Acetylenes: cytochrome P450 oxidation and mechanism-based enzyme inactivation

Abstract

The oxidation of carbon-carbon triple bonds by cytochrome P450 produces ketene metabolites that are hydrolyzed to acetic acid derivatives or are trapped by nucleophiles. In the special case of 17α-ethynyl sterols, D-ring expansion and de-ethynylation have been observed as competing pathways. The oxidation of acetylenic groups is also associated with mechanism-based inactivation of cytochrome P450 enzymes. One mechanism for this inactivation is reaction of the ketene metabolite with cytochrome P450 residues essential for substrate binding or catalysis. However, in the case of monosubstituted acetylenes, inactivation can also occur by addition of the oxidized acetylenic function to a nitrogen of the heme prosthetic group. This addition reaction is not mediated by the ketene metabolite, but rather occurs during oxygen transfer to the triple bond. In some instances, a detectable intermediate is formed that is most consistent with a ketocarbene-iron heme complex. This complex can progress to the N-alkylated heme or revert back to the unmodified enzyme. The ketocarbene complex may intervene in the formation of all the N-alkyl heme adducts, but is normally too unstable to be detected.

Keywords: Acetylene oxidation; cytochrome P450 inactivation; ethynylsterols; iron–carbene complexes; ketene formation; oxirene.

Figure 1.

In vivo conversion of phenylacetylene to phenylacetic acid and its phenylaceturic acid derivative.

Figure 2:

Cytochrome P450 catalyzed oxidation of 4-ethynylbiphenyl (R = H) or its 2’-fluoro substituted analogue (R = F) to the acetic acid metabolites.

Figure 3.

Theoretical mechanistic alternatives for oxidation of the triple bond.

Figure 4.

Oxidation of erlotinib to a ketene that can react with a protein (Protein-XH) or be trapped by reaction with 4-bromobenzylamine.

Figure 5.

Oxidation of selegiline to a hydroxylated derivative (R = H -> R = OH) is followed by oxidation of the acetylenic group to a ketene that can be trapped by glutathione (GSH) or can acylate the P450 protein (Protein-XH).

Figure 6.

Oxidation of a terminal triple bond with concomitant migration of the hydrogen via a transition state that does not generate an oxirene intermediate. The Fe in a box of nitrogens represents the activated species of cytochrome P450.

Figure 7.

D-Homoannulation of 17α-ethynylestradiol (Abdel-Aziz and Williams 1969; Schmid et al. 1983) and a revised mechanism for this transformation that does not invoke an oxirene intermediate. The heme and activated oxygen of P450 are represented by the square of nitrogens with an iron atom.

Figure 8.

Cytochrome P450-catalyzed oxidation of efavirenz to a ring-expanded metabolite by a concerted mechanism not involving formation of an oxirene metabolite. The cytochrome P450 activated species is represented as in Figure 6.

Figure 9.

De-ethynylation of (top) norethindrone and (bottom) 17α-ethynylestradiol.

Figure 10:

A possible mechanism for the de-ethynylation of 17-hydroxy-17-ethynyl sterols.

Figure 11.

Oxidation of 1-biphenyl-1-propyne to 2-biphenylylpropionic acid by CYP1A1 and CYP1A2.

Figure 12.

Oxidation of mifepristone to a glutathione conjugate (GSH = glutathione).

Figure 13.

In vivo oxidation of dichloroacetylene to dichloroacetic acid and the mechanism proposed for its formation.

Figure 14.

Oxidative desilylation of a proinsecticide to an insecticidal terminal acetylene, where Pr stands for propyl.

Figure 15.

The structure of the adduct formed with propyne (Ortiz de Montellano et al. 1981b) after demetallation and esterification of the carboxyl groups. The four pyrrole rings of the porphyrin are labeled A-D. In this instance, the covalent bond was formed exclusively with pyrrole ring A, but other pyrrole rings can be alkylated in other adducts (e.g., Ortiz de Montellano et al. 1982a).

Figure 16.

Schematic mechanism for autocatalytic alkylation of the heme prosthetic group of cytochrome P450 in the oxidation of a terminal acetylene. Delivery of the reactive oxygen to the substituted acetylene terminus leads to heme alkylation rather than ketene formation.

Figure 17.

Structures of tBA and tBMP, and a structure proposed for the reversible intermediate formed in the reaction of the CYP2E1 T303A mutant with tBA and tBMP (Blobaum 2006).

Figure 18.

The theoretically accessible manifold of intermediates in the oxidation of a triple bond.

Figure 19.

Proposed formation of a carbene-iron complex in the oxidation of tBA (R = tert-butyl) by the CYP2E1 T303A mutant. With proton donation, the complex migrates to give a nitrogen alkylated heme adduct, but in its absence it can decompose to regenerate the parent heme.

Figure 20.

Structures of 11-dodecynoic acid (11-DDYA) and 10-undecynoic acid (10-UDYA) and the diacid metabolites formed from them by cytochrome P450.

Figure 21.

Oxidation of disubstituted acetylenes to pregnenolone by CYP11A1.