Cytochrome aliphatic hydroxylation

The metabolism of foreign compounds (xenobiotics) often takes place in two consecutive reactions, classically referred to as phases one and two. Phase I is a functionalization of the lipophilic compound that can be used to attach a conjugate in Phase II. The conjugated product is usually sufficiently water-soluble to be excretable into the urine. The most important biotransformations of Phase I are aromatic and aliphatic hydroxylations catalyzed by cytochromes P450. Other Phase I enzymes are for example epoxide hydrolases or carboxylesterases. Typical Phase II enzymes are UDP-glucuronosyltrans-ferases, sulfotransferases, N-acetyltransferases and methyltransferases e.g. thiopurin S-methyltransferase. 

The numerous biotransformations catalyzed by cytochrome P450 enzymes include aromatic and aliphatic hydroxylations, epoxidations of olefinic and aromatic structures, oxidations and oxidative dealkylations of heteroatoms and as well as some reductive reactions. Cytochromes P450 of higher animals may be classified into two broad categories depending on whether their substrates are primarily endogenous or xenobiotic substances. Thus, CYP enzymes of families 1-3 catalyze... 

Groves JT, McClusky GA. Aliphatic hydroxylation by highly purified liver microsomal cytochrome P-450. Evidence for a carbon radical intermediate. Biochem Biophys Res Commun... 

Groves, J. T., McClusky, G. A., White, R. E., and Coon, M. J., 1978, Aliphatic hydroxylation by highly purified liver microsomal cytochrome P-450. Evidence for a carbon radical intermediate, Biochem. Biophys. Res. Commun. 81 154nl60. 

Finally, Fig. 4.88 presents two examples of such radical-type mechanisms catalyzed by cytochromes P450 and leading to aliphatic hydroxylation, through the so-called oxygen-rebound mechanism, and epoxidation, respectively. 

Fig. 4.88. Proposed radical-type reaction mechanisms for (a) aliphatic hydroxylation and (b) epoxidation by the high-valent iron-oxo porphyrin intermediate of cytochromes P450. The mechanism depicted in (a) is called the "oxygen-reboimd" mechanism.

Loida, P.J. and S.G. Sligar (1993). Engineering cytochrome P-450cam to increase the stereospecificity and coupling of aliphatic hydroxylation. Protein Eng. 6, 207-212. 

In addition to epoxidation, Tabushi s system also catalyzes aromatic and aliphatic hydroxylation, as well as dealkylation of tertiary amines [81], i.e. all of the important types of reactions that are catalyzed by cytochrome P-450. A practical disadvantage of this artificial P-450 catalytic system is that the active oxometal species is to a considerable extent (typically 80-90%) consumed for water production via interaction with the reducing agent Pt/H. ... 

White RE, Miller JR Favreau LV, Bhattacharyaa A (1986) Stereochemical dynamics of aliphatic hydroxylation by cytochrome P-450. J Am Chem Soc 108 6024-6031... 

The Use of Primary Kinetic Isotope Effects to Probe the Mechanism of Aliphatic Hydroxylation by Iron(III) Porphyrins One reaction that is uncommon in organic chemistry but is common in biological systems in the hydroxylation of alkanes to form alcohols. Cytochrome P-450, a heme containing enzyme, catalyzes this reaction. Large primary isotope effects are found, as well as an absence of carbocation-like skeletal rearrangements and loss of stereochemistry. These observations led researchers to conclude that a radical cage mechanism is operative. 

Aliphatic Oxidation and O-Dealkylation - Cytochrome P-450 monoxygenases appear to be involved in a variety of aliphatic hydroxylations. One common pathway involves an initial w-hydroxylation as with pentobarbital, followed by further metabolism to the aldehyde and acid by dehydrogenases. Hydroxylation also frequently occurs at the penultimate carbon as in amobarbital l, at benzylic and allylic positions and at carbons bearing a heteroatom. Many examples of such oxidations are cited in a recent review.Hydroxylation at carbon bearing a heteroatom such as oxygen, nitrogen or sulfur leads to the so-called dealkylation reactions so common in drug metabolism. 

The mechanism of aliphatic hydroxylation by cytochrome monoxygenases would appear to Involve a direct oxygen-insertion with retention of configuration. For exan le, when S-(+)-l- H-ethylbenzene was incubated with liver microsomes, the 1-phenylethanol produced in the reaction consisted of 92% of the R isomer and only 87. of the S isomer.83 pheno-barbital pretreatment diminished the stereoselectivity of this reaction. 63, 64 ihe reaction of the a a-dideutero-derivative exhibited an isotope effect of 1.8 when compared to the undeuterated substrate, consistent with an oxygen-insertion mechanism. In certain examples of aliphatic hydroxylation, however, it appears from the absence of an isotope effect with labeled substrates that the insertion reaction is not the rate-limiting step.85-67... 

Cytochrome P-450. Cytochrome P-450 enzymes consist of a large number of haem-containing mono-oxygenases which catalyze aliphatic and aromatic hydroxylations, epoxidations, as well as other oxidation reactions thus, these enzymes are able to cleave aromatic C-H bonds and also... 

In mammalian liver microsomes, cytochrome P-450 is not specific and catalyzes a wide variety of oxidative transformations, such as (i) aliphatic C—H hydroxylation occurring at the most nucleophilic C—H bonds (tertiary > secondary > primary) (ii) aromatic hydroxylation at the most nucleophilic positions with a characteristic intramolecular migration and retention of substituents of the aromatic ring, called an NIH shift,74 which indicates the intermediate formation of arene oxides (iii) epoxidation of alkenes and (iv) dealkylation (O, N, S) or oxidation (N, S) of heteroatoms. In mammalian liver these processes are of considerable importance in the elimination of xenobiotics and the metabolism of drugs, and also in the transformation of innocuous molecules into toxic or carcinogenic substances.75 77... 

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