Heme N-alkylation

Dexter A, Hager L. Transient heme N-alkylation of chloroperoxidase by terminal alkenes and alkynes. J Am Chem Soc 1995 117 817-818. 

Chloramphenicol and secobarbital exhibit properties similar to those of tienilic acid, but they have not been studied in humans (11). Oxidative dechlorination of chloramphenicol with formation of reactive acyl chlorides appears to be an important metabolic pathway for irreversible inhibition of CYP. Chloramphenicol binds to CYP, and subsequent substrate hydroxylation and product release are not impaired. The inhibition of CYP oxidation and the inhibition of endogenous NADPH oxidase activity suggest that some modification of the CYP has taken place, which inhibits its ability to accept electrons from the CYP reductase (11). Secobarbital completely inactivates rat CYP2B1 functionally, with partial loss of the heme chromophore. Isolation of the N-alkylated secobarbital heme adduct and the modified CYP2B1 protein revealed that the metabolite partitioned between heme N-alkylation, CYP2B1 protein modification, and epoxidation. A small fraction of the prosthetic heme modifies the protein and contributes to the CYP2B1 inactivation (12). 

P-450 has been shown to catalyze epoxidation with retention of the olefin configuration (114). Ortiz de Montellano and co-woiicers have shown that heme N-alkylation accompanies epoxidation when terminal olefins are oxidized by P-450 (775). Further, the oxidation of 1,1,2-trichloroethylene is known to give trichloroacetaldehyde along with epoxide (776, 777). A mechanism that explains simultaneous epoxidation, heme alkylation, and halogen migration is depicted in Scheme XVI (777). In this process, initial electron transfer affords a transient rr-radical cation that can collapse with C-0 bond formation to give either radical or cation intermediates. 

The details of heme N-alkylation by alkyl radicals are unclear because no intermediates have been detected in the reaction. However, the fact that reaction... 

Collman, J.P., P.D. Hampton, and J.l. Brauman (1990). Suicide inactivation of cytochrome P-450 model compounds by terminal olefins. Part I A mechanistic study of heme N-alkylation and epoxidation. J. Am. Chem. Soc. 112, 2977-2986. 

N-substituted iron porphyrins form upon treatment of heme enzymes with many xenobiotics. The formation of these modified hemes is directly related to the mechanism of their enzymatic reactivity. N-alkyl porphyrins may be formed from organometallic iron porphyrin complexes, PFe-R (a-alkyl, o-aryl) or PFe = CR2 (carbene). They are also formed via a branching in the reaction path used in the epoxidation of alkenes. Biomimetic N-alkyl porphyrins are competent catalysts for the epoxidation of olefins, and it has been shown that iron N-alkylporphyrins can form highly oxidized species such as an iron(IV) ferryl, (N-R P)Fe v=0, and porphyrin ir-radicals at the iron(III) or iron(IV) level of metal oxidation. The N-alkylation reaction has been used as a low resolution probe of heme protein active site structure. Modified porphyrins may be used as synthetic catalysts and as models for nonheme and noniron metalloenzymes. 

In the early 1970s it was discovered that P-450 cytochromes are irreversibly inhibited during the metabolism of xenobiotics (1). The formation of a modified heme prosthetic group is associated with enzyme inhibition and subsequent studies have identified these modified complexes as N-alkylated protoporphyrin-IX (2). The chemistry of N-sub-stituted porphyrins was comprehensively reviewed by Lavallee in 1987 (3). Since that time, there have been many significant contributions to this field by several groups. The goal of this chapter is to summarize some of this work as it relates to the mechanism of formation and reactivity of iron N-alkyl porphyrins. Biomimetic model complexes have played an important role in elucidating the chemistry of N-alkyl hemes in much the same way that synthetic iron tetraarylporphyrins have aided... 

The N-alkylation reaction represents a bifurcation of the normal alkene epoxidation reaction cycle and, therefore, N-alkylation is a suicide event that leads to catalytic inhibition in the native system. With synthetic tetraarylporphyrins that mimic the N-alkylation reaction, the use of halogen-substituted catalysts that are stable toward oxidative degradation (26, 27) provide the most useful model systems because the heme model remains intact for a significantly greater number of turnovers than the partition number. The partition number is the ratio of epoxidation cycles to N-alkylation cycles, i.e., N-alkyl porphyrins are formed before the heme is oxidatively destroyed. 

Traylor (38) has also shown that biomimetic iron N-alkylporphyrins themselves are competent catalysts for epoxidation of alkenes with a rate constant of about 104 M-1 s-1. On the basis of these observations and rearrangement reactions of specific alkenes, Traylor has proposed the reaction sequence outlined in Scheme 3 as representative of the oxidation and N-alkylation reactions of the P-450 model systems. In this scheme, the epoxide and the N-alkylated heme are derived from a common, electron-transfer intermediate (caged ferrylporphyrin-alkene cation radical). Collman and co-workers (28, 29) prefer a concerted mechanism (or a short-lived, acyclic intermediate) for epoxidation and N-alkylation reactions. Both authors note that the reactions catalyzed by cytochrome P-450 (and biomimetic reactions) probably can not be ascribed to any single mechanism. 

Oxidation of ]V-MeTTPFenCl (46, 52). Catalytic alkene oxidation by iron N-alkylporphyrins requires that the modified heme center can form an active oxidant, presumably at the HRP compound I level of oxidation. To show that iron N-alkyl porphyrins could form highly oxidized complexes, these reactive species were generated by chemical oxidation and examined by NMR spectroscopy. Reaction of the (N-MeTTP)FenCl with chlorine or bromine at low temperatures results in formation of the corresponding iron(III)-halide complex. Addition of ethyl- or t-butyl-hydroperoxide, or iodosylbenzene, to a solution of N-MeTTPFenCl at low temperatures has no effect on the NMR spectrum. However, addition of m-chloroperoxybenzoic acid (m-CPBA) results in the formation of iron(III) and iron(IV) products as well as porphyrin radical compounds that retain the N-substituent. 

Carbenes constitute a further class of neutral reactive species that provide a route to alkylation of the prosthetic heme group. The oxidation of sydnones by cytochrome P-450, which yields an alkyl diazonium product, results in inactivation of the enzyme (Ortiz de Montellano and Grab, 1986 Grab et al., 1988). This loss of activity is associated with the formation of (V-alkyl heme adducts best rationalized by addition of a catalytically generated carbene or carbene equivalent to the heme group (Fig. 32). Model studies have shown that diazo compounds do react with reduced iron porphyrins to give N-alkylated products (Komives et al., 1988). The details of the enzymic N-alkylation reactions remain to be defined, but evidence exists for two distinct reaction pathways. The first is... 

Grab, L.A., B.A. Swanson, and P.R. Ortiz de Montellano (1988). Cytochrome P-450 inactivation by 3-alkylsydnones Mechanistic implications of N-alkyl and N-alkenyl heme adduct formation. Biochemistry 27, 4805 814. 

Terminal Olefins and Acetylenes - The first recognized and most studied of the suicide substrates for cytochrome P-450 is 2-isopropyl-4-pentenamide (AIA). The Tt-bond of AIA is normally oxidized to the epoxide but approximately once in every 200 catalytic events oxidation of the double bond results in covalent attachment of the substrate to the prosthetic heme group.Early work showed that this alkylation yields protoporphyrin IX with AIA bound to one of the nitrogen atoms.Chemical and mass spectrometrlc evidence identified the N-alkyl group as the five- or six-membered lactone expected from intramolecular cycllzatlon of, respectively, the y- or 6-hydroxylamide obtained if an oxygen atom adds to one end and a porphyrin nitrogen to the other end of the TT-bond... 

AIA and novonal are oxidized by cytochrome P-450 to lactone metabolites that are structurally analogous to the N-alkyl group in the corresponding heme adducts.The lactone metabolite of AIA, as shown by l 0-studles, arises by intramolecular attack of the amide carbonyl on an initially formed epoxide metabolite.The oxygen incorporated into the lactone function in the heme adduct, however, derives from molecular oxygen and thus must be introduced by the catalytic action of the... 

Horseradish peroxidase does not give an iron-phenyl complex with phenylhydrazine but does react with cyclopropanone and nitromethane to yield meso alkylated products (the meso positions are marked by dots in Scheme 1),50,56,57 j io meso-alkylated heme adducts have been reported for cytochrome P-450. The factors that favor meso alkylation in horseradish peroxidase but N-alkylation in myoglobin, hemoglobin, catalase, and cytochrome P-450 remain to be elucidated. 

Halogenated Hydrocarbons - The destruction of cytochrome P-450 by CCl, first attributed to lipid peroxidation, has been shown to occur even under conditions where lipid peroxidation is not detectable.one possible explanation for this inactivation is that the trlchloromethyl radical or a related species obtained by reduction of the halocarbon reacts with the heme moiety or the apoprotein. The ill-defined radio-labeled porphyrins reported in Incubations of labeled CCI4 with hepatic microsomes would provide support for a heme alkylation mechanism were it not for the conflicting report that fluorescent N-alkylated porphyrins similar to those obtained with AIA are not isolated from CCl -incubated microsomes by procedures that result in isolation of the AIA adducts. ... 

The epoxidation of terminal alkenes is accompanied by the mechanism-based ( suicide") N-alkylation of the heme-porphyrin ring. If the TT-complex attaches to the alkene at the internal carbon, the terminal carbon of the double bond can irreversibly N-alkylate the pyrrole nitrogen of the porphyrin ring (32,33). The heme adduct formation is mostly observed with monosubstituted, unconjugated alkenes (i.e., 17a-ethylenic steroids and 4-ene metabolite of valproic acid). 

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