is formed on the high-spin reaction pathway. In the latter case, the carbon radical may attack the nearby heme nitrogen and modify the heme covalently. This reaction is also an important inactivation pathway of cytochromes P450 during oxidative transformations of terminal double and triple bonds. 

For the enantioselective preparations of chiral synthons, the most interesting oxidations are the hydroxylations of unactivated saturated carbons or carbon-carbon double bonds in alkene and arene systems, together with the oxidative transformations of various chemical functions. Of special interest is the enzymatic generation of enantiopure epoxides. This can be achieved by epoxidation of double bonds with cytochrome P450 mono-oxygenases, w-hydroxylases, or biotransformation with whole micro-organisms. Alternative approaches include the microbial reduction of a-haloketones, or the use of haloperoxi-dases and halohydrine epoxidases [128]. The enantioselective hydrolysis of several types of epoxides can be achieved with epoxide hydrolases (a relatively new class of enzymes). These enzymes give access to enantiopure epoxides and chiral diols by enantioselective hydrolysis of racemic epoxides or by stereoselective hydrolysis of meso-epoxides [128,129]. 

Oxidative attack on unsaturated aliphatic systems. Carbon-carbon double bonds are oxidized by cytochromes P450 to reactive epoxides. Thus, vinyl chloride yields epoxychlor-ethane, an alkylating metabolite which can for example alkylate nucleic acids (Fig. 31.10). 

The cytochrome P450-catalyzed oxidation of nonaromatic carbon-carbon double bonds usually, but not always, results in formation of the corresponding epoxide. Epoxidation, as demonstrated by early experiments on the oxidation of olefins such as cw-stilbene [170], oleic acid [171], and ti-a 5 -[l- H]-l-octene [172], invariably proceeds with retention of the olefin ste-reochentistiy. To date, no example is known of a P450-catalyzed epoxidation that does not proceed with retention of stereochemistry. This retention of stereochentistiy argues for a mechanism in which the transition state involves interactions of... 

Scheme 10.8 Biosynthesis of epothilone. Individual PKS domains are represented as circles and individual NRPS domains as hexagons. Acyl carrier proteins (ACPs) and thiola-tion domains (T) are posttranslationally modified by a phos-phopantetheinyl group to which the biosynthetic intermediates are covalently bound throughout the chain assembly. The thioesterase domain (TE) cyclizes the fully assembled carbon chain to give the 16-membered lactone. Following dehydration of Cl 2—Cl 3 to give epothilones C and D, the final step in epothilone biosynthesis is the epoxidation of the C12=C13 double bond by the cytochrome P450 enzyme P450epol<. KS ketosyn-thase KS(Y) active-site tyrosine mutant of KS AT acyltransfer-ase C condensation domain A adenylation domain ...

The hybridization of the carbon in an alkene makes it even more difficult to break the carbon-hydrogen bond of a vinylic carbon than of a saturated carbon. As a consequence, cytochrome P450, rather than abstracting a hydrogen atom, catalyzes the addition of an oxygen atom to the double bond leading to the formation of an epoxide as shown in Figure 4.72. 

A less common reactive species is the Fe peroxo anion expected from two-electron reduction of O2 at a hemoprotein iron atom (Fig. 14, structure A). Protonation of this intermediate would yield the Fe —OOH precursor (Fig. 14, structure B) of the ferryl species. However, it is now clear that the Fe peroxo anion can directly react as a nucleophile with highly electrophilic substrates such as aldehydes. Addition of the peroxo anion to the aldehyde, followed by homolytic scission of the dioxygen bond, is now accepted as the mechanism for the carbon-carbon bond cleavage reactions catalyzed by several cytochrome P450 enzymes, including aromatase, lanosterol 14-demethylase, and sterol 17-lyase (133). A similar nucleophilic addition of the Fe peroxo anion to a carbon-nitrogen double bond has been invoked in the mechanism of the nitric oxide synthases (133). 

Enzyme complexes occur in the endoplasmic reticulum of animal cells that desaturate at A5 if there is a double bond at the A8 position, or at A6 if there is a double bond at the A9 position. These enzymes are different from each other and from the A9-desaturase discussed in the previous section, but the A5 and A6 desaturases do appear to utilize the same cytochrome b5 reductase and cytochrome b5 mentioned previously. Also present in the endoplasmic reticulum are enzymes that elongate saturated and unsaturated fatty acids by two carbons. As in the biosynthesis of palmitic acid, the fatty acid elongation system uses malonyl-CoA as a donor of the two-carbon unit. A combination of the desaturation and elongation enzymes allows for the biosynthesis of arachidonic acid and docosahexaenoic acid in the mammalian liver. As an example, the pathway by which linoleic acid is converted to arachidonic acid is shown in figure 18.17. Interestingly, cats are unable to synthesize arachidonic acid from linoleic acid. This may be why cats are carnivores and depend on other animals to make arachidonic acid for them. Also note that the elongation system in the endoplasmic reticulum is important for the conversion of palmitoyl-CoA to stearoyl-CoA. 

From Iron(III) Tetraarylporphyrins and Alkenes. N-alkyl porphyrins are formed via side reactions of the normal catalytic cycle of cytochromes P-450 with terminal alkenes or alkynes. N-alkylpor-phyrins formed from terminal alkenes (with model iron porphyrin catalysts under epoxidation conditions) usually have a covalent bond between the terminal carbon atom of the alkene and a pyrrole nitrogen. The double bond is oxidized selectively to an alcohol at the internal carbon. Mansuy (23) showed that, in isolated examples, terminal alkenes can form N-alkylated products in which the internal carbon is bound to the nitrogen and the terminal carbon is oxidized to the alcohol. Internal alkenes may also form N-alkyl porphyrins (24, 25). 

In many of the haemoproteins we shall be discussing, the protoporphyrin IX group is held to the polypeptide chain only by hydrogen bonding, Van der Wools forces and iron-protein bonds. In several other cases, notably in cytochrome-c and its related compounds, the haem is covalently linked to the protein via substituents at the pyrrole carbon atoms. Cyto-chrome-c can be regarded as an iron protoporphyrin IX group with the addition of a protein cysteine side-chain across the vinyl double bonds giving two thio-ether links (Fig. 3). 

Free radicals from certain halogenated xenobiotics Incorporate Into phospholipid. Trudell et al. (81,82) have shown that free radicals from carbon tetrachloride and halothane add to the double bonds of fatty acyl chains of phospholipids In the membrane surrounding cytochrome P-450. Oleic acid moieties were converted... 

Desaturation of fatty acids involves a process that requires molecular oxygen (O2), NADH, and cytochrome dj. The reaction, which occurs in the endoplasmic reticulum, results in the oxidation of both the fatty acid and NADH (Fig. 33.18). The most common desaturation reactions involve the placement of a double bond between carbons 9 and 10 in the conversion of palmitic acid to palmitoleic acid (16 1, A ) and the conversion of stearic acid to oleic acid (18 1, A ). Other positions that can be desaturated in humans include carbons 4, 5, and 6. 

Insertion of the C5,6 double bond (A -> A ) involves removal of the 5a (ex 4-pro-R H of MVA) and 6a (ex 5-pro-S H of MVA) hydrogens. In rat liver and yeast microsomal preparations the reaction requires aerobic conditions and NADH or NADPH and both of the eliminated hydrogens are found in HjO. This suggests a mechanism in which hydroxyla-tion at CS or C6 is followed by dehydration across these two carbons or, alternatively, a fatty acid-type desaturation. There is evidence of the former in yeast, whereas the latter seems more likely in rat liver where the multienzyme complex catalysing it consists of the A -desaturase itself, cytochrome and an NAD(P)H-dependent flavoprotein the A -desaturase is a monooxygenase whieh uses electrons derived from NAD(P)H, via the flavoprotein and cytochrome i>5, and O2 to effect the removal of the two hydrogens from the sterol. 

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