850
Sun et al.
MS/MS fragmentation. Theoretically, GSH could conjugate should clarify the dehydrogenation aromatase mechanism by
at different positions such as C-5 or C-6 of the indoline selective P450 enzymes, through the investigation of the
aromatic ring, but the mass spectral fragmentation pattern structural and conformational characteristics of a series of
did not permit us to establish the location of the GSH adduct. indoline derivatives, as well as the spatial and electronic
NMR could be used for this purpose, but the amounts of the
GSH adduct were too small to collect enough adduct for this
purpose.
parameters of active sites of these enzymes. In particular, it
will be important to determine whether dehydrogenation
proceeds through initial C-H bond hydrogen atom abstrac-
tion from the C-2 or C-3 positions or by nitrogen one-electron
oxidation mechanisms.
Metabolism of indoline by FMO3 produced three more me-
tabolites. Pig liver FMO N-oxidized desmethylpromethazine
to a secondary hydroxylamine that additionally formed a
nitrone (Clement et al., 1993). A similar metabolic pathway
was proposed for the oxidation of N-hydroxynorzimeldine
(Cashman et al., 1990). In those studies, the nitrone inter-
mediate decomposed to a primary hydroxylamine and an
aldehyde. Others (Rodriguez et al., 1999; Cerny and Hanzlik,
2005) have also reported the same hydroxylamine to nitrone
pathway of FMO-catalyzed oxidation of N-benzyl-N-cyclopro-
pylamine and N-deacetylketoconazole. The nitrone interme-
diates from both compounds decomposed to the primary hy-
droxylamines and the aldehydes. Our studies of the FMO3-
catalyzed oxidation of indoline also showed a sequential
N-oxidation pathway (i.e., the sequential formation of M3, a
hydroxylamine, to M2, the tautomerized nitrone). However,
instead of the nitrone decomposing to a hydroxylamine and
an aldehyde as the final products, oxidation of N-hydroxyin-
doline led to the nitrone that tautomerized to N-hydroxyin-
dole, a more stable aromatized structure.
Incubations of indoline with only FMO3 (without presence
of P450 enzymes) also produced an oxidized dimer of indoline
nitrone, [1,4,2,5]dioxadiazino[2,3-a:5,6-aЈ]diindole. This me-
tabolite (M4) was identified and characterized in this study.
A typical reaction of nitrone is the cycloaddition to other
1,3-diploes (either homo- or hetero-) (Hamer and Macaluso,
1964; Breuer, 1989). It has been known that a six-membered
cyclic nitrone, 3,4,5,6-tetrahydropyridine N-oxide, could be
dimerized by this cycloaddition mechanism as a result of
dipole-dipole interaction (Hamer and Macaluso, 1964;
Breuer, 1989). This symmetrical dimer formed quickly with-
out the help of any catalyst. We postulate that the indoline
nitrone metabolite could form a dimeric precursor to M4,
6a,7,13a,14-tetrahydro-[1,4,2,5]dioxadiazino[2,3-a:5,6-aЈ]di-
indole, which subsequently forms M4 by an additional oxida-
tion reaction. The extended conjugation of the aromatic ring
system of M4 is consistent with its visible absorbance spec-
trum. Unlike indigo or indirubin, which were formed through
oxidized indoxyl and isatin in the oxidation of indole by P450
enzymes (Gillam et al., 2000), the formation of M4 proceeds
through an indoline nitrone pathway in the oxidation of
indoline by FMO3. The M4 metabolite that was characterized
in the current work would introduce a new pigment that was
produced by FMO3 oxidation of indoline and may have po-
tential applications, such as a new method in commercial dye
production.
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